Antisense oligonucleotides for the treatment of hurler syndrome

Abstract

Described herein are antisense oligonucleotides which are capable of causing the target adenosine specific deamination to inosine in a target RNA molecule in a eukaryotic cell, wherein the oligonucleotide is for treating MPS 1, preferably Hurler syndrome, and more preferably for deaminating a portion of a premature stop codon present in a human IDUA pre-mRNA or IDUA mRNA - a target adenosine.

Description

[0001] Cross-references and citations of related applications

[0002] This PCT application claims priority to U.S. Provisional Application No. 63 / 622,296, filed January 18, 2024, the entire contents of which are incorporated herein by reference.

[0003] Citation of a sequence list submitted electronically

[0004] The contents of the electronic ST.26 sequence list (XML format; name: 0070WO01ORD_20250115xmL.xml; size: 1,064,699 bytes; creation date: January 16, 2025) submitted with this application are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to the medical field. More specifically, it relates to diseases caused by genetic defects, such as mucopolysaccharidosis type I (MPS 1), the most severe form of which is Hurler syndrome. This disclosure relates to using RNA editing technology to target (pre-)mRNA to deaminate target adenosine present in the target RNA to inosine using an antisense oligonucleotide (ASO), said antisense oligonucleotide enabling RNA editing when hybridizing with the target (pre-)mRNA. Background Technology

[0006] MPS 1 is a hereditary metabolic disorder characterized by dysfunction of α-L-iduronidase (hereinafter referred to as "iduronidase") encoded by the IDUA gene, leading to the accumulation of glycosaminoglycans (GAGs) dermatin sulfate and heparan sulfate as oligosaccharides in lysosomes. The IDUA gene has over 100 known mutations in individuals with the disease. Due to the diverse range of possible genotypes, a broad spectrum of clinical phenotypes exists, with or without neurological damage. The classic and most severe form is called Hurler syndrome (also known as MPS-IH), the intermediate form is called Hurler-Scheie, and the mildest form is called Schieie syndrome. Individuals with Hurler syndrome exhibit progressive developmental delay, corneal opacity, airway obstruction, heart disease, organ enlargement, severe joint limitation, and an untreated life expectancy of 10 years. Individuals with MPS 1 require a coordinated team of specialists to manage the symptoms that arise.

[0007] Currently, there are two clinically available treatments for MPS 1: (1) enzyme replacement therapy using laronidase and (2) hematopoietic stem cell transplantation (HSCT). Laronidase is a human recombinant enzyme administered intravenously into the bloodstream and endocytosed into cells to break down glycosaminoglycans (GAGs) that accumulate in lysosomes. Laronidase has limitations, such as its association with the blood-brain barrier, leading to the continuous progression of neurological disease, the need for lifelong repeated administration, and high cost. HSCT is an effective approach when successful, but it has a high morbidity and mortality associated with surgery, requires preparative chemotherapy, and carries the risk of graft failure. Unfortunately, neither treatment completely eliminates the clinical manifestations of the disease, and individuals with MPS 1 may still require intervention through surgery or a symptom-specific specialist.

[0008] Gene therapy (including gene editing) is a third approach and has also been explored as a potential treatment for this disease, using retroviral vectors, lentiviral vectors, and adeno-associated virus (AAV) vectors. Another therapy under investigation utilizes the CRISPR / Cas9 gene editing tool for gene therapy due to its targeting accuracy in mediating gene integration. Two mouse studies using CRISPR / Cas9 to integrate Idua cDNA with liposome complexes have shown varying degrees of success. In the first study, a liposome complex containing a CRISPR / Cas9 and Idua cDNA vector was injected into the superficial temporal vein and retained in the lungs, heart, and liver. Significant iduronate activity was detected in serum, and GAG was reduced in several organ systems, but not in the brain (Schuh et al. 2018, J. Control. Release 288:23-33). The second study using these compounds reported that treated mice showed improved facial bone structure and some recovery of respiratory function, but limited overall improvement in cardiovascular pathology (Schuh et al. 2020, GeneTher. 27:74-84).

[0009] It is generally believed that current clinical treatments for MPS1 are inadequate, resulting in residual disease burden, disability, and premature death in treated individuals. Preclinical gene therapy studies of MPS1 have demonstrated therapeutic efficacy in restoring iduronate activity, but long-term restoration of iduronate activity is required in both systemic and CNS compartments, and certain vector-based methods have limitations due to safety concerns. However, the success of gene editing in achieving the necessary iduronate concentrations to produce broad therapeutic effects in the brain and in more difficult-to-treat parts of the body, such as heart valves, spinal meninges, and cartilage, remains to be seen.

[0010] In Western societies, the incidence of Hurler syndrome is 1 in 100,000. One of the most common mutations is the guanosine-to-adenosine mutation at codon 402 of the human IDUA coding sequence, where the TGG triplet (encoding tryptophan) is mutated to TAG, which determines premature termination in the transcript. This mutant variant is also known as c.G1205A, G1205A, or c.1205G>A of the coding sequence, and is commonly referred to as the protein variants W402X, p.W402X, p.W402*, or p.Trp402Ter (see NCBI reference variant NM_000203.5), accounting for 40% of patients. In human IDUA mRNA (see SEQ ID NO: 135), position 1205 is mutated from guanosine to adenosine, which is the intermediate nucleotide of the tryptophan codon at position 402.

[0011] Notably, mutations from guanosine to adenosine allow for another therapeutic approach known as “RNA editing,” in which ASOs (also called AONs, or, when used for RNA editing, RNA editing oligonucleotides (EONs)) target and hybridize into (pre-)mRNA regions containing the mutated nucleotides. The oligonucleotides work synergistically with adenosine deaminase (ADAR) enzymes that act on RNA, either administered to the cell but preferably endogenously present within it, causing the target adenosine to deaminate to inosine (A-to-I), which is subsequently read as guanosine by the translation machine. In the case of Hurler syndrome caused by the c.1205G>A mutation, this reversal results in the production of wild-type iduronate protein.

[0012] RNA editing, in its basic form, is a natural process by which eukaryotic cells alter the sequence of their RNA molecules, typically in a site-specific and precise manner, thereby increasing the library of genome-encoded RNA 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 crucial role in managing cellular homeostasis in metazoans ranging from the simplest life forms, such as *Caenorhabditis elegcins*, to humans. Examples of RNA editing include the aforementioned A-to-I conversion and the conversion of cytidine (C) to uridine (U). The most extensively studied RNA-editing system is the ADAR enzyme.

[0013] ADARs (e.g., ADAR1; ADAR2) are multidomain proteins containing—depending on the enzyme in question—two to three double-stranded RNA recognition domains and one catalytic domain. The recognition domains recognize specific double-stranded RNA (dsRNA) sequences and / or conformations, while the catalytic domain converts adenosine at a predetermined position in the target RNA to inosine via nucleotide deamination. As mentioned above, inosine is read as guanosine by the cell's translational machinery, meaning that if the edited adenosine is located in the coding region of mRNA or pre-mRNA, it can re-encode the protein sequence. The c.1205G>A (W402X) mutation is not a natural target of ADARs.

[0014] Early attempts to force ADAR to target editing required the use of genetically engineered ADARs covalently bound to guide RNA (Montiel-Gonzalez et al. 2013, Proc Natl Acad Sci USA 110:18285-18290; Vogel et al. 2014, Angewandte Chemie Int Ed 53:267-271). While intriguing, the practical difficulty of delivering bulky modified ADAR proteins into patient cells and the uncertainty surrounding the immunogenicity of administering such proteins to patients made this system unlikely for clinical use. Another scientific publication (Woolf et al. 1995, Proc Natl Acad Sci USA 92:8298-8302) disclosed a simpler approach using relatively long single-stranded antisense RNA oligonucleotides (25-52 nucleotides in length), where the longer oligonucleotides (34-mer and 52-mer) can facilitate the editing of the target RNA through endogenous ADAR due to the double-stranded nature of the target RNA and the hybrid oligonucleotide. However, oligonucleotides appear to work only via microinjection into cell extracts or amphibian (Xenopus) oocytes, and there is a serious lack of specificity; almost all adenosine in the target RNA strand complementary to ASO is edited.

[0015] Recent developments have established that carefully crafted ASOs can be used to successfully redirect naturally occurring intracellular ADAR to edit the c.1205G>A (W402X) mutation. Early attempts using purified reagents, as described below, resulted in significant levels of RNA editing. However, for clinical availability, ASOs must survive in the in vivo environment, both inside and outside the target cells, after administration and still achieve clinically relevant levels of editing. Criteria associated with overall clinically achieved editing levels include: the ASO should possess appropriate nuclease resistance; it should hybridize to the target (pre-)mRNA with appropriate strength; it should be able to attract and bind ADAR with appropriate strength; it should rapidly achieve editing before being replaced from the target (pre-)mRNA via intracellular splicing events; and it should survive long enough to edit new (pre-)mRNA transcribed intracellularly. Work is still needed to identify ASOs with a suitable balance of these properties to ultimately achieve clinically relevant editing levels. Exemplary disclosures in this field to date include, but are not limited to, the following.

[0016] International patent application publication number WO 2017 / 220751 represents an early example of using ASO to redirect native ADAR in a Hurler syndrome mouse model. It discloses a study conducted by transfecting mRNA and ASO into immortalized mouse embryonic fibroblast (MEF) cells, culturing for 48 hours, lysing the cells, separating the supernatant, and measuring the idurolactamase activity of the supernatant. It was determined that the specific placement of the mismatch between ASO and target RNA led to improved enzyme activity, representing an increase in mRNA editing.

[0017] International Patent Application Publication No. WO 2018 / 041973 discloses a study on chemical modifications in ASOs targeting mouse Idua mRNA. Various ASOs containing sugar and / or base modifications in the central triplet (nucleotides directly opposite the target adenosine) underwent stability and activity assays. DNA nucleotides, 2'-fluorine (2'-F) modifications, and phosphate thioester (PS) bonds within the central triplet were shown to be beneficial.

[0018] International patent application publication number WO 2019 / 158475 discloses the use of computer modeling of the ASO / IDUA / ADAR complex to predict nucleotide sites on ASO that may be resistant to 2'-O-methoxyethyl (2'-MOE) modification without disrupting the complex. Several ASOs were prepared based on the predictions and tested against IDUA (pre-)mRNA.

[0019] International patent application publication number WO 2019 / 219581 discloses the use of computer modeling of ASO / IDUA / ADAR complexes to predict which bonds in ASO can tolerate PS modification without destroying the complex.

[0020] International patent application publication number WO 2020 / 165077 discloses the use of computer modeling of the ASO / IDUA / ADAR complex to predict which bonds in ASO can tolerate phosphonoacetate modification without destroying the complex, and to predict which nucleotides in ASO can tolerate unlocking nucleic acid (UNA) modification without destroying the complex.

[0021] International Patent Application Publication No. WO 2020 / 201406 discloses the use of ASOs containing methylphosphonate (MP) bond modifications. An optimized ASO (IDUA268) was found to edit up to 1.4% of RNA in mouse embryonic fibroblast assays (see Figure 8 and its related description).

[0022] International patent application publication number WO 2020 / 252376 discloses the use of cytidine analogs in ASO. Optimized sequences with C analogs (such as Benner bases) achieved up to 12% editing in primary mouse liver fibroblasts (see Figure 10 and its related description).

[0023] RNA editing of the c.1205G>A mutation in human IDUA using 111nt-long ADAR recruiting oligonucleotides has also been published (Qu et al. 2019, Nat. Biotechnol. 37:1059-1069), although some hybrid editing was observed.

[0024] Despite the aforementioned disclosures, predicting whether an ASO sequence (with or without modification) exists that could provide a balance between stability and activity, thereby producing higher activity in cells, remains challenging. Such an ASO could potentially be useful in the further development of drugs for the treatment of Hurler syndrome. Summary of the Invention

[0025] This disclosure relates to an RNA editing antisense oligonucleotide (ASO) that forms a double-stranded complex with a human IDUA RNA molecule, wherein the RNA molecule contains a target adenosine at position 1205 of SEQ ID NO: 135, optionally wherein the ASO is 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 long, wherein the ASO comprises the following nucleotide sequence:

[0026] ,

[0027] in:

[0028] i) m5Ce is a 5-methyl-cytidine containing 2'-O-methoxyethyl (2'-MOE) ribose substitution;

[0029] ii) Zd is a deoxyribonucleoside that is directly opposite to the target adenosine and contains the Benner base;

[0030] iii) Ad is deoxyadenosine;

[0031] iv) Gm is a guanosine nucleoside containing a 2'-O-methyl (2'-OMe) ribose substitution;

[0032] v) Af is an adenosine nucleoside containing a 2'-fluoro(2'-F) ribose substitution;

[0033] vi) Ge is a guanosine containing a 2'-MOE ribose substitution;

[0034] vii) This represents the nucleoside bond between phosphodiester (PO);

[0035] viii) This indicates the nucleoside bond between phosphate thioesters (PS); and

[0036] ix) This represents the internucleotide bond between methylphosphonate (MP) nucleotides.

[0037] The nucleoside position numbering makes Z a nucleoside position 0, and the inter-nucleoside bond numbering makes the 5' bond of Z a bond number 0. In ASO, the nucleoside position and bond position increase positively (+) towards the 5' end and negatively (-) towards the 3' end.

[0038] In one implementation, the ASO disclosed herein comprises a nucleotide sequence:

[0039]

[0040] Wherein X and Y are nucleotides containing adenine, guanine, thymine, uracil, hypoxanthine, or cytosine bases, or derivatives or analogs of any of these nucleobase moieties as discussed herein.

[0041] In one embodiment, the ASO disclosed herein consists of the following nucleotide sequence:

[0042] ,

[0043] Wherein X and Y are nucleotides containing adenine, guanine, thymine, uracil, hypoxanthine, or cytosine bases, or any derivatives or analogs of these nucleobase moieties, as discussed herein.

[0044] This disclosure relates to an ASO capable of forming a double-stranded complex with a portion of a human IDUA transcript molecule in a human cell, wherein the double-stranded complex can recruit an endogenous (=naturally occurring) ADAR enzyme present in the cell, wherein the portion contains adenosine as a target for deamination by the ADAR enzyme, and wherein the ASO contains the nucleotide sequence disclosed herein.

[0045] In one embodiment, the ASO disclosed herein comprises: i) a nucleotide at position +2 that forms a swing base pair with the RNA molecule; ii) 2'-F or 2'-OMe ribose substitution at nucleotide positions +2, +3, +4, +5, +6, +7, +8, +10, -5, -6, -7, -8, -9, -10, -11, and / or -13; iii) 2'-OMe substitution at nucleotide positions +9 and / or -12; iv) 2'-OMe or 2'-MOE substitution at nucleotide positions +11, +12, +13, +14, and / or +15; v) a PS bond at bond positions +1, +2, +3, +4, +5, +6, +7, +8, +9, -5, -6, -7, -9, -10, -11, and / or -12; vi) The PS or PNdmi key at key position -8 and / or -13; vii) the PS or PO key at key positions +10, +11, +12 and / or +13; and / or viiii) the PS, PNdmi or PO key at key position +14.

[0046] In one implementation, the ASO disclosed herein comprises a nucleotide sequence:

[0047] (SEQ ID NO: 136).

[0048] In one embodiment, the ASO disclosed herein comprises at least one nucleotide, said nucleotide including one or more additional non-naturally occurring chemical modifications in the ribose, bond, or base portion, and said one or more additional ribose modifications are selected from: deoxyribose (DNA), unlocked nucleic acid (UNA), and 2'-F.

[0049] In one embodiment, the one or more additional modifications are bond modifications selected from the group consisting of PS, 3'-methylenephosphonate, 5'-methylenephosphonate, 3'-phosphatidyl ester, and 2'-5'-PO.

[0050] In one implementation, ASO One or more nucleotides outside the motif contain additional modifications, which are mono- or di-substituted substitutions at the 2', 3', and / or 5' positions of the sugar, selected from the group consisting of: -OH; -F; substituted or unsubstituted, straight-chain or branched lower (C1-C2) nucleotides. 10 Alkyl, alkenyl, alkynyl, alkylaryl, allyl or aralkyl, which may be intercalated with one or more heteroatoms; -O-, S- or N-alkyl (e.g., -O-methyl); -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.

[0051] In one implementation, the ASO disclosed herein comprises a nucleotide sequence:

[0052] (SEQ ID NO: 213), where:

[0053] i) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, wherein the uracil nucleobase is optionally 5-methyluracil (thymine) nucleobase;

[0054] ii) The nucleotides at positions +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, -7, -8, -9, -10, -11, and -13 each independently contain a 2'-OMe, 2'-MOE, or 2'-F ribose substitution;

[0055] iii) The nucleotides at positions +9 and -12 contain a 2'-OMe ribose substitution; and

[0056] iv) "~" indicates a nucleoside inter-bond containing a PS bond, PNdmi bond, or PO bond.

[0057] In one implementation, the ASO disclosed herein comprises a nucleotide sequence:

[0058] (SEQ ID NO: 214), where:

[0059] i) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, wherein the uracil nucleobase is optionally 5-methyluracil (thymine) nucleobase;

[0060] ii) The nucleotides at positions +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10, and -11 each independently contain either a 2'-MOE or a 2'-F ribose substitution;

[0061] iii) The nucleotides at positions +10, +11, +12, and +13 each independently contain either a 2'-OMe or a 2'-MOE ribose substitution;

[0062] iv) The nucleotides at positions +9, -12, and -13 contain 2'-OMe ribose substitutions;

[0063] v) “=" indicates a nucleoside internucleotide bond containing a PO or PS bond; and

[0064] vi) "%" indicates a nucleoside inter-bond containing a PS or PNdmi bond.

[0065] In one embodiment, the ASO disclosed herein comprises or consists of a nucleotide sequence and chemical modification of any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 180, 181, 182, 183, 184, 185, 191, 194, or 195.

[0066] In one embodiment, the ASO disclosed herein includes a delivery portion. A preferred delivery portion is N-acetylgalactosamine (GalNAc). In one embodiment, the GalNAc is of formula I, II, III(b), IV, or V. In a preferred embodiment, the GalNAc is of formula I:

[0067]

[0068] Formula I

[0069] The ASO is connected to the connection point E of Formula I, optionally via a connector, as disclosed herein.

[0070] In one embodiment, the ASO disclosed herein comprises or consists of a nucleotide sequence and chemical modifications thereof from any of SEQ ID NO: 67, 68, 69, 70, 71, 72, 99, 100, 101, 102, 103, 104, 110, 113, 114, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, or 179. These ASOs comprise a GalNAc portion, which can be any GalNAc portion known to those skilled in the art, and preferably the GalNAc portion disclosed herein (e.g., Formula I also referred to as "L004"), optionally coupled to the ASO via a connector, which can be any suitable connector known to those skilled in the art, and preferably the connector disclosed herein. The GalNAc portion can be coupled to the ASO at its 5' end and / or its 3' end, and preferably at its 3' end. The GalNAc portion is preferably a three-antennae GalNAc portion.

[0071] In one embodiment, the ASO comprises or consists of a nucleotide sequence and chemical modifications of any one of SEQ ID NO: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 or 149.

[0072] In one embodiment, the ASO comprises or is composed of a nucleotide sequence and chemical modifications of any of SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99. These ASOs comprise a GalNAc portion, which can be any GalNAc portion known to those skilled in the art, and preferably a GalNAc portion disclosed herein (e.g., Formula I also known as “L004”), optionally conjugated to the ASO via a linker, which can be any suitable linker known to those skilled in the art, and preferably a linker disclosed herein. The GalNAc portion may be conjugated to the ASO at its 5' end and / or its 3' end, and preferably at its 3' end. The GalNAc portion is preferably a triantennae GalNAc portion.

[0073] In one embodiment, the ASO disclosed herein comprises the nucleotide sequence of SEQ ID NO: 16 and chemical modifications. In a preferred embodiment, the ASO disclosed herein consists of the nucleotide sequence of SEQ ID NO: 16 and chemical modifications. In one embodiment, the ASO according to SEQ ID NO: 16 is conjugated to a delivery portion. In one embodiment, the conjugation is at its 3' end. In one embodiment, the conjugation is at its 3' end and via a linker. In some embodiments, the delivery portion is GalNAc, such as, but not limited to, GalNAc of Formula I (see above).

[0074] In one implementation, the ASO disclosed herein includes Figure 27 The structure of formula (A) disclosed in the document may be composed of it.

[0075] In one implementation, the ASO disclosed herein includes this document and Figure 28 The structure of formula (B) disclosed in the document may be composed of it.

[0076] In one implementation, the ASO disclosed herein includes Figure 29 The structure of formula (C) disclosed in the document is or is composed of it.

[0077] Figure 1A SEQ ID NO: 1 is a portion of a human IDUA transcript containing the c.1205G>A mutation. The ASO of this disclosure can form a double-stranded complex with this portion of the human IDUA transcript (which may be pre-mRNA or mRNA) and deamination adenosine at position 1205 (in SEQ ID NO: 135) to inosine by recruiting an endogenous (also referred to as “naturally occurring” or “naturally occurring”) enzyme with deamination activity (such as ADAR2) that recognizes and binds to the double-stranded complex.

[0078] This disclosure also relates to pharmaceutical compositions comprising the ASO disclosed herein and a pharmaceutically acceptable carrier.

[0079] This disclosure also relates to the ASO or pharmaceutical composition disclosed herein for the treatment of mucopolysaccharidosis type 1 (MPS 1), preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene.

[0080] This disclosure also relates to the use of the ASO or pharmaceutical composition disclosed herein in the preparation of a medicament for treating MPS 1, preferably Hurler syndrome caused by the c.1205G>A mutation in the human IDUA gene.

[0081] This disclosure also relates to a method for treating MPS 1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene, comprising administering a therapeutically effective amount of the disclosed ASO or the disclosed pharmaceutical composition to a patient in need. In one embodiment, the administration step is via subcutaneous delivery. In one embodiment, the administration step is to the central nervous system, optionally via intrathecal delivery. In one embodiment, the administration step is via intrathecal delivery.

[0082] This disclosure also relates to a method for deamination of a target adenosine present in a target RNA molecule in a cell, wherein the target RNA molecule is human IDUA pre-mRNA or mRNA or a portion thereof, wherein the target adenosine is the c.1205G>A mutation in SEQ ID NO: 135, the method comprising the steps of: i) contacting the cells with the ASO disclosed herein or the pharmaceutical composition disclosed herein under conditions suitable for: a) cell uptake of the ASO; b) annealing of the ASO with the target RNA molecule; and c) deamination of the target adenosine in the target RNA molecule to inosine by a naturally occurring mammalian ADAR enzyme present in the cell; and ii) optionally treating the cellular pre-mRNA or mRNA to determine the presence of inosine at the target adenosine site in the target RNA molecule. In one implementation, optional step (ii) includes: a) determining the sequence of the target RNA molecule; b) assessing the presence of functional, extended, full-length, and / or wild-type iduronate protein; or c) using a functional readout, wherein the deamination of the target RNA encodes a functional, full-length, extended, and / or wild-type iduronate protein. Attached Figure Description

[0083] The advantages, effects, features, and purposes beyond those described above will become more apparent upon reading the following detailed description. This detailed description refers to the following accompanying figures, in which:

[0084] Figure 1AA partial sequence of the target human IDUA RNA molecule carrying the c.1205G>A mutation that causes Hurler syndrome is shown (top sequence; 3' to 5'; target adenosine A is shown in bold; SEQ ID NO: 1). The ASO sequence disclosed herein is shown below SEQ ID NO: 1 (5' to 3'). The chemical modifications are as follows: Ae and Ge are 2'-MOE-modified adenosine and guanosine, respectively; Cm, Am, Um, and Gm are 2'-OMe-modified cytidine, adenosine, uridine, and guanosine, respectively; Gf, Cf, Af, and Uf are 2'-F-modified guanosine, cytidine, adenosine, and uridine, respectively; m5Ce is 2'-MOE-modified 5-methylcytidine; Zd (orphan nucleotide), shown in bold, is a deoxynucleotide carrying a Benner base (a deoxycytidine analog); Ad is deoxyadenosine; Te is 2'-MOE-modified thymidine (equivalent to 5-methyluridine with 2'-MOE substitution, also represented as m5Ue here and elsewhere); "!" indicates a PNdmi bond; "Refers to the MP key;" "Refers to the PS bond. All other internucleotide bonds are PO bonds. The basic sequence of ASO shown in SEQ ID NO: 2, 5 to 37, 67 to 72, 86 to 98 and 99 to 113, without any chemical modification of sugars or bonds, is 5'- T GCGACACUUCGGUCZAGAGCUGCUCCUC-3' (SEQ ID NO:64), where the underlined T can be referred to as T or U depending on the modification, as described above, and where the bold Z is a nucleotide carrying a Benner base. The basic sequence of ASO shown as A-26, without any chemical modifications of sugar or bonds, is 5'-CGACACUUCGGUCZAGAGCUGCUCCUC-3' (SEQ ID NO:65), where the bold Z is a nucleotide carrying a Benner base. The basic sequence of ASO shown as A-32, without any chemical modifications of sugar or bonds, is 5'-C T CGACACUUCGGUCZAGAGCUGCUCCUC-3' (SEQ ID NO:66), wherein the underlined T may be referred to as T or U depending on the modification, as described above, and wherein the bold Z is a nucleotide carrying a Benner base. Figure 1B Showing with Figure 1AThe ASO provided is from the same group as described above, except that a GN is attached to the 3' end. The GN is a delivery portion, preferably an N-acetylgalactosamine (“GalNAc” or “GN”) portion, more preferably a tri-antennae GalNAc portion (e.g., but not limited to those disclosed in International Patent Application Publication No. WO 2022 / 271806). Other non-limiting embodiments of the GN are described in International Patent Application Publications Nos. WO 2009 / 073809, WO 2014 / 025805, WO 2015 / 006740, WO 2019 / 053661, WO 2014 / 179620, WO 2021 / 261998 and U.S. Patent Application Publication No. 2019 / 0256849. Figure 1C The mouse Idua-targeting ASO ASO mIdua-85-A to -M are shown, corresponding to A-50 (targeting human IDUA) carrying the GalNAc moiety and containing a chiral pure PS bond (represented here by "@", which can be Rp or Sp), as disclosed in International Patent Application No. WO 2019 / 219581. ASOmIdua-85-A to -M correspond to A-50 GN-A to M of the depicted human IDUA-targeting transcripts. Figure 1D Displayed is SEQ ID NO: 135, which represents the full-length mRNA sequence (5' to 3') of the human IDUA gene with the W402X UAG stop codon (underlined), which contains the c.1205G>A mutation, where the uppercase A is indicated in bold. Figure 1E The sequences of ASO A-50-A to -M are shown, which have the same ASO sequences and modifications as A-50 GN-A to -M (see...). Figure 1C ), but does not include the GalNAc(GN) part.

[0085] Figure 2A , Figure 2B , Figure 2C and Figure 2D It shows that there is Figure 1A The results of in vitro biochemical editing assays were used to evaluate the percentage of target human IDUA edited under several ASO conditions specified in the study.

[0086] Figure 3A , Figure 3B and Figure 3C The results of cell-based assays were shown, and the assays were evaluated by... Figure 1A The percentage of target human IDUA edited by naturally occurring ADAR in cells recruited by ASO in a live-cell environment.

[0087] Figure 4A portion of the mouse Idua transcript (SEQ ID NO: 134; 3' to 5') is shown, which is similar to Figure 1A and Figure 1D The region of the human IDUA transcript containing the G>A mutation that causes Hurler syndrome, shown here, is also in bold. Below the target sequence is the ASO sequence (5' to 3') used in in vivo mouse experiments. Chemical modifications are as follows: Figure 1A As described above. GN represents the 3'-binding triantennae GalNAc portion as described in International Patent Application Publication No. WO 2022 / 271806. Scr1 is a disordered oligonucleotide used as an editing negative control.

[0088] Figure 5A The application of such Figure 4 The percentage of edits in the liver of W392X mice after fourteen ASOs with GalNAc portions and out-of-order ASOs (with GN) is shown. The numbers on the X-axis represent... Figure 4 The ASO number. Calculate the percentage and relative to the wild-type level set to 100%. Figure 5B This study showed a recovery of idurolactamase activity in the same experiment (day 14), with an increase in activity to approximately 4.5% of wild-type activity compared to wild-type mice. This represents a more or less significant improvement compared to the residual activity observed in mutant mice (represented by the Scr control). Figure 5A The same overall results are shown, with ASO mIdua-75GN, mIdua-89GN, mIdua-88GN, mIdua-85GN, and mIdua-87GN showing the best performance. Figure 5C It showed the recovery of iduronate activity in the plasma of the same mice, showing the same... Figure 5A and Figure 5B The overall pattern is similar to that shown.

[0089] Figure 6 The in vivo study dosing regimens using different schemes and different amounts of mIdua-75 and mIdua-85 ASO (both partially conjugated to GalNAc (GN)) are shown.

[0090] Figure 7A Group 1 was shown (see Figure 6 The average percentage of edits in mice. Figure 7B The average idurolactone enzyme activity of this group over 4 weeks is shown. The y-axis on the right shows the percentage of activity compared to the wild type (100%).

[0091] Figure 8AThe mean percentage of edits at 4 weeks is shown for mice in group 2 (administered 10 mg / kg) and group 3 (administered 30 mg / kg). EON 11 refers to mIdua-85GN, see Table 1. Figure 8B The mean idurolactamase activity of these groups over 4 weeks is shown. The y-axis on the right shows the percentage of activity compared to the wild type (100%).

[0092] Figure 9A The mean percentage of edits was shown for mice in group 3 (receiving 30 mg / kg mIdua-85GN (EON 11)) and group 4 (receiving 30 mg / kg mIdua-75GN (EON 6)) after 4 weeks. Figure 9B The mean idurolactamase activity of these groups over 4 weeks is shown. The y-axis on the right shows the percentage of activity compared to the wild type (100%).

[0093] Figure 10A The mean percentage of edits was shown in group 2 (receiving 10 mg / kg mIdua-85GN (EON 11) twice weekly) and group 5 (receiving 10 mg / kg mIdua-85GN (EON 11) once weekly) mice after 4 weeks. Figure 10B The mean idurolactamase activity of these groups over 4 weeks is shown. The y-axis on the right shows the percentage of activity compared to the wild type (100%).

[0094] Figure 11A The mean percentage of edits per dose of 10 mg / kg mIdua-85GN (EON 11) is shown for mice in Group 2 (autopsy at 4 weeks after dosing twice weekly), Group 6 (autopsy at 8 weeks after dosing twice weekly for the first two weeks, and then at 8 weeks after dosing every two weeks from week 3 to week 8). Figure 11B The average idurolactamase activity of these groups at the end of the protocol is shown. The y-axis on the right shows the percentage of activity compared to the wild type (100%).

[0095] Figure 12A The levels of GAG in the livers of all seven groups of mice at the time of autopsy are shown. NT represents untreated mutant mice as controls; WT represents the GAG ​​levels observed in wild-type mice, and WT(NT) represents the GAG ​​levels observed in untreated wild-type mice. Figure 12B The table shows GAG levels in urine (µg / mg tissue) across all groups at the end of the protocol. 8 (WT) represents the GAG ​​levels observed in the urine of a group of wild-type mice. NT shows the GAG ​​levels in untreated mutant mice.

[0096] Figure 13The percentage of human IDUA target adenosine edited in human iPSC-derived hepatocytes after incubation with six specified ASOs (bound to the GalNAc moiety (GN) or unbound) for 2 days (12 bars on the left, squares), 5 days (12 bars in the middle, triangles), and 7 days (12 bars on the right; dots). The sequences and modifications of the specified ASOs used are shown in [link to ASO list]. Figure 1A .

[0097] Figure 14A The HPLC chromatograms of A-50GN before and after treatment with various nucleases are shown in the biochemical stability assay. Figure 14B The peaks in the top chromatogram show the control sample treated with an inactive biostability assay, while the bottom chromatograms show the same sample after the biostability assay treatment. Figure 14A The peak with a retention time of 9.10 minutes in the top chromatogram is A-50GN. Figure 14A The bottom chromatogram showed the same peak at a retention time of 9.12 minutes, but more degradation products were observed after treatment. Figure 14B The chromatograms are shown in the same order and under the same treatment, but this experiment was performed using A-78GN. This indicates that the molecule is less susceptible to degradation than A-50GN.

[0098] Figure 15 The HPLC chromatograms generated after the stability determination show the percentage stability of A-50 GN (A-50 here), A-78 GN (A-78 here), mIdua-85, and mIdua-88 as a bar chart.

[0099] Figure 16A A set of ASOs based on A-50 GN (SEQ ID NO: 69) is shown, with varying bond modifications to determine the stability of the ASOs based on the presence of PO and PS bonds at different positions. Different positions of A-50 were also replaced with PNdmi bonds to determine the stability introduced by such bond modifications. Chemical modifications such as... Figure 1A 。 " " indicates a PO bond; "#" indicates a PNMS bond; Cd is deoxycytidine. L004 is associated with... Figure 4 The same GalNAc portion (GN) described herein, and may be replaced by any preferred GalNAc portion disclosed herein. Figure 16B Showing with Figure 16A ASOs in the same group, but represented by the '-X' symbol, and both lack the GalNAc part at the 3' end.

[0100] Figure 17The percentage of editing in primary human W402X fibroblasts (GM00798) after transfection with the specified ASO, as determined by ddPCR, is shown. A-50 (SEQ ID NO: 16), A-50 GN (SEQ ID NO: 69), and A-78 GN (SEQ ID NO: 70) served as positive controls. Simulated transfection served as a negative control. The specific chemical modifications and sequence of the ASO are shown in Figure 16.

[0101] Figure 18 The in vivo study dosing regimens using mIdua-85, mIdua-75, and mIdua-89 ASO (unconjugated and partially conjugated with GalNAc (GN)) are shown; see also Figure 6 The dosage of ASO lacking the GalNAc portion was adjusted in mg / kg, as shown in the figure. Control group 1 received no administration, but urine / blood samples were collected at 1 week (-1), weeks 2, 4, and 6, and at the final autopsy (week 8). Group 2 received only the carrier solution. Group 3 received a randomized control ASO conjugated with the GalNAc portion. Group 18 received mIdua-85 ASO formulated in lipid nanoparticles (LNPs). Administration to groups 2 through 17 was subcutaneous (SC), while group 18 received the LNP formulation intravenously (IV). Group 1 consisted of wild-type C57BL / 6j mice, and groups 1 through 18 consisted of Idua-W392X model mice.

[0102] Figure 19A The percentage of mouse Idua target transcripts edited at necropsy in mouse livers at 4 weeks (group 5), 8 weeks (group 4), 12 weeks (group 6), 16 weeks (group 7), and 20 weeks (group 8) after administration of mIdua-85GN, and 8 weeks (groups 9 and 10) after administration of mIdua-75GN and mIdua-89GN, respectively, is shown compared with the wild-type case set at 100% (although it was not edited itself) (group 1) and two other negative control groups at 8 weeks. Figure 19B The results are shown in the arm that does not exist in GalNAc (here referred to as 'naked', meaning only the arm lacking the GalNAc portion). Figure 19C Showing with Figure 19A The mean Idua enzyme activity in the liver at autopsy of the same GalNAc group is shown. Figure 19D Showing with Figure 19B The mean Idua enzyme activity in the liver at autopsy of the same group without GalNAc (here, 'naked'). Figure 19E Showing with Figure 19AThe images show GAG levels in the liver at autopsy in the same GalNAc group. Low levels of GAG accumulation in wild-type mice (group 1) are shown as hollow bars. The right axis shows the percentage reduction in GAG levels compared to the disordered control. Figure 19F Showing with Figure 19B The same data were shown for the GAG ​​levels in the liver at autopsy in the GalNAc (here, 'naked') group.

[0103] Figure 20A The percentage of mouse Idua target transcripts edited at necropsy in mouse kidneys at 4 weeks (group 5), 8 weeks (group 4), 12 weeks (group 6), 16 weeks (group 7), and 20 weeks (group 8) after administration of mIdua-85GN, and 8 weeks (groups 9 and 10) after administration of mIdua-75GN and mIdua-89GN, respectively, is shown compared with the wild-type case set at 100% (although it was not edited itself) (group 1) and two other negative control groups at 8 weeks. Figure 20B The results are shown in the arm that does not exist in GalNAc (here referred to as 'naked', meaning only the arm lacking the GalNAc portion). Figure 20C Showing with Figure 20A The mean Idua enzyme activity in the kidneys of the same GalNAc group at autopsy time is shown. Figure 20D Showing with Figure 20B The mean Idua enzyme activity in the kidneys at autopsy of the same group without GalNAc (here, 'naked'). Figure 20E Showing with Figure 20A The figures show GAG levels in the kidneys at autopsy in the same GalNAc group. Low levels of GAG accumulation in wild-type mice (group 1) are shown as hollow bars. The right axis shows the percentage reduction in GAG levels compared to the disordered control. Figure 20F Showing with Figure 20B The same GalNAc (here, 'naked') group showed GAG levels in the kidneys at autopsy.

[0104] Figure 21A The percentage of mouse Idua target transcripts edited at necropsy in mouse spleens at 4 weeks (group 5), 8 weeks (group 4), 12 weeks (group 6), 16 weeks (group 7), and 20 weeks (group 8) after administration of mIdua-85GN, and 8 weeks (groups 9 and 10) after administration of mIdua-75GN and mIdua-89GN, respectively, is shown compared with the wild-type case set at 100% (although it was not edited itself) (group 1) and two other negative control groups at 8 weeks. Figure 21B The results are shown in the arm that does not exist in GalNAc (here referred to as 'naked', meaning only the arm lacking the GalNAc portion).

[0105] Figure 22AThe percentage of mouse Idua target transcripts edited at necropsy in mouse lungs at 4 weeks (group 5), 8 weeks (group 4), 12 weeks (group 6), 16 weeks (group 7), and 20 weeks (group 8) after administration of mIdua-85GN, and 8 weeks (groups 9 and 10) after administration of mIdua-75GN and mIdua-89GN, respectively, is shown compared with the wild-type case set at 100% (although it was not edited itself) (group 1) and two other negative control groups at 8 weeks. Figure 22B The results are shown in the arm that does not exist in GalNAc (here referred to as 'naked', meaning only the arm lacking the GalNAc portion). Figure 22C Showing with Figure 22A and Figure 22B The mean Idua enzyme activity in the lungs at autopsy of the same GalNAc and 'naked' (GalNAc-free) groups is shown. Figure 22D Showing with Figure 22C GAG levels in the lungs of the same groups at autopsy. Low levels of GAG accumulation in wild-type mice (group 1) are shown as hollow bars. The right axis shows the percentage reduction in GAG levels compared to the disordered control.

[0106] Figure 23A The percentage of mouse Idua target transcripts edited at necropsy in mouse hearts at 4 weeks (group 5), 8 weeks (group 4), 12 weeks (group 6), 16 weeks (group 7), and 20 weeks (group 8) after administration of mIdua-85GN, and 8 weeks (groups 9 and 10) after administration of mIdua-75GN and mIdua-89GN, respectively, is shown compared with the wild-type case set at 100% (although it was not edited itself) (group 1) and two other negative control groups at 8 weeks. Figure 23B The results are shown in the arm that does not exist in GalNAc (here referred to as 'naked', meaning only the arm lacking the GalNAc portion). Figure 23C Showing with Figure 23A and Figure 23B The mean Idua enzyme activity in the heart at autopsy was shown in the same GalNAc and 'naked' (GalNAc-free) groups. Figure 23D Showing with Figure 23C GAG levels in the heart at autopsy in the same groups are shown. Low levels of GAG accumulation in wild-type mice (group 1) are shown as hollow bars. The right axis shows the percentage reduction in GAG levels compared to the disordered control.

[0107] Figure 24AThe percentage of mouse Idua target transcripts edited at necropsy in the quadriceps femoris muscle of mice at 4 weeks (group 5), 8 weeks (group 4), 12 weeks (group 6), 16 weeks (group 7), and 20 weeks (group 8) after administration of mIdua-85GN, and 8 weeks (groups 9 and 10) after administration of mIdua-75GN and mIdua-89GN, respectively, is shown compared with the wild-type case set at 100% (although it was not edited itself) (group 1) and two other negative control groups at 8 weeks. Figure 24B The results are shown in the arm that does not exist in GalNAc (here referred to as 'naked', meaning only the arm lacking the GalNAc portion). Figure 24C Showing with Figure 24A and Figure 24B The mean Idua enzyme activity in the quadriceps femoris muscle at autopsy is shown in the same GalNAc and 'naked' (GalNAc-free) groups.

[0108] Figure 25 The mean Idua enzyme activity in mouse plasma is shown at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8) after administration of mIdua-85GN, and at 8 weeks (Groups 9 and 10, respectively) after administration of mIdua-75GN and mIdua-89GN, compared with the wild-type case set at 100% (although not edited itself) (Group 1) and two other negative control groups at 8 weeks. Results in the arm without GalNAc (referred to here as 'naked', meaning only lacking the GalNAc portion) are also shown.

[0109] Figure 26A The levels of GAG in urine samples treated with ASO containing GalNAc in the designated group at the time of autopsy are shown. Figure 26B The same ASOs are shown, but for those lacking the GalNAc portion. Wild-type levels (hollow bars) represent GAG levels compared to the disordered control, with the average level set to 100%. RM4870 = mIdua-85GN, RM5306 = mIdua-75GN, RM4772 = mIdua-89GN, RM4347 = mIdua-85, RM4337 = mIdua-75, and RM4351 = mIdua-89. Figure 26C This displays the time progression of GAG levels from the start (T=0) to week 20 after application of mIdua-85GN. The levels are compared to an out-of-order level set to 100% (not shown).

[0110] Figure 27 The structure of ASO A-36 GN (SEQ ID NO: 67; Formula (A)) is shown.

[0111] Figure 28 The structure of ASO A-50 GN (SEQ ID NO: 69; Formula (B)) is shown.

[0112] Figure 29 The structure of ASO A-94 GN (SEQ ID NO: 72; Formula (C)) is shown. Detailed Implementation

[0113] This disclosure relates to ASOs (also referred to herein and elsewhere as AONs or EONs) and their use in the treatment of diseases, particularly MPS1, and more particularly Hurler syndrome. It should be noted that the oligonucleotides described herein do not edit RNA themselves, although this name is sometimes used. When an oligonucleotide attaches to its target region, it provides an environment in which an endogenous ADAR enzyme (preferably ADAR2) can bind to the double-stranded RNA complex and specifically deamination the target A to I, simply because the oligonucleotides described herein hybridize / bind to their target sequence. The ASOs described herein target specific A's in human IDUA pre-mRNA or mRNA and cause these A's to be deamination to I, which is read as G in translation. This disclosure, for example, but not limited to, is applicable to editing the W402X mutation in human IDUA transcripts, restoring an early termination codon to a codon encoding tryptophan. Repairing (or deaminating) the A in the W402X mutation to I will result in the translation of a wild-type protein.

[0114] The ASO described herein does not contain the ADAR recruitment portion as described in International Patent Application Publication No. WO 2016 / 097212. Nor does the ASO described herein contain portions that can form intramolecular stem-loop or hairpin structures that are not complementary to the target sequence, as seen in oligonucleotides used in CRISPR / Cas9 systems (e.g., International Patent Application Publication No. WO 2019 / 005884). The ASO described herein is not covalently linked to an enzyme of the deamination target nucleotide before entering the target cell, but can form a complex with such enzyme entities (preferably ADAR) present in the target cell (thus recruiting, interacting, and forming a complex with endogenous ADAR enzymes). The ASO described herein is shorter than such oligonucleotides in the prior art, making them less expensive to produce, easier to use (likely due to increased and more efficient cell entry and transport), and easier to manufacture. International patent applications WO 2017 / 220751 and WO 2018 / 041973 disclose ASO complementary to target RNA for deamination of target A present in the target RNA sequence complementary to ASO, but lack a recruitment component while still being able to utilize ADAR enzymes present in the cell to edit target A. This disclosure utilizes this knowledge to address the problem of targeting Hurler syndrome mutations, preferably c.1205G>A (W402X) mutations.

[0115] As described above, this disclosure relates to an ASO that forms a double-stranded complex with a human IDUA RNA molecule, wherein the RNA molecule contains a target adenosine at position 1205 of SEQ ID NO: 135, SEQ ID NO: 135 representing a c.G1205A mutant variant of the human IDUA mRNA sequence, optionally wherein the ASO is 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 length, wherein the ASO comprises the following nucleotide sequence:

[0116] ,

[0117] in:

[0118] i) m5Ce is a 5-methyl-cytidine containing 2'-O-methoxyethyl (2'-MOE) ribose substitution;

[0119] ii) Zd is a deoxyribonucleoside that is directly opposite to the target adenosine and contains the Benner base;

[0120] iii) Ad is deoxyadenosine;

[0121] iv) Gm is a guanosine nucleoside containing a 2'-O-methyl (2'-OMe) ribose substitution;

[0122] v) Af is an adenosine nucleoside containing a 2'-fluoro(2'-F) ribose substitution;

[0123] vi) Ge is a guanosine containing a 2'-MOE ribose substitution;

[0124] vii) This represents the nucleoside bond between phosphodiester (PO);

[0125] viii) This indicates the nucleoside bond between phosphate thioesters (PS); and

[0126] ix) This represents the internucleotide bond between methylphosphonate (MP) nucleotides.

[0127] The nucleoside position numbering makes Z a nucleoside position 0, and the inter-nucleoside bond numbering makes the 5' bond of Z a bond number 0. In ASO, the nucleoside position and bond position increase positively (+) towards the 5' end and negatively (-) towards the 3' end.

[0128] It has been surprisingly discovered that this " "Modal ASO showed improved editing activity."

[0129] As used herein, the term "central triplet" refers to the three nucleotides opposite target A in the target RNA, with the middle nucleotide of the central triplet directly opposite target A. The central triplet is not necessarily located at the center of the ASO, as it can be located closer to the 3' end of the ASO or towards the 5' end, whichever is preferred for a particular target. Therefore, in this respect, the term "central" refers more to the triplet located at the center of catalytic activity when chemical modification and targeting target A are involved. It should also be noted that the ASO is sometimes depicted from 3' to 5', especially when the target sequence is shown from 5' to 3'. However, whenever the sequence of nucleotides within the ASO is discussed herein, it is always from 5' to 3' of the ASO. Positions can also be indicated based on specific nucleotides within the ASO, while still following the 5' to 3' directionality, in which case the other nucleotides at 5' are labeled as positive and the 3' as negative.

[0130] In this disclosure, the central triplet contains the motif " c -Z*A”, where Z is the Benner base (also known as “2’-deoxyBenner base Z” or “dZ”; see Yang et al. (2006) Nucl Acid Res 34:6095-6101). The Benner base is a cytidine analog. The Benner dZ nucleotide (6-amino-5-nitro-3-yl-2(1’-β-d-2’-deoxyfuranose)-2(1H)-pyridone) has been shown to provide the optimal hydrogen bonding mode for interacting with glutamate residues present on the ADAR flip ring, resulting in an increased in vitro deamination rate (Doherty et al. (2021) J. Am. Chem. Soc. 143:6865-6876). The structure of the Benner base is shown below, where the lower link is a 1-position link to the ribose ring:

[0131]

[0132] In this disclosure, the nucleosides constituting ASO are numbered by position, wherein the nucleoside position numbering makes Z (i.e., the nucleoside directly opposite target A in the target RNA molecule) nucleoside position 0 (orphan nucleotide), and the inter-nucleoside bond numbering makes the 5' bond of Z bond number 0, wherein the nucleoside position and bond position in ASO increase positively (+) toward the 5' end and negatively (-) toward the 3' end.

[0133] This document discloses an ASO, wherein the ASO comprises or is composed of the following nucleotide sequences: , where X and Y are nucleotides containing adenine, guanine, thymine, uracil, hypoxanthine, or cytosine bases.

[0134] In some embodiments, the ASO further comprises at least one modified nucleoside interbond and at least one nucleotide comprising a modified sugar moiety.

[0135] In one implementation, the ASO disclosed herein includes:

[0136] i) Nucleosides at position +2 that form a swinging base pair with RNA molecules;

[0137] ii) 2'-F or 2'-OMe ribose substitution at nucleoside positions +2, +3, +4, +5, +6, +7, +8, +10, -5, -6, -7, -8, -9, -10, -11 and / or -13;

[0138] iii) 2'-OMe substitution at nucleoside positions +9 and / or -12;

[0139] iv) 2'-OMe or 2'-MOE substitution at nucleoside positions +11, +12, +13, +14 and / or +15;

[0140] v) The PS key at key positions +1, +2, +3, +4, +5, +6, +7, +8, +9, -5, -6, -7, -9, -10, -11 and / or -12;

[0141] vi) The PS or PNdmi key at key position -8 and / or -13;

[0142] vii) The PS or PO key at key positions +10, +11, +12 and / or +13; and / or the PS, PNdmi or PO key at key position +14.

[0143] In one implementation, the ASO disclosed herein comprises a nucleotide sequence:

[0144] (SEQ ID NO: 136).

[0145] In one implementation, the ASO disclosed herein comprises a nucleotide sequence:

[0146] (SEQ ID NO: 213), wherein: i) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) the nucleotides at positions +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, -7, -8, -9, -10, -11 and -13 each independently contain a 2'-OMe, 2'-MOE or 2'-F ribose substitution; iii) the nucleotides at positions +9 and -12 contain a 2'-OMe ribose substitution; and iv) "~" indicates a nucleoside internucleotide bond containing a PS bond, a PNdmi bond or a PO bond.

[0147] In one implementation, the ASO disclosed herein comprises a nucleotide sequence: (SEQ IDNO: 214), wherein: i) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) the nucleotides at positions +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10 and -11 each independently contain a 2'-MOE or 2'-F ribose substitution; iii) the nucleotides at positions +10, +11, +12 and +13 each independently contain a 2'-OMe or 2'-MOE ribose substitution; iv) the nucleotides at positions +9, -12 and -13 contain a 2'-OMe ribose substitution; v) "=" indicates a nucleoside inter-bond containing a PO bond or a PS bond; and vi) "%" indicates a nucleoside inter-bond containing a PS or PNdmi bond.

[0148] This disclosure relates to an ASO targeting human IDUA transcripts or portions thereof, wherein the ASO comprises chemical modifications and / or nucleotide sequences selected from the group consisting of: SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 180, 181, 182, 183, 184, 185, 191, 194, and 195.

[0149] In one embodiment, the ASO disclosed herein consists of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 180, 181, 182, 183, 184, 185, 191, 194, or 195.

[0150] In one embodiment, the ASO disclosed herein includes a delivery portion. Preferably, the delivery portion is an N-acetylgalactosamine (GalNAc) portion. Preferred GalNAc portions are tri-antennae GalNAc portions as shown in Formulas I, II, III, and VI (hereinafter). Preferred mono-antennae GalNAc portions are shown in Formulas IV and V (hereinafter).

[0151] Formula I, including connection point E (see WO2022 / 271806):

[0152] .

[0153] Formula II includes the connector and the connection point F for assembling with the ASO (see WO2014179620):

[0154] .

[0155] Formulas III(a) and III(b) include the connector and the connection point F for fusion with the ASO (see WO2009 / 073809):

[0156] Formula III(a);

[0157]

[0158] Formula III(b).

[0159] Formula IV:

[0160] .

[0161] Formula V :

[0162] .

[0163] Formula VI (see WO2011104169), where the wavy line indicates the connection point of Formula VI to the ASO, optionally via a connector and / or spacer:

[0164] .

[0165] When applying the GalNAc portion of Formula I, the GalNAc portion is preferably joined to the ASO at its 3' end via the connection point E of Formula I, optionally via a connector and / or spacer, as depicted, for example, as shown in Formula VII (hereinafter).

[0166] Equation VII:

[0167] .

[0168] When applying the GalNAc portion of Formula II, the GalNAc portion is preferably connected to the ASO at its 5' end via the connection point F of Formula II, optionally via a connector and / or spacer.

[0169] This disclosure relates to an ASO targeting human IDUA transcripts or portions thereof, wherein the ASO comprises chemical modifications and / or nucleotide sequences selected from the group consisting of: SEQ ID NO: 67, 68, 69, 70, 71, 72, 99, 100, 101, 102, 103, 104, 110, 113, 114, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, and 179. In one embodiment, the ASO disclosed herein consists of SEQ ID NO: 67, 68, 69, 70, 71, 72, 99, 100, 101, 102, 103, 104, 110, 113, 114, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, or 179.

[0170] This disclosure relates to an ASO targeting human IDUA transcripts or portions thereof, wherein the ASO comprises or is composed of the sequence of SEQ ID NO: 16 and modifications thereof. Preferably, the ASO comprising or is composed of the sequence of SEQ ID NO: 16 and modifications thereof further comprises a delivery portion, preferably wherein the delivery portion is a GalNAc portion, more preferably a triantennae GalNAc portion, and even more preferably the GalNAc portion of Formula I (above).

[0171] This disclosure relates to an ASO as disclosed herein that targets human IDUA transcripts or portions thereof, wherein the ASO includes or comprises the structure of formula (B), see also Figure 28 :

[0172] Formula (B)

[0173] This disclosure relates to an ASO as disclosed herein that targets human IDUA transcripts or portions thereof, wherein the ASO comprises the structure of SEQ ID NO: 64, wherein the ASO is chemically modified by means of a choice of ribose, bond or nucleobase moiety chemical modification as taught herein, and preferably wherein the ASO comprises a GalNAc moiety at the 3' end.

[0174] In one embodiment, this disclosure relates to ASO as provided in structure (A), see Figure 27 .

[0175] In one embodiment, this disclosure relates to an ASO as provided in structure (B), see Figure 28 .

[0176] In one embodiment, this disclosure relates to ASO as provided in structure (C), see Figure 29 .

[0177] In one embodiment, the portion of the human IDUA transcript molecule contains the sequence of SEQ ID NO: 1, wherein the target adenosine represents the c.1205G>A mutation.

[0178] In one embodiment, the human IDUA transcript molecule is based on the sequence of SEQ ID NO: 135, where the adenosine at position 1205 represents the c.1205G>A mutation. It should be understood that the sequence of SEQ ID NO: 135 is a mutant human IDUA mRNA sequence that results in Hurler syndrome.

[0179] In one embodiment, at least one nucleotide in the ASO disclosed herein contains one or more additional non-naturally occurring chemical modifications in the ribose, bond, or base portion, wherein the one or more additional ribose modifications are selected from: deoxyribose (DNA), unlocked nucleic acid (UNA), and 2'-F.

[0180] In one embodiment, the one or more additional modifications are bond modifications selected from the group consisting of PS, 3'-methylenephosphonate, 5'-methylenephosphonate, 3'-phosphatidyl ester, and 2'-5'-PO.

[0181] In one implementation, ASO One or more nucleotides outside the motif contain additional modifications, which are mono- or di-substituted sugars at the 2', 3', and / or 5' positions, selected from: -OH; -F; substituted or unsubstituted, straight-chain or branched lower (C1-C2) nucleotides. 10Alkyl, alkenyl, alkynyl, alkylaryl, allyl or aralkyl, which may be intercalated with one or more heteroatoms; -O-, S- or N-alkyl (e.g., -O-methyl); -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.

[0182] This article also discloses pharmaceutical compositions comprising the ASO disclosed herein and a pharmaceutically acceptable carrier.

[0183] This article also discloses ASO or pharmaceutical compositions as disclosed herein for the treatment of mucopolysaccharidosis type 1 (MPS 1), preferably Hurler syndrome caused by the c.1205G>A mutation in the human IDUA gene.

[0184] This document also discloses the use of ASOs or pharmaceutical compositions as disclosed herein in the preparation of a medicament for treating MPS1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene.

[0185] Also disclosed is a method for treating MPS 1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene, comprising administering a therapeutically effective amount of ASO or a pharmaceutical composition as disclosed herein to a patient in need. In a preferred embodiment, the administration step is via subcutaneous delivery. In another preferred embodiment, the administration step is to the central nervous system, optionally via intrathecal delivery. In one embodiment, the administration step is via intrathecal delivery.

[0186] A method for deamination of target adenosine present in a target RNA molecule in a cell is also disclosed, wherein the target RNA molecule is human IDUA pre-mRNA or mRNA or a portion thereof, wherein the target adenosine is the c.1205G>A mutation in SEQ ID NO: 135, the method comprising the steps of: (i) contacting the cells with the ASO disclosed herein or the pharmaceutical composition disclosed herein under conditions suitable for: a) cell uptake of ASO; b) annealing of ASO with the target RNA molecule; and c) deamination of the target adenosine in the target RNA molecule to inosine by a naturally occurring mammalian ADAR enzyme present in the cell; and ii) optionally treating the cellular pre-mRNA or mRNA to determine the presence of inosine at the target adenosine site in the target RNA molecule. The optional steps include: a) determining the sequence of the target RNA molecule; b) assessing the presence of functional, extended, full-length, and / or wild-type iduronate protein; or c) using a functional readout, wherein the deamination of the target RNA encodes a functional, full-length, extended, and / or wild-type iduronate protein. Preferably, the target adenosine is a c.1205G>A mutation in the human IDUA gene.

[0187] This paper discloses an ASO consisting of the following sequence:

[0188] (SEQ ID NO: 213), where:

[0189] i) Chemical modification as shown in Figure 1, Figure 4 And as shown in Figure 16;

[0190] ii) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, where uracil can also be 5-methyluracil (or thymine) nucleobases;

[0191] iii) The nucleotides at positions +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, -7, -8, -9, -10, -11, and -13 each independently contain a 2'-OMe, 2'-MOE, or 2'-F ribose substitution;

[0192] iv) The nucleotides at positions +9 and -12 contain a 2'-OMe ribose substitution; and

[0193] v) "~" indicates a nucleoside inter-bond containing a PS bond, PNdmi bond, or PO bond.

[0194] This paper discloses an ASO consisting of the following sequence: (SEQ IDNO: 214), where:

[0195] i) Chemical modification as shown in Figure 1, Figure 4 And as shown in Figure 16;

[0196] ii) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, where uracil can also be 5-methyluracil (or thymine) nucleobases;

[0197] iii) The nucleotides at positions +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10, and -11 each independently contain either a 2'-MOE or a 2'-F ribose substitution;

[0198] iv) The nucleotides at positions +10, +11, +12, and +13 each independently contain a 2'-OMe or 2'-MOE ribose substitution;

[0199] v) The nucleotides at positions +9, -12, and -13 contain a 2'-OMe ribose substitution;

[0200] vi) “=" indicates a nucleoside internucleotide bond containing a PO or PS bond; and

[0201] vii) "%" indicates a nucleoside inter-bond containing a PS or PNdmi bond.

[0202] This document also discloses ASOs as disclosed herein, wherein at least one nucleotide contains one or more additional non-naturally occurring chemical modifications in the ribose, bond, or base moiety, provided that Z does not contain a 2'-OMe ribose substitution.

[0203] In one embodiment, the ASO according to this disclosure includes selected from Figure 1A , Figure 1B , Figure 1C , Figure 1E , Figure 16A and Figure 16B The nucleotide sequence (including specified chemical modifications) of any one of the human IDUA c.1205G>A targets or is composed of the human IDUA c.1205G>A target ASO.

[0204] In one embodiment, the ASO according to this disclosure comprises or consists of nucleotide sequences selected from the group consisting of (including specified chemical modifications): SEQ ID NO: 16, 34, 35, 10, 8, 13, 14, 7, 37, 36, 33, 25, 6, 21 and 18, representing ASO A-50, A-91, A-92, A-40, A-38, A-43, A-44, A-37, A-94, A-93, A-90, A-82, A-36, A-78 and A-56, respectively, and their respective chemical modifications.

[0205] In one embodiment, the ASO according to this disclosure comprises or consists of sequences selected from the group consisting of (including specified chemical modifications): SEQ ID NO: 69, 99, 72, 68, 71, 67, 70, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 116, 120, 123, 124, 125, and 126, representing ASO A-50 GN, A-104, A-94 GN, A-38 GN, A-93 GN, A-36 GN, and A-78 GN, respectively. GN, A-105, A-106, A-107, A-108, A-109, A-110, A-111, A-112, A-113, A-114, A-1 15. A-116, A-117, A-118, A-121, A-125, A-128, A-129, A-130 and A-131, and their respective chemical modifications.

[0206] In one embodiment, the ASO according to this disclosure comprises or consists of sequences selected from the group consisting of (including specified chemical modifications) the following: SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 and 98, representing ASOA-50 GN-A to A-50 GN-M, respectively, and their respective chemical modifications (see [link to relevant documentation]). Figure 1C This disclosure provides, for example, Figure 1B The variant of A-50 GN ASO (SEQ ID NO: 69) described herein contains one or more additional non-naturally occurring chemical modifications in the sugar, (chiral pure) bond, or base moiety, provided that the one or more additional chemical modifications are in a different sugar, bond, or base moiety modification category than any modification described herein.

[0207] In a preferred embodiment, the ASO according to this disclosure comprises or consists of the sequence of SEQ ID NO: 113 (including the specified chemical modifications) and contains a 2'-OMe modified guanosine residue (Gm) at position +10 instead of the 2'-OMe modified cytidine residue (Cm) at position +10 in A-50 (SEQ ID NO: 16).

[0208] In a preferred embodiment, the ASO according to this disclosure comprises or consists of the sequence of SEQ ID NO: 194 (including the specified chemical modifications) and contains a 2'-OMe modified guanosine residue (Gm) at position +10 instead of the 2'-OMe modified cytidine residue (Cm) at position +10 in A-50GN (SEQ ID NO: 69).

[0209] Those skilled in the art will know that oligonucleotides, such as RNA oligonucleotides, are typically composed of repeating monomers. The most common of these monomers are nucleotides or nucleotide analogs. The most common naturally occurring nucleotides in RNA are A, C, G, and U. They consist of a pentose sugar, a ribose sugar, a 5'-linked phosphate group linked by a phosphate ester, and a 1'-linked base. The sugar linking the base and the phosphate is therefore often referred to as the "backbone" of the nucleotide. Modifications to the pentose sugar are therefore often referred to as "backbone modifications." In severe modifications, the original pentose sugar can be completely replaced by another similarly linked base and phosphate group. Therefore, it should be understood that while the pentose sugar is often the backbone, the backbone is not necessarily a pentose sugar.

[0210] Bases, sometimes called nucleobases, are typically adenine, cytosine, guanine, thymine, or uracil, or their derivatives. C, T, and U are pyrimidine bases, usually linked to the backbone via their 1-nitrogen group. A and G are purine bases, usually linked to the backbone via their 9-nitrogen group.

[0211] Nucleotides are typically linked to adjacent nucleotides through the condensation of their 5'-phosphate moiety with the 3'-hydroxy moiety of the adjacent nucleotide monomer. Similarly, their 3'-hydroxy moiety is typically linked to the 5'-phosphate moiety of the adjacent nucleotide monomer. This forms a PO bond. The PO and backbone form an alternating copolymer. Bases are grafted onto this copolymer, i.e., grafted onto the backbone moiety. Due to this property, the alternating copolymer formed by the linking monomers of oligonucleotides is often referred to as the "backbone" of the oligonucleotide. Because the PO bonds link adjacent monomers together, they are often referred to as "backbone bonds." It should be understood that when the phosphate group is modified to a similar moiety such as PS, such a moiety is still referred to as the backbone bond of the monomer. This is called "backbone modification." Generally, the backbone of an oligonucleotide contains alternating backbones and backbone bonds.

[0212] In one respect, the nucleobases in the ASO of this paper are A, C, G, T, or U. In another respect, the nucleobases are modified forms of A, C, G, or U. In yet another respect, the modified nucleobases are hypoxanthine (the nucleobase in inosine), pseudouracil, pseudocytosine, 1-methylpseudouracil, orotic acid, agrobacterioside, lysine, 2-thiouracil, 2-thiothymine, 5-halouracil, 5-halomethyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-formyluracil, 5-aminomethylcytosine, and 5-formylcytosine. 5-Hydroxymethylcytosine, 7-deazoguanine, 7-deazoadenine, 7-deazo-2,6-diaminopurine, 8-aza-7-deazoguanine, 8-aza-7-deazoadenine, 8-aza-7-deazo-2,6-diaminopurine, pseudoisocytosine, N4-ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2-aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamp, Super A, Super T, Super G, amino-modified nucleobases or their derivatives; and degenerate or universal bases, such as 2,6-difluorotoluene, or those without a base site (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, azaribose). As used herein, the terms 'adenine', 'guanine', 'cytosine', 'thymine', 'uracil', and 'hypoxanthine' refer to the nucleobases themselves. The terms 'adenosine', 'guanosine', 'cytosine', 'thymidine', 'uridine', and 'inosine' refer to the nucleobases linked to (deoxy)ribosyl sugars.

[0213] The term 'nucleoside' refers to a nucleobase linked to a (deoxy)ribosyl sugar and without a phosphate group. A 'nucleotide' consists of a nucleoside and one or more phosphate groups. Therefore, the term 'nucleotide' refers to each nucleobase-(deoxy)ribosyl-phosphate linker, and any chemical modification of the ribose moiety or phosphate group. Thus, the term will include nucleotides containing a locked ribosyl moiety (containing a 2'-4' bridge, containing a methylene or any other group), UNA, threonine (TNA), or a linker containing PO, phosphonoacetate, triphosphate, PS, dithiophosphate, MP, methylthiophosphonate, phosphoramide ester, etc. Sometimes the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymine / uridine, inosine and hypoxanthine are used interchangeably to refer to the nucleobases on one side and the nucleoside or nucleotide on the other. Thymine (T) is also called 5-methyluracil (m). 5U), is a uracil (U) derivative; thymine, 5-methyluracil, and uracil are used interchangeably in this text. Similarly, thymidine, also known as 5-methyluridine, is a uridine derivative; thymidine, 5-methyluridine, and uridine are used interchangeably in this text. Sometimes the terms nucleobase, nucleoside, and nucleotide are used interchangeably unless the context explicitly requires otherwise, such as when a nucleoside is linked to an adjacent nucleoside and the bond between these nucleosides is modified. As described herein, a nucleotide is a nucleoside with one or more phosphate groups. The terms 'ribonucleoside' and 'deoxyribonucleoside', or 'ribose' and 'deoxyribose', are used as in the art.

[0214] Whenever oligonucleotide, oligo, ON, ASO, oligonucleotide composition, antisense oligonucleotide, AON, (RNA) editing oligonucleotide, EON, and RNA (antisense) oligonucleotide are mentioned, unless the context otherwise specifies, they refer to oligonucleotides and deoxyoligonucleotides. Oligonucleotides may be completely lacking in naturally occurring RNA or DNA nucleotides and may consist entirely of modified nucleotides. Whenever 'oligonucleotide' is mentioned, it may contain the bases A, G, C, U, or I. Whenever 'deoxyoligonucleotide' is mentioned, it may contain the bases A, G, C, T, or I. However, the ASO of this disclosure may contain a mixture of ribonucleosides and deoxyribonucleosides. When deoxyribonucleosides are used, and therefore there is no modification at the 2' position of the sugar, the nucleotide is usually abbreviated as dA, dC, dG, or T, where 'd' represents the deoxynival nature of the nucleotide, while ribonucleosides that are normal RNA or modified at the 2' position are usually abbreviated without 'd' and are often abbreviated by their respective modifications, as explained herein.

[0215] When referring to nucleotides in ASO, the terms include cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine; when referring to adenine, the terms include N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine, and 7-methyladenine; when referring to uracil, the terms include dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudouracil, 4-thiouracil, and 5-hydroxymethyluracil; and when referring to guanine, the terms include 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. Whenever nucleosides or nucleotides are mentioned, this includes furanose derivatives such as 2'-deoxy, 2'-hydroxy, and 2'-O-substituted variants such as 2'-O-methyl, as well as other modifications, including 2'-4' bridging variants. Whenever oligonucleotides are mentioned, the bond between two mononucleotides can be a PO bond and its modifications, including phosphonoacetate, phosphate triester, PS, dithiophosphate, MP, phosphoramide ester linker, phosphorylguanidine, thiophosphorylguanidine, sulfonylphosphoramide, etc.

[0216] In one aspect, the ASO of this invention comprises a 2'-substituted PS monomer, preferably a 2'-substituted PS RNA monomer, a 2'-substituted phosphate RNA monomer, or a mixture of phosphate / PS monomers containing 2'-substituted monomers. It should be noted that, with respect to 2' substitution, DNA is considered an RNA derivative. The ASO of this disclosure comprises at least one 2'-substituted RNA monomer linked by a PS or phosphate backbone bond or a mixture thereof. The 2'-substituted RNA is preferably 2'-F, 2'-H (DNA), 2'-O-methyl, or 2'-O-(2-methoxyethyl). 2'-O-methyl is often abbreviated as "2'-OMe", and the 2'-O-(2-methoxyethyl) portion is often abbreviated as "2'-MOE". More preferably, the 2'-substituted RNA monomer in the ASO of this disclosure is a 2'-OMe monomer, which, except for monomers opposite to the target adenosine, should not carry a 2'-OMe substitution, as further outlined herein. A preferred aspect in this regard is provided in an ASO according to the present disclosure, wherein the 2'-substituted monomer can be a 2'-substituted RNA monomer, such as a 2'-F monomer, a 2'-NH2 monomer, a 2'-H monomer (DNA), a 2'-O-substituted monomer, a 2'-OMe monomer, or a 2'-MOE monomer, or a mixture thereof. Preferably, the monomer opposite to the target adenosine is a 2'-H monomer (DNA), but it can also be a monomer that allows deamination of the target adenosine, rather than a 2'-OMe monomer. Preferably, any other 2'-substituted monomer within the ASO is a 2'-substituted RNA monomer, such as a 2'-OMe RNA monomer or a 2'-MOE RNA monomer, which may also appear in combination within the ASO.

[0217] Throughout this disclosure, the 2'-OMe monomer within the ASO can be replaced by 2'-OMe PS RNA, 2'-OMe phosphate RNA, or 2'-OMe phosphate / PS RNA. Throughout this disclosure, the 2'-MOE monomer can be replaced by 2'-MOE PS RNA, 2'-MOE phosphate RNA, or 2'-MOE phosphate / PS RNA. Throughout this disclosure, oligonucleotides consisting of 2'-OMe RNA monomers linked or coupled via PS, phosphate, or mixed phosphate / PS backbone bonds can be replaced by oligonucleotides consisting of 2'-OMe PS RNA, 2'-OMe phosphate RNA, or 2'-OMe phosphate / PS RNA. Throughout this disclosure, oligonucleotides consisting of 2'-MOE RNA monomers linked or coupled via PS, phosphate, or mixed phosphate / PS backbone bonds can be replaced by oligonucleotides consisting of 2'-MOE PS RNA, 2'-MOE phosphate RNA, or 2'-MOE phosphate / PS RNA.

[0218] In addition to specific preferred chemical modifications at certain positions in the compounds described herein, the compounds may further contain or consist of one or more (additional) modifications to or of the nucleobases, backbone, and / or main chain bonds. These additional modifications may or may not be present in the same monomer, for example, at the 3' and / or 5' positions. Backbone modifications refer to the presence of modified versions of the ribosyl moiety (i.e., the pentose moiety) naturally present in RNA, such as bicyclic sugars, tetrahydropyran, hexoses, morpholino, 2'-modified sugars, 4'-modified sugars, 5'-modified sugars, and 4'-substituted sugars. Examples of suitable modifications include, but are not limited to, 2'-O-modified RNA monomers, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, such as 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-MOE, 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propynyl, 2'-O-allyl, 2'-O-(2-aminopropyl), 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkyl)methyl, such as 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] 2'-(2-N-methylcarbamoyl)ethyl] (MOCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halogenated, e.g., 2'-F, FANA; 2'-O-[2-(methylamino)-2-oxoethyl] (NMA); bicyclic or bridging nucleic acid (BNA) backbone modifications, such as conformation-restricted nucleotide (CRN) monomers, locked nucleic acid (LNA) monomers, xylose-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 monomer, (2'-O,4'-C)-confined methoxyethyl (cMOE) BNA monomer, 2',4'-BNA NC (NH) monomer, 2',4'-BNA NC (NMe) monomer, 2',4'-BNA NC(NBn) monomers, ethylene glycol-bridged nucleic acid (ENA) monomers, carbo-LNA (cLNA) monomers, 3,4-dihydro-2H-pyranoic acid (DpNA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, oxo-CBBN monomers, heterocyclic-bridged BNA monomers (e.g., triazole or tetraazole linkages), 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, α-terminal isomeric bicyclic DNA (abcDNA) monomers, oxadiazine nucleotide monomers, locked PMO monomers derived from 2'-amino-LNA, guanidine-bridged nucleic acid (GuNA) monomers, spirocyclopropene-bridged nucleic acid (scpBNA) monomers and their derivatives; cyclohexenyl nucleic acid (CeNA) monomers, atroitol nucleic acid (ANA) monomers, hexitol nucleic acid (HNA) monomers, fluorinated HNA (F-HNA) monomers, pyranosyl-RNA (p-RNA) monomers, 3'-deoxypyranosyl DNA (p-DNA), UNA; and reverse versions of any of the above monomers. All of these modifications are known to those skilled in the art.

[0219] "Main chain modification" indicates the presence of a modified version of the ribosome moiety naturally present in RNA ("backbone modification"), as described above, and / or a modified version of PO naturally present in RNA ("main chain modification"). Examples of nucleotide inter-bond modifications are PS, chiral pure PS, Rp PS, and Sp. PS, dithiophosphate (PS2), phosphonoacetate (PACE), thiophosphonoacetate, phosphonoacetamide (PACA), thiophosphonoacetamide, PS prodrug, S-alkylated PS, H-phosphonate, MP, methyl thiophosphonate, methyl phosphate, methyl thiophosphate, ethyl phosphate, ethyl thiophosphate, boran phosphate, boran thiophosphate, methyl boran phosphate, methyl boran thiophosphate, methyl boran phosphonate, methyl boran phosphonophosphate, phosphoroguanidine (PGO), methyl sulfonyl phosphoramide, phosphoramide, phosphoramide, N3'→P5' phosphoramide, N3'→P5' thiophosphonamide, phosphoridamide, thiophosphonidamide, aminosulfonate, dimethylene sulfoxide, sulfonate, triazole, oxaloyl, carbamate, methylene imino (MMI) and thioacetamido (TANA); and their derivatives. The ASO disclosed herein may also include a PNdmi bond between the terminal nucleotides at the 5' and / or 3' ends and the penultimate nucleotide of each of these ends. The PNdmi bond preferably used in the ASO has the following structure:

[0220] PNdmi key .

[0221] The ASO disclosed herein may also include a bond portion selected from PS, phosphonoacetate, dithiophosphate, methylphosphonate (MP), sulfonylphosphatase, or PNdmi nucleoside internucleotides.

[0222] As disclosed in this article, ASO may also include one or more key modifications according to the following structure:

[0223]

[0224] in:

[0225] X = O or S; and

[0226] R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C 20 Alkyl, substituted C1-C 20 Alkyl, C1-C6 alkenyl, C1-C6 substituted alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugated groups. In a preferred embodiment, X = O and R = methyl, and this bond modification is called methanesulfonylphosphatamide ester, MsPA, or PNms.

[0227] In one aspect, the ASO disclosed herein contains at least one MP nucleoside inter-bond according to the following structure:

[0228]

[0229] As noted in the art, the preferred position of the MP bond in an ASO is bond position -2, thereby linking the nucleoside at position -1 to the nucleoside at position -2. In one aspect, in an ASO as disclosed herein, this position contains the PNms bond modification as disclosed above, rather than an MP bond. International Patent Application Publication No. WO 2020 / 201406 discloses the use of MP bond modifications at certain positions around orphan nucleotides in the first nucleic acid strand. Although the presence of MP bonds is compatible with RNA editing by human ADAR enzymes, introducing MP bonds during oligonucleotide manufacturing is challenging due to the additional manufacturing (purification) steps involved in coupling and uncoupling. In one aspect, an ASO does not contain MP bonds.

[0230] The ASO described herein does not contain 5'-terminal O6-benzylguanosine or 5'-terminal amino modification, and is not covalently linked to a SNAP-tag domain (an engineered O6-alkylguanosine-DNA-alkyltransferase). In one embodiment, the ASO described herein contains 0, 1, 2, or 3 wobble base pairs with the target sequence, and / or contains 1, 2, 3, 4, 5, 6, 7, or 8 mismatched base pairs with the target RNA sequence. The target adenosine in the target sequence forms a mismatched base pair with a nucleotide in the ASO containing a Benner base and directly opposite the target adenosine. The ASO disclosed herein does not contain a boxB RNA hairpin sequence. The ASO of this disclosure can specifically edit the target adenosine in the target RNA sequence using endogenous cellular pathways and naturally available ADAR enzymes. The ASO of this disclosure is capable of recruiting ADAR and forming a complex with it, then allowing deamination of the target adenosine nucleotide in a (single) specific target RNA sequence. Ideally, only one adenosine is deaminated. When the ASO of this disclosure forms a complex with ADAR, it preferably contains a deaminoated monoadenosine target.

[0231] Analysis of the natural targets of ADAR enzymes has shown that these typically involve mismatches between the two strands that form the RNA helix edited by ADAR1 or 2. These mismatches have been proposed to enhance the specificity of the editing response (Stefl et al. (2006) Structure 14:345-355; and Tian et al. (2011) Nucleic Acids Res. 39:5669-5681). Characterizing the optimal pattern of paired / mismatched nucleotides between ASO and target RNA is also crucial for developing effective ADAR-based ASO therapies.

[0232] The ASO described in this paper uses specific nucleotide modifications at predetermined sites to ensure stability and proper ADAR binding and activity, as described above. These modifications may vary and may include modifications to the ASO backbone, the nucleotide sugar moiety, and nucleobases or PO bonds, as detailed above. They may also be variable in the ASO sequence. Specific modifications may be required to support the interactions of different amino acid residues within the ADAR enzyme's RNA-binding domain and deaminase domain. For example, internucleotide PS bonds or 2'-OMe or 2'-MOE modifications may be permissible in some parts of the ASO, while should be avoided in others to prevent disruption of critical interactions between the enzyme and phosphate and 2'-OH groups. Specific nucleotide modifications may also be necessary to enhance editing activity on the substrate RNA when the target sequence is not the optimal sequence for ADAR editing.

[0233] Previous work has identified certain sequence contexts that are more conducive to editing. For example, the target sequence 5'-UAG-3' (with target A in the middle) contains the most preferred nearest-neighbor nucleotide of ADAR2, while the 5'-CAA-3' target sequence is unfavorable (Schneider et al. (2014) Nucleic Acids Res. 42:e87). Structural analysis of the ADAR2 deaminase domain suggests that careful selection of nucleotides opposite the target trinucleotide may enhance editing. For example, the 5'-CAA-3' target sequence, which pairs with the 3'-GCU-5' sequence on the opposite side chain (forming an AC mismatch in the middle), is unfavorable because the guanosine base creates a spatial conflict with the amino acid side chain of ADAR2.

[0234] The ASO described herein (when not expressed via a viral vector) contains one or more nucleotides with one or more sugar modifications. Therefore, a single nucleotide in the ASO may have one or more sugar modifications. Within the ASO, one or more nucleotides may have such sugar modifications. Another aspect of this disclosure is that the nucleotide in the ASO corresponding to the target A to be edited does not contain 2'-OMe or 2'-MOE modifications. Typically, the direct 3' and 5' nucleotides ("adjacent nucleotides") of this nucleotide in the ASO also lack such chemical modifications, although it is not necessary for both adjacent nucleotides to not contain 2'-O-alkyl (such as 2'-OMe). Any one or two adjacent nucleotides, or all three nucleotides of the central triplet, may carry 2'-OH.

[0235] As outlined in this paper, the nucleotides outside the central triplet are typically modified with 2'-OMe or 2'-MOE. However, this is not a strict requirement for the ASO in this paper. Using these 2' substitutions ensures proper stability of those parts of the ASO, but other modifications may also be applied.

[0236] This disclosure also relates to sequences containing " "As a central triplet, ASO consists of m5Ce, a 5-methyl-cytidine with a 2'-MOE ribose group, Zd, a Benner base (i.e., a cytidine analog located at the target A position), and Ad, an A with a 2'-H ribose group. The bond between the cytidine and the Benner base is a PO bond, and the bond between the Benner base and A is a PS bond."

[0237] In another preferred aspect, the sugar modification is selected from the group consisting of deoxyribose (DNA), UNA, and 2'-fluororibose. In a particularly preferred aspect, this disclosure relates to an ASO comprising a central triplet of three consecutive nucleotides, wherein the nucleotide directly opposite the target adenosine is the intermediate nucleotide of the central triplet, and wherein two nucleotides in the central triplet are DNA nucleotides, to make the ASO more stable and / or more effectively induce deamination of target A. In another preferred aspect, the remainder of the ASO consists of RNA nucleotides, which preferably (but not necessarily) are substituted at the 2' position of the sugar, preferably having 2'-OMe, 2'-F, or 2'-MOE modifications. Other ribose modifications quite compatible with the targeted editing according to this disclosure are LNA and 2'-NH2. Different combinations of sugar modifications may be applied. On the other hand, the ASO of this invention contains at least one non-naturally occurring internucleotide bond modification selected from the group consisting of: PS, 3'-methylenephosphonate (i.e., 3'-O-MP internucleotide bond), 5'-methylenephosphonate (i.e., 5'-O-MP internucleotide bond), 3'-phosphatidyl ester (i.e., N-3'-phosphatidyl ester internucleotide bond), and 2'-5'-PO (i.e., 2'-5'-PO internucleotide bond). The PS bond is particularly preferred.

[0238] The ASO disclosed herein can also be administered in the context of aids to increase ASO entry into target cells and / or endosome escape once inside the cell. A portion suitable for such applications is, for example, a group of compounds (typically purified from nature) referred to as “saponins” or “triterpenoid glycosides.” A preferred saponin suitable for use in the methods disclosed herein is AG1856, disclosed in International Patent Application Publication No. WO 2021 / 122998, and further described for use with the RNA editing production of oligonucleotides disclosed in GB Patent Application No. 2300865.9 (unpublished). Such saponins can be administered separately from or together with ASO, and are preferably conjugated to ASO, for example at the 5' or 3' end, using a suitable linker if desired.

[0239] The term "comprising" encompasses both "including" and "consisting of," for example, a composition "comprising X" may consist of only X or may contain additional elements (e.g., X + Y). The term "about" is optional when referring to the numerical value x, and refers to, for example, x... +10%. The term “substantially” does not exclude “completely” (e.g., a composition that is “substantially Y-free” can be completely Y-free). Where applicable, the term “substantially” may be omitted from the definition of this disclosure. The term “downstream” in relation to nucleic acid sequences refers to a point further along the sequence in the 3' direction; the term “upstream” is the opposite. Thus, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand but downstream of the stop codon in the antisense strand. References to “hybridization” generally refer to specific hybridization and exclude non-specific hybridization. Specific hybridization may occur under selected experimental conditions using techniques well known in the art to ensure that most stable interactions between the probe and target occur when the probe and target have at least 70%, preferably at least 80%, more preferably at least 90% sequence complementarity. When the ASO and target sequences are 100% complementary, it means that there are no mismatched or wobble base pairs. Target A in the target RNA molecule is typically not perfectly complementary to an orphan nucleotide, which, as outlined herein, is usually a C or a deoxyribonucleotide carrying a Benner base Z, an analog of C, and therefore not a perfect complementary match to target A, and should be considered a “mismatch.” Therefore, the ASO disclosed herein is not 100% complementary to the target sequence. The term “mismatch” is used herein to refer to an opposing nucleotide in a double-stranded RNA complex that does not form a perfect base pair according to the Watson-Crick base pairing rule. Historically, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the first nucleic acid strand of this disclosure contains fewer than four mismatches with the target sequence, such as 0, 1, or 2 mismatches. “Wag base pairs” are GU, IU, IA, and IC base pairs. Although G:G pairing will be considered a mismatch, this does not necessarily mean that the interaction is unstable. This means that the term "mismatch" may be somewhat outdated based on the current content of this disclosure, where Hoogsteen base pairing can be considered a mismatch based on nucleotide origin but still relatively stable. Isolated G:G pairings in double-stranded RNA may, for example, be quite stable but are still defined as mismatches. In one embodiment, the ASO contains 1, 2, 3, 4, 5, 6, 7, or 8 mismatches, which include a base pair between the target adenosine and a nucleotide in the ASO containing a Benner base (i.e., directly opposite to target A). In one embodiment, the ASO contains 0, 1, 2, or 3 wobble base pairs.

[0240] Various chemistry and modifications are known in the field of oligonucleotides and can be readily used according to this disclosure, as described above. Conventional internucleotide bonds between nucleosides can be modified by mono- or dithiolated modification of the PO bond to produce PS esters or dithiophosphate esters, respectively. Other modifications to the internucleotide bonds are possible, including amidation and peptide linker groups. In one embodiment, the ASO comprises 1, 2, 3, 4, or more PS bonds between the terminal nucleotides of the ASO (thus at the 5' and 3' ends), meaning that in the case of 4 PS bonds, the final 5 nucleotides are linked accordingly. Those skilled in the art will understand that the number of such bonds at each end can vary depending on various factors, such as toxicity.

[0241] In one embodiment, the ASO comprises a substitution of one of the non-bridging oxygen atoms in the PO bond. This modification slightly disrupts base pairing but significantly increases resistance to nuclease degradation. Preferred nucleotide analogs comprise PS, phosphonoacetate, dithiophosphate, phosphate triester, aminoalkyl phosphate triester, H-phosphonate, methyl and other alkylphosphonates, including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, hypophosphonates, phosphoramidites, including 3'-aminophosphatates and aminoalkylphosphatates, thiophosphatates, thioalkylphosphonates, thioalkyl phosphate triesters, selenophosphates, or boron phosphates.

[0242] Particularly preferred are modifications to include nucleotide inter-bonds containing PS. Many of these non-naturally occurring bond modifications, such as PS, are chiral, meaning that Rp and Sp configurations exist, as is known to those skilled in the art. In a preferred aspect, the chirality of the PS bond is controlled, meaning that each bond is either Rp or Sp configuration, whichever is preferred. The selection of Rp or Sp configuration at a specified bond position may depend on the target sequence and the binding and induction efficiency provided for RNA editing. However, unless specifically required, the composition may contain an ASO having Rp and Sp configurations at the specified bond positions as the active compound. Mixtures of such ASOs are also feasible, wherein some positions preferably have either configuration, while others are irrelevant. International Patent Application Publication No. WO 2019 / 219581 discloses specific disallowed and permitted positions of Rp and Sp configurations of PS bonds when computer-modeling known structural features of ADAR2 binding to RNA using mouse Idua mRNA as a model target sequence. This knowledge applies to the ASOs disclosed herein, as well as anticipated ASO sequences with their chemical modifications, including permitted Rp and Sp bond positions. These Rp and Sp specificities apply to preferred ASOs (A-50; SEQ ID NO: 16 and 69 (with GN)) targeting human IDUA transcripts. These ASOs (with GN) are... Figure 1C As given in the A-50 GN-A to -M series, those without GN are... Figure 1EIn the middle, key positions +5, +6, +7, +8, -4, and -5 are PO keys here because International Patent Application Publication No. WO 2019 / 219581 states that Rp or Sp PS keys are not allowed in these positions. Keys +1, +2, +3, +9, +11, +12, +13, -7, -9, -10, -11, and -12 are stereo random PS keys (with no preference for Sp or Rp configurations), while keys 0, +14, -2, -8, and -13 remain unchanged compared to A-50.

[0243] In one embodiment, the ASO disclosed herein comprises the sequence and chemical modifications (including stereochemical bonds) of ASO A-50 GN-A, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, or -M, preferably A-50 GN-A (SEQ ID NO: 86). Figure 1C The description.

[0244] Those skilled in the art will recognize that various single-antennae and triple-antennae GalNAc portions are available in the art. Therefore, the GalNAc portion (GN) with the ASO position indicated in the accompanying drawings (primarily at the 3' end) can also be replaced by alternative single-antennae or triple-antennae GalNAc portions if required for the (therapeutic) application of the ASO. Preferred GalNAc portions are given in Formulas I, II, III, and IV herein.

[0245] In one embodiment, ASO comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3', and / or 5' positions, such as: -OH; -F; substituted or unsubstituted, straight-chain or branched lower (C) l -C 10 Alkyl, alkenyl, alkynyl, alkylaryl, allyl, or aralkyl, which may be inter-doped with one or more heteroatoms; -O-, S-, or N-alkyl (e.g., -O-methyl); -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.

[0246] The sugar moiety can be pyranose or a derivative thereof, or deoxypyranose or a derivative thereof, preferably ribose or a derivative thereof, or deoxyribose or a derivative thereof. A preferred derived sugar moiety comprises an LNA, wherein the 2'-carbon atom is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. A preferred LNA comprises a 2'-O,4'-C-ethylene glycol-bridged nucleic acid. These substitutions endow the nucleotide analog with RNase H and / or nuclease resistance and increase affinity for target RNA.

[0247] In one embodiment, the nucleotide analog within the ASO comprises one or more base modifications or substitutions. Modified bases comprise synthetic and natural bases such as inosine, xanthine, hypoxanthine, and other thioalkyl derivatives of -aza, denitrogenated, -hydroxyl, -halogenated, -thio, thiol, -alkyl, -alkenyl, -ynyl, pyrimidine, and purine bases, which are known or to be known in the art. Purine nucleobases and / or pyrimidine nucleobases may be modified to alter their properties, for example, through amination or deamination of heterocycles. The exact chemistry and form can vary from oligonucleotide construct to oligonucleotide construct and from application to application, and can be formulated according to the wishes and preferences of those skilled in the art. It is understood in the art that four or more consecutive DNA nucleotides in an oligonucleotide create a so-called "gap body" that induces RNase H cleavage of the target RNA when annealed to its RNA homologous sequence. According to this disclosure, RNase H cleavage of the target RNA should generally be avoided as much as possible.

[0248] All chemical modifications listed herein that can be used with the ASO disclosed herein can also be used with the positive strand complementary to the ASO when the ASO and the positive strand form a so-called heteroduplex RNA editing oligonucleotide (HEON) complex, as described in International Patent Application No. WO2024 / 084048, except that the opposite positive strand does not have a so-called “orphan nucleotide,” which is a nucleotide in the ASO located at a position directly opposite to target A in the IDUA transcript. Therefore, modifications associated with orphan nucleotides are only related to the ASO, while all other modifications are related to the ASO and any (protective) positive oligonucleotide that can be used with the ASO in pharmaceutical products. This includes the use of hydrophobic moieties (such as tocopherol and cholesterol) and cell-specific ligands (such as the GalNAc moieties), which are also described in detail in International Patent Application No. WO 2024 / 084048, which can bind to the ASO or its opposite strand or both.

[0249] In some embodiments, the ASO comprises a cell-specific ligand as a delivery portion. In one non-limiting embodiment, the delivery portion is a GalNAc portion. In one embodiment, the GalNAc portion is attached to the 3' end of each oligonucleotide. Non-limiting examples of the GalNAc portion are disclosed in the following international patent application publication numbers: WO 2022 / 271806; WO2009 / 073809; WO 2014 / 025805; WO 2015 / 006740; WO 2019 / 053661; WO 2014 / 179620; WO2021 / 261998; U.S. Patent Application Publication No. 2019 / 0256849; WO2011104169; WO2018013525; WO2023034719; WO2023114746; WO2018185241; WO2018185240; WO2019 / 128611; WO2020093061; WO2021127214; WO2023045995; WO2022076922; WO2022 / 189861; and WO2016100401.

[0250] In one embodiment, the GalNAc portion is based on the structure of Formula I in International Patent Application Publication No. WO 2022 / 271806. This embodiment of the GalNAc portion is reproduced herein as Formula I (above).

[0251] In some implementations, any ASO described herein is coupled to connection point E of Formula I, optionally via a connector.

[0252] In one embodiment, the GalNAc portion is attached to the 3' end of each oligonucleotide, and the GalNAc portion has the structure of Formula I in International Patent Application Publication No. WO 2022 / 271806.

[0253] Mutagenesis of human ADAR2 has shown that a single mutation at residue 488, from glutamate to glutamine (E488Q), increases the deamination rate constant by 60-fold compared to the wild-type enzyme (Kuttan & Bass (2012) Proc Natl AcadSci USA 109:3295-3304). During deamination, ADAR2 flips the edited base out of its RNA duplex and into the enzyme's active site. When ADAR2 edits A in a preferred context (A:C mismatch), the nucleotide opposite the target A is typically referred to as an orphan nucleotide (or, depending on the case, 'orphan cytidine'), as described above. Crystal structures of ADAR2 E488Q bound to double-stranded RNA show that the glutamine (Gln;Q) side chain at position 488 can form a hydrogen bond with the N3 position of orphan cytidine, leading to the increased catalytic rate of ADAR2 E488Q. In the wild-type enzyme, position 488 contains glutamate (or glutamate; Glu; E) instead of glutamine (Gln), where the amide group is absent and a carboxylic acid is present. To achieve the same contact between orphan cytidine and the E488Q mutant, protonation is required in the wild-type case to enable this contact. Maximizing the editing efficiency of the wild-type ADAR2 enzyme present in cells is crucial for utilizing endogenously expressed ADAR2 to correct disease-related mutations. International Patent Application Publication No. WO 2020 / 252376 discloses the use of AONs with modified RNA bases, particularly at the orphan nucleotide position, to mimic the hydrogen bonding pattern observed in the E488Q ADAR2 mutant. By replacing the nucleotide opposite target A in the AON with a cytidine analog that acts as a hydrogen bond donor at N3, it is expected that the homologous contact, which is thought to provide an increased catalytic rate for the mutant enzyme, can be stabilized. Two cytidine analogues have attracted particular attention: pseudocytidine (also known as 'piC'; Lu et al. (2009) J.Org. Chem. 74:8021-8030; Burchenal et al. (1976) Cancer Res 36:1520-1523) and Benner base Z (also known as 'dZ' or 'Zd'; Yang et al. (2006) Nucl Acid Res 34:6095-6101), which were initially chosen because they provide a hydrogen bond donor at N3 while causing minimal interference with the shape of the nucleobase. Benner base is also known as 6-amino-5-nitro-2(1H)-pyridone. The presence of cytidine analogues in ASO can be independent of modification of the 2' group of the ribose. The ribose 2' group in orphan nucleotides can be independently selected from 2'-H (i.e. DNA), 2'-OH (i.e. RNA), 2'-OMe, 2'-MOE, 2'-F or 2'-4'-linked (i.e. bridging nucleic acids such as locked nucleic acids (LNA)), or other 2' substitutions.The 2'-4' links are selected from linkers known in the art, such as methylene linkers or constrained ethyl linkers.

[0254] The orphan nucleotides in the ASO described herein are preferably cytidine or its analogues (such as nucleotides carrying a Benner base) or uridine or its analogues (such as isouridine). The orphan nucleotide, whether cytidine or its analogues, or uridine or its analogues, preferably contains deoxyribose (2'-H; = DNA) but may also contain a diF modification at the 2' position of the sugar (see International Patent Application No. PCT / EP2023 / 069609; not disclosed). In one embodiment, at least one, and in another embodiment, the two (i.e., directly adjacent) nucleotides flanking the orphan nucleotide do not contain a 2'-OMe modification. The oligonucleotides wherein all nucleotides have a 2'-OMe modification (including the orphan nucleotide), a complete modification of the native base resulting in an oligonucleotide that is nonfunctional in terms of RNA editing (known in the art), presumably because it inhibits the activity of ADAR at the target site. Typically, adenosine in target RNA can be protected from editing by providing a contrasting nucleotide with a 2'-OMe group (at least when there are no other chemical substitutions or modifications within the nucleotide), or by providing guanine or adenine as contrasting bases, since these two nucleobases also reduce the editing of contrasting A.

[0255] The ASO disclosed herein is typically longer than 10 nucleotides, preferably more than 11, 12, 13, 14, 15, or 16 nucleotides, and still more preferably more than 17 nucleotides. In one aspect, the ASO herein is longer than 20 nucleotides. The ASO herein is preferably shorter than 100 nucleotides, still more preferably shorter than 60 nucleotides, and still more preferably shorter than 50 nucleotides. In a preferred aspect, the ASO herein contains 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, and even more preferably 18 to 50 nucleotides. Thus, in a particularly preferred aspect, the ASO herein contains 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 implementation, the length of the ASO is 27, 28, 29, or 30 nucleotides.

[0256] In another preferred aspect, reverse deoxy-T or dideoxy-T nucleotides may be incorporated into either end or both ends of the ASO described herein.

[0257] It is known in the art that RNA editing entities (such as human ADAR enzymes) edit double-stranded RNA structures with varying degrees of specificity, depending on several factors. One important factor is the degree of complementarity of the two strands constituting the double-stranded RNA sequence. Perfect complementarity of the two strands generally results in the catalytic domain of hADAR deaminating adenosine in an indiscriminate manner, reacting more or less with any adenosine it encounters. The specificity of hADAR1 and 2 can be enhanced by ensuring the presence of a certain number of mismatches in the double-stranded RNA, which presumably helps to localize the double-stranded RNA-binding domain in a manner not yet clearly defined. Furthermore, the deamination reaction itself can be enhanced by providing an ASO containing a mismatch opposite to the adenosine to be edited. In this disclosure, this is achieved by using a nucleoside containing a Benner base. After deamination of the adenosine in the target strand, the target strand will acquire inosine, which, for most biochemical processes, is “read” as G by the cell’s biochemical machinery. Therefore, those skilled in the art will understand that after the A-to-I conversion, the mismatch is resolved because I is able to perfectly pair with the opposing C (or cytidine analog) in the target portion of the oligonucleotide construct. However, after deamination, it is clear that ASO hybridization with the target sequence is no longer necessary. With the mismatch resolved by editing, the substrate is released, and the oligonucleotide construct-editing entity complex is released from the target RNA sequence, which can then be used for downstream biochemical processes such as splicing and translation.

[0258] The desired level of specificity for editing target RNA sequences can vary from application to application. Following the guidance in this disclosure, those skilled in the art will be able to design complementary oligonucleotides as needed and obtain the desired results through trial and error.

[0259] Pharmaceutical Compositions and Uses

[0260] This disclosure also relates to pharmaceutical compositions comprising the ASO described herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier may simply be an aqueous saline solution. This may advantageously be isotonic or hypotonic, particularly for pulmonary delivery. The pharmaceutical composition may be sterile. This disclosure also provides a delivery device (e.g., a syringe) comprising the pharmaceutical composition described herein.

[0261] The ASO described herein is suitable for administration in aqueous solution or suspension, and optionally includes additives, excipients, and / or other ingredients compatible with the pharmaceutical use. The pharmaceutical composition may contain ASO at a concentration of about 1 ng / ml to about 1 g / ml, preferably about 10 ng / ml to about 500 mg / ml, more preferably about 100 ng / ml to about 100 mg / ml. The dosage for patients may suitably range from about 1 µg / kg to about 100 mg / kg, preferably about 10 µg / kg to about 10 mg / kg, more preferably about 100 µg / kg to about 1 mg / kg.

[0262] Administration to patients may be performed by inhalation (e.g., via nebulization), intranasal, oral, injection or infusion, intravenous, subcutaneous, intradermal, intracranial, intravitreal, intramuscular, intratracheal, intraperitoneal, rectal, or central nervous system, such as intrathecal. Administration may be in solid form, powder form, pills, gel, eye drops, or any other form compatible with human use.

[0263] Treatment methods and therapeutic uses

[0264] As described above, this disclosure provides an ASO for forming a double-stranded complex with human IDUA RNA molecules in human cells. Therefore, the therapeutic effect is preferably achieved in human cells in vivo. Of course, the method can also be performed in vitro or ex vivo.

[0265] This disclosure provides an ASO or pharmaceutical composition thereof for treating a disease. This disclosure also provides the use of the ASO or pharmaceutical composition thereof in the preparation of a medicament for treating a disease. This disclosure further provides a method of treating a patient's disease, comprising administering a therapeutically effective amount of the ASO or pharmaceutical composition thereof. Preferably, the disease is MPS1. More preferably, the disease is Hurler syndrome, which is caused by a c.1205G>A mutation in the IDUA gene of a patient. The ASO is preferably administered therapeutically, rather than prophylactically.

[0266] The therapeutically effective amount, dosage, and administration regimen of the ASO to be administered may vary depending on cell type, disease to be treated, target population, route of administration (e.g., systemic vs. local), disease severity, and acceptable levels of side effects, but these can be assessed through trial and error in in vitro studies, preclinical, and clinical trials. Testing is particularly direct when the modified sequence results in easily detectable phenotypic changes. Higher doses of ASO may competitively bind intracellular ADAR, thereby depleting the amount of free entities involved in RNA editing, but standard dose testing will reveal any such effects of a given ASO and a given target.

[0267] "Effective dose" refers to the amount (over a period of time and by the method of administration) necessary to achieve the desired therapeutic outcome. The effective dose of ASO and / or ASO-GalNAc conjugates can vary depending on factors such as disease state, age, sex, individual weight, and the individual's ability to elicit the desired response. The effective dose is also the amount by which any toxic or adverse effects are outweighed by the beneficial therapeutic effect. "Therapeutic effective dose" is the amount of a pharmaceutical composition that, when administered according to the desired treatment regimen, effectively produces some of the desired therapeutic effect in proportion to a reasonable benefit / risk ratio.

[0268] A suitable assay technique involves delivering the ASO of this paper to a cell line or test organism and then collecting biopsy samples at subsequent time points. The sequence of the target RNA in the biopsy sample can be assessed, and the proportion of cells with the modification can be easily tracked. Once this assay is performed, knowledge is retained, and future deliveries can be made without collecting biopsy samples. Therefore, the method disclosed herein may include a step of identifying the presence of a desired change in the cellular target RNA sequence, thereby verifying that the target RNA sequence has been modified. This step would typically involve sequencing the relevant portion of the target RNA or a copy of its cDNA (or, if the target RNA is pre-mRNA, a copy of its splice product's cDNA), as described above, and thus the sequence change can be easily verified. Alternatively, the change can be assessed at the protein level (length, glycosylation, function, etc.) or by some functional readout, such as when the protein encoded by the target RNA sequence is an ion channel, for example, an (inducible) current.

[0269] Following RNA editing in a cell, the modified RNA can be diluted over time, for example, due to cell division, the limited half-life of the edited RNA, etc. Therefore, in practical therapeutic applications, the approach described herein could involve repeated delivery of the ASO described herein until a sufficient amount of the target RNA has been modified to provide tangible benefits to the patient and / or maintain those benefits over time.

[0270] Deamination method

[0271] This disclosure also provides a method for deamination of at least one specific target adenosine present in a target RNA molecule in a cell, wherein the target RNA molecule is human IDUA pre-mRNA or mRNA or a portion thereof, the method comprising the steps of: (i) providing the cell with the ASO or the pharmaceutical composition herein; (ii) allowing the cell to take up the ASO; (iii) allowing the ASO to anneal with the target RNA molecule; (iv) allowing a mammalian ADAR enzyme containing a natural double-stranded RNA-binding domain, such as that found in wild-type enzymes, to deaminate the target adenosine in the target RNA molecule to inosine; and optionally (v) identifying the presence of inosine in the target RNA molecule. Such methods can be performed in vitro, in vivo, or ex vivo. The identification in step (v) can be performed by: (a) sequencing the target RNA molecule; (b) assessing the presence of a functional, extended, full-length, and / or wild-type iduronate protein; or (c) using a functional readout, wherein the deaminated target RNA encodes a functional, full-length, extended, and / or wild-type iduronate protein.

[0272] Functional assessments of each disease mentioned herein will generally be performed according to methods known to those skilled in the art. A highly suitable method for determining the presence of target adenosine after deamination and sequencing is, of course, RT-PCR and sequencing, using methods well known to those skilled in the art. In one embodiment, the editing rate can be calculated by comparing the percentage of adenosine detected at the target site after editing to that of guanosine. In one embodiment, the ASO of this disclosure provides editing rates of 30%, 35%, 40%, 45%, 50%, 55%, 60%, or higher when measured in mammalian cells, such as patient-derived fibroblasts.

[0273] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Database entries and electronic publications disclosed in this disclosure are incorporated herein by reference in their entirety. The most recent version of database entries or electronic publications publicly available at the time of filing of this application is incorporated herein by reference. Database entries disclosed in this application corresponding to gene or protein identifiers (e.g., genes or proteins identified by accession numbers or database identifiers of public databases such as Genbank, Refseq, or Uniprot) are incorporated herein by reference in their entirety. The incorporated information related to genes or proteins is not limited to sequence data contained in database entries. Information incorporated by reference includes the complete content of the most recent version of the databases publicly available at the time of filing of this application. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0274] Example

[0275] Example 1. A-to-I editing of human IDUA target RNA using chemically modified oligonucleotides in an in vitro biochemical editing assay.

[0276] To obtain the human IDUA target RNA, PCR was performed using hIDUA G-block (IDT), which contains the T7 promoter and a partial human IDUA sequence as a template, using the forward primer 5'-CTC GAC GCA AGC CAT AAC AC-3' (SEQ ID NO: 38) and the reverse primer 5'-TGG ACC GAC TGG AAA CGT AG-3' (SEQ ID NO: 39). The PCR product was then used as a template for in vitro transcription. This reaction was performed using the MEGAscript T7 transcription kit. The RNA was purified on a urea gel and then extracted with 50 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.1% SDS, and 0.3 M NaCl buffer, followed by phenol-chloroform purification. The purified RNA was used as a target in biochemical editing assays.

[0277] Guide oligonucleotides A-6, 26, 32, 35, 36, 37, 38, 39, 40, 42, 43, 44, 50, and 55 were annealed with human IDUA target RNA in a 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 (200 nM target RNA and 600 nM oligonucleotides). The samples were heated at 95°C for 3 minutes and then slowly cooled to 4°C. Next, the editing reaction was performed. The annealed oligonucleotides / target RNA were mixed with a protease inhibitor (COP) TMThe mixture was prepared with EDTA-free Protease I (Sigma-Aldrich), RNase inhibitors (RNasin (Promega), poly A (Qiagen), tRNA (Invitrogen), and editing reaction buffer (15 mM Tris-Cl pH 7.4, 1.5 mM EDTA, 3% glycerol, 60 mM KCl, 0.003% NP-40, 3 mM MgCl2, and 0.5 mM DTT) to a final concentration of 6 nM oligonucleotides and 2 nM target RNA. The reaction was initiated by adding 6 nM of purified ADAR2 (GenScript) to the mixture and incubated at 37°C at predetermined time points (0 sec, 30 sec, 1 min, 2 min, 4 min, 8 min, and 16 min). Each reaction was terminated by adding 95 µl of boiling 3 mM EDTA solution. The 6 µl aliquot of the termination reaction mixture was then used as a template for cDNA synthesis using the Maxima Reverse Transcriptase Kit (Thermo Fisher) and random hexamer primers (Thermo Fisher Scientific). Initial RNA denaturation was performed at 95°C for 5 min in the presence of primers and dNTPs, followed by slow cooling to 10°C. First-strand synthesis was then performed in a total volume of 20 µl using an extension temperature of 62°C, according to the manufacturer's instructions. The product was amplified by PCR for pyrosequencing analysis using the Amplitaq Gold 360 DNA Polymerase Kit (Applied Biosystems), following the manufacturer's instructions, using 5 µl of cDNA as a template. The following primers were used at a concentration of 10 µM: Pyroseq Fwd hIDUA 5'-TAC CAC CCG CAC CCC TTC-3' (SEQ ID NO: 40) and Pyroseq Rev hIDUA Biotin, 5'- / 5BiosG / CAC CGT GTG GTT GCT GTC C-3' (SEQ ID NO: 41). PCR was performed using the following thermal cycling program: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 7 min.

[0278] Because inosine pairs with cytidine during cDNA synthesis in reverse transcription, the nucleotide incorporated at the editing site during PCR will be guanosine. The percentage of guanosine (edited) to adenosine (unedited) was determined by pyrosequencing. Pyrosequencing of the PCR products and data analysis were performed using a PyroMark Q48 Autoprep instrument (QIAGEN) according to the manufacturer's instructions, using 10 µl of PCR product input and sequencing primers of less than 4 µM: hIDUA-Seq, 5'-GCT GGC GCT GCT GGA T-3' (SEQ ID NO: 42). The setup specifically defined for this target RNA chain included two sets of sequence information. The first set defined the sequence to be analyzed by the instrument, where specific positions may contain adenosine or guanosine, indicated by " / ": GAG GAG CAG CTC TA / G GGCCGA AGT GTC GCA GGC CGG GAC C (SEQ ID NO: 43). The allocation order defined for this analysis is as follows: CGAGAG CAG CTC GTA GCG AGT GTC GCA GAC GCA C (SEQ ID NO: 44). The analysis performed by the instrument will provide the results of the selected nucleotides as the percentage of adenosine and guanosine detected at that position, so the degree of A-to-I editing at the selected position will be measured by the percentage of guanosine at that position.

[0279] Figure 2A , Figure 2B , Figure 2C and Figure 2D The results shown indicate that all tested guide oligonucleotides can edit the target hIDUA RNA.

[0280] Example 2. Specific A-to-I editing of human IDUA target RNA using modified oligonucleotides in primary human patient fibroblasts.

[0281] Next, we investigated whether the modified oligonucleotides from the previous examples edited the target adenosine in human IDUA RNA in cells carrying the c.1205G>A mutation. For this purpose, primary human W402X fibroblasts (GM00798) were used. 24 hours prior to transfection, approximately 75,000 cells were seeded per 24-well plate. Transfection was performed using 100 nM guide oligonucleotides and Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions (ratio 1:2, 1 µg oligonucleotide to 2 µl Lipofectamine 2000). 48 hours post-transfection, RNA was extracted from the cells using the Direct-zol RNA MicroPrep (Zymo Research) kit according to the manufacturer's instructions, and cDNA was prepared using the Maxima reverse transcriptase kit (Thermo Fisher) according to the manufacturer's instructions, using a combination of random hexamer and oligo-dT primers. The cDNA was used as a template for digital droplet PCR (ddPCR), with 50 ng of RNA introduced per reaction. Absolute quantification of nucleic acid target sequences was performed using the BioRad QX-200 Droplet Digital PCR system by ddPCR. 4 µl of 2x diluted cDNA obtained from the RT cDNA synthesis reaction was added to a total 21 µl reaction mixture containing ddPCR Supermix for Probes no dUTP (BioRad), Taqman SNP genotyping assay, and the following forward and reverse primers and gene-specific probes:

[0282] Forward primer: 5'-CTG TTG CGC AAG CCG GTG-3' (SEQ ID NO: 45)

[0283] Reverse primer: 5'-CCA CCG TGT GGT TGC TGT C-3' (SEQ ID NO: 46)

[0284] Wild-type probe (FAM NFQ labeled): 5'- / 56-FAM / AG CTC T+G+G +GCC GAA GTG T / 3IABkFQ / -3' (SEQ ID NO: 47)

[0285] Mutant probe (HEX NFQ labeled): 5'- / 5HEX / AG +CTC T+A+G +GCC GAA GTG T / 3IABkFQ / -3' (SEQ ID NO: 48)

[0286] Using a multichannel pipette, add a total volume of 21 µl of PCR mixture, including cDNA, to the middle row of the ddPCR cartridge (BioRad). Reproduced samples are dispensed using two cartridges. The bottom row is filled with 70 µl of droplet-generating oil (BioRad) for probes. After replacing the rubber gasket, generate droplets in the QX200 droplet generator. Transfer 42 µl of the oil emulsion from the top row of the cartridge to a 96-well PCR plate. Seal the PCR plate with foil at 170°C for 4 seconds using a PX1 plate sealer, then perform the following PCR program: 1 cycle of enzyme activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 30 sec and annealing / extension at 63°C for 1 min, 1 cycle of enzyme inactivation at 98°C for 10 min, followed by storage at 8°C. After PCR, read and analyze the plate using a QX200 droplet reader. Figure 3A , Figure 3B and Figure 3C The results shown indicate that all tested oligonucleotides induced editing.

[0287] Example 3. In vivo use of modified oligonucleotides for specific A-to-I editing of mouse Idua target RNA and restoration of protein activity.

[0288] In the next experiment, we investigated whether A-to-I editing could also occur in the livers of mice with the W392X mutation (a known Hurler mouse model; see, for example, International Patent Application Publication No. WO 2018 / 041973) using oligonucleotides carrying the GalNAc (GN) ligand motif. Figure 4 The names, SEQ ID NO, and sequences of the fourteen mouse Idua sequence-specific ASOs initially tested, as well as one out-of-order oligonucleotide (Scr1), are shown, all containing the GalNAc moiety. The GalNAc moiety is linked to the 3' end of each oligonucleotide and conforms to the structure of Formula I in International Patent Application Publication No. WO 2022 / 271806. In addition to editing efficiency (percentage), the reversibility of idurolactamase protein activity after oligonucleotide treatment was investigated. For this purpose, four mice (each group; one ASO per group) were subcutaneously injected with 10 mg / kg ASO on days 0, 4, 8, and 12. Animals were sacrificed on day 14. RNA editing in the liver was determined using ddPCR to quantify the percentage of target RNA edited, generally as described above, using the following primer and probe sets:

[0289] Forward primer: 5'- ctcacagtcatggggctc-3' (SEQ ID NO: 130)

[0290] Reverse primer: 5'- cactgtatgattgctgtccaac-3' (SEQ ID NO: 131)

[0291] Wild-type probe (FAM NFQ labeled): 5'- / 56-FAM / A+CAACTC+T+GGGCAGAGG+T / 3IABkFQ / -3' (SEQ ID NO: 132)

[0292] Mutant probe (HEX NFQ labeled): 5'- / 5HEX / A+CAACTC+T+A+GGCAGAGG+T / 3IABkFQ / -3' (SEQ ID NO: 133)

[0293] Iduuronidase activity was analyzed in liver tissue using an activity assay generally described in WO2018 / 041973 to quantify enzyme activity after treatment with a specific ASO. Out-of-order oligonucleotides (4 animals) and the untreated (NT) group (2 animals) served as negative controls, while wild-type animal samples served as positive controls (set as 100%) in both assays.

[0294] The results show Figure 5A and Figure 5B middle. Figure 5A The percentage of edits on day 14 is displayed. Figure 5B Showing the use of Figure 5A Iduuronidase activity in the same liver samples from the same mice treated with the guide oligonucleotides shown. The highest percentage of editing was determined after treatment with the oligonucleotides mIdua-75GN, -89GN, -88GN, -85GN (one animal showed 8% editing), and -87GN (SEQ ID NO: 49, 58, 57, 55, and 56). Those skilled in the art will understand that RNA renewal can be highly dynamic, can be very rapid, and can vary depending on the tissue, cell, and target RNA, and the availability of the guide oligonucleotides to induce editing adds another layer of complexity to this assessment. This makes it possible that the percentage of RNA editing may differ at different time points, and a particular percentage at a strict time point may not be entirely translated into the amount of protein translated from edited RNA versus unedited RNA, not to mention the possibility of degradation differences between edited and unedited target RNA. Rather than strictly assessing the percentage of editing, the activity of the iduronidase protein (absent in W392X mice) reveals an additional indication of whether the edited RNA was translated into wild-type protein. Figure 5BIt is clearly shown that after ASO treatment, iduronate activity was measured to increase by nearly 6 times (approximately 4.5% activity) compared to levels observed with out-of-order oligonucleotides, with mIdua-75GN, -89GN, -88GN, -85GN, and -87GN again showing the best performance, although with slight variations in efficiency. It can be concluded that when observing the percentage of edits, the best-performing guide oligonucleotides also provide the highest percentage of protein recovery in vivo. Figure 5C The study showed iduronate activity in the plasma of the same animals on day 14, clearly demonstrating that the aforementioned best-performing oligonucleotides (in terms of RNA editing and hepatic iduronate activity) also exhibited the highest plasma iduronate activity levels, providing an excellent biomarker tool for future evaluation.

[0295] Example 4. In vivo use of mIdua-75GN and mIdua-85GN ASO for specific A-to-I editing of mouse Idua target RNA and restoration of protein activity.

[0296] Next, guide oligonucleotides mIdua-75GN and mIdua-85GN (see Example 3 and...) Figure 4 The results of these ASOs in mice were used in experiments similar to those shown in Example 3. However, the dosage and protocol schedule were changed, and sampling was conducted at different time points. Furthermore, editing and protein activity assays were performed at 4 and 8 weeks post-sacrifice, respectively. All mice were subcutaneously injected with ASO. Each group contained 4 animals, except for group 1 which contained 20 animals, with 4 animals sacrificed at 12 hours, 48 ​​hours, 1 week, 2 weeks, and 4 weeks post-dose. The seven groups were set up as follows: Figure 6 As shown.

[0297] Figure 7A The results for Group 1 are shown, indicating that the edit level was between 1% and 2% at 48 hours, 1 week, and 2 weeks, and appeared to decrease after 4 weeks. Interestingly, however, iduronate activity was also examined at these same time points, and it appeared to increase over time. Figure 7B It can be seen that enzyme activity may even increase four weeks after a single dose on day 0. These experiments suggest that proteins produced from edited transcripts, and the activities performed by these proteins, may follow the editing of the transcripts, which could be a highly dynamic process where new RNA is transcribed, old RNA is degraded, and available ASO can induce editing when the transcript is targetable. Proteins translated from these edited transcripts may actually have a longer half-life and exist for much longer than each edited transcript itself. Further investigation is needed to clarify whether this is indeed the case.

[0298] Groups 2 and 3 differed only in the dosage administered; Group 2 received 10 mg / kg, and Group 3 received 30 mg / kg. Both groups received the medication twice weekly. Figures 8A-8B (ASO mIdua-85GN is referred to as EON 11) shows the percentage of edited liver at autopsy (4 weeks after day 0), indicating a dose-dependent effect, with 3.5% edited in mice administered 30 mg / kg and 2.5% in mice administered 10 mg / kg. It is unclear why these percentages are lower than those described in the previous example 3. Importantly, however, iduronate activity increased by an average of approximately 13% in mice receiving 30 mg / kg and 5% in mice receiving 10 mg / kg, indicating a strong dose-dependent effect on protein activity levels.

[0299] Groups 3 and 4 differ only in the ASO applied. ASO mIdua-75GN is also known as EON 6, and mIdua-85GN is also known as EON 11. Figures 9A-9B The results showed that mIdua-85GN (EON 11) was superior to mIdua-75GN (EON 6) in both RNA editing percentage and idurolactamase activity, which is consistent with... Figures 5A-5C The findings shown are similar.

[0300] Groups 2 and 5 differed only in their regimen; Group 2 received mIdua-85GN (EON 11) (10 mg / kg) twice weekly for 4 weeks, while Group 5 received the same dose (10 mg / kg) once weekly only. There were no differences in edit percentage and idurouronase activity between these groups, indicating that ASO overdose was available during the experiment. Results showed... Figure 10A middle.

[0301] The difference between Group 2 and Group 6 is that the same dosing regimen (twice a week, 10 mg / kg) was administered either for 4 weeks until autopsy (Group 2) or 8 weeks until autopsy (Group 6), with ASO administered as mIdua-85GN. Figure 11A-11B The results showed that with prolonged administration, the percentage of edits and iduronate activity increased. However, as... Figure 11A-11B It was also shown that changing the dosing regimen to twice a week initially, followed by once every two weeks (group 7) did not provide an improvement. Figure 11A and 11B The second item in the text represents group 2. Figure 11A and 11B The third item in the text represents group 6. Figure 11A and 11B The fourth item in the text represents group 7.

[0302] Example 5. In vivo A-to-I editing of mouse Idua target RNA using mIdua-75GN and mIdua-85GN ASO resulted in a reduction of GAG.

[0303] GAG content was tested in samples (liver at sacrifice and urine samples collected during the process) from all mice in the seven groups described in Example 4. The most severe form of MPS 1 is Hurler syndrome. MPS 1 is a hereditary metabolic disorder characterized by dysfunction of iduronate, encoded by the IDUA gene, leading to the accumulation of GAGs dermatan sulfate and heparan sulfate as oligosaccharides in lysosomes. Since the RNA editing disclosed herein restores iduronate activity, it should result in a reduction in GAG content in various tissues, including the liver and circulation (represented by excreted urine). Therefore, all mice in all groups described in Example 4 were sampled, and the GAG ​​content of the samples was tested using the Blyscan GAG Assay Kit (Blyscan, No. B1000). 20 mg to 50 mg of liver tissue was homogenized with a pestle and digested in 1 mL of papain extraction reagent at 65°C for 24 h. The total GAG amount in each sample was measured according to the manufacturer's protocol. Finally, the measured GAG was normalized to mg of tissue. Urine samples were centrifuged and filtered using Ultrafree-MC HV Centrifugal Filters (Cat# UFC30HV00, Millipore). GAG levels in the urine were also measured using a Blyscan GAG Assay Kit (Blyscan, No. B1000). The total GAG amount in each sample was measured according to the manufacturer's protocol. Finally, the measured GAG was normalized to creatinine levels using a creatinine assay kit (Cat# MAK080, Sigma) according to the manufacturer's protocol.

[0304] Figure 12A-12B The results showed that in groups 2, 3, 4, 5, 6, and 7, GAG levels were significantly lower compared to untreated mice. Figure 12A This shows the levels in the liver. Figure 12B The levels in urine were shown, and the levels achieved were comparable to those in the wild-type case where GAG ​​levels were close to zero. Group 1, with a single dose, did not show a reduction (data not shown).

[0305] These results collectively demonstrate that RNA editing of mouse Idua transcripts containing the W392X mutation, which is similar to the mutation that causes Hurler syndrome in humans, resulted in a significant increase in idurouronase activity over an extended period, followed by a significant decrease in GAG levels in the liver and urine. This suggests that RNA editing and the restoration of protein activity may have a significant impact when human Hurler syndrome patients are treated with the oligonucleotides disclosed herein using an appropriate dosing regimen.

[0306] Example 6. Stability study.

[0307] The efficacy of ASO in producing RNA editing on specific target sequences depends on a number of characteristics, including the ability to interact (and re-release) with complementary portions of the target sequence, the ability to recruit endogenous ADAR as a double-stranded complex with the target molecule, and the general stability of ASO, as it is always susceptible to degradation by cellular components such as nucleases once it enters the target cell and moves toward the target molecule.

[0308] To investigate the GalNAc-conjugated A-50 (A-50GN) (which targets human IDUA transcripts) and its counterpart A-78 (also conjugated to GalNAc; A-78GN) (which shares the same sequence and chemical modifications as A-50GN, but contains a PS bond instead of a PO bond at the 5' end) (see [link to relevant documentation]). Figure 1B And the corresponding ASO for targeting mouse Idua target molecules: mIdua-85GN and mIdua-88GN conjugated with GalNAc. Figure 4 The stability of A-50GN was determined using a biochemical stability assay. For this purpose, the following mixtures were used to determine the stability of each A-50GN at a final concentration of 10 µM. Figure 15 (abbreviated as A-50), A-78GN ( Figure 15 (abbreviated as A-78), mIdua-85GN and mIdua-88GN: i) Snake venom phosphodiesterase (6.5 x 10⁻⁶) -5 units / µl; Sigma; cat# P3243-1VL); ii) BAL-31 (2.6x10 -2 units / µl; Takara; cat# 2510A); iii) DNAse I (2.6x10 -2 units / µl; ThermoFisher Scientific; cat# EN0531); and iv) RNAse A (1.3x10 -1The ASO solution (µg / µl; Biolabs; cat#M0303L) was dissolved in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 1 mM EDTA, and 50% glycerol buffer, for a total volume of 50 µL. As a control, the same ASO solution was diluted in PBS to a final concentration of 10 µM, in a volume of 50 µL. After incubation at 37°C for 24 hours, the reaction was terminated with 50 µL of 8.0 M guanidine hydrochloride buffer (Thermo Fisher Scientific; cat# 24115) and heated at 95°C for 2 minutes. Samples were stored at -20°C until further analysis.

[0309] HPLC analysis was performed using ion-pair reversed-phase (IPRP) UPLC combined with UV detection and mass spectrometry (MS). IPRP is based on the electrostatic interaction between oligonucleotides and ion-pairing reagents (such as tri- or tetraalkylamine salts) added to the mobile phase, and the hydrophobic interaction between the ion-pairing reagents and the nonpolar stationary phase. For this method, triethylamine (TEA) and hexafluoroisopropanol (HFIP) were used as ion-pairing reagents, and methanol was used as an organic modifier. UV detection at 260 nm and MS detection for identification were used. Figure 14A The chromatogram of A-50GN is shown. Figure 14B The chromatogram of A-78GN is shown, clearly demonstrating that A-78GN has higher stability than A-50GN, with more degradation peaks visible in the A-50GN chromatogram. MS spectra of the peaks in the chromatogram of the degraded A-50GN sample indicate cleavage at bond position +10 between 2'-OMe-modified cytidine (Cm) at position +10 and 2'-MOE-modified adenosine (Ae) at position +11. Figure 15 The stability percentages are shown in bar charts relative to the untreated sample, indicating that A-78GN has a stability of 96%, mIdua-85GN 91%, mIdua-88GN 88%, and A-50GN 48%. Unbound by theory, mIdua-85GN is more stable than A-50GN, even though it contains the same chemical modification at the same position within the nucleotide sequence as A-50GN. This can be explained by the fact that mIdua-85GN contains a 2'-OMe modified guanosine instead of cytidine at nucleotide position +10.

[0310] The stability of ASO (e.g., A-50 conjugated with GalNAc (A-50GN)) can be measured by any other assay. A non-limiting assay involves exposing ASO to a liver homogenate extract and analyzing the stability of the ASO by HPLC.

[0311] Example 7. Specific A-to-I editing of human IDUA target RNA using modified oligonucleotides in primary human patient fibroblasts.

[0312] Those skilled in the art know that PS bonds improve the stability of ASOs, while PO bonds are more likely to be targets for nucleases. However, PO bonds are preferred over PS bonds because they are closer to the natural state (where RNA becomes double-stranded and recruits ADAR for RNA editing) and due to potential toxicity and manufacturing issues. Therefore, it was investigated whether replacing the PO bonds at the 5' end of A-50 with PS bonds (but not as many as used in A-78) would improve the stability of A-50 to a level comparable to that observed in A-78, particularly regarding the bond between positions +9 and +10. To investigate this and determine the effect observed at position +10, a further set of ASOs was designed, all with a 3'-linked GalNAc portion, as shown in Figure 16, where multiple positions at the 5' end of the ASO are either PS or PO bonds. The only difference between A-50 GN and A-118 (SEQ ID NO: 113) is that A-118 contains a Gm nucleotide (underlined in Figure 16) at position +10 instead of Cm. This change provided ASO with approximately 35% more stability than A-50 GN in stability measurements (data not shown), indicating that by changing Cm at position +10 to Gm, a cut between Cm at position +10 and Ae at position +11 can be prevented.

[0313] All ASOs in Figure 16 were tested for RNA editing in primary human W402X fibroblasts (GM00798), followed by RNA isolation and ddPCR analysis as described in Example 2 above, and comparisons with A-50, A-50GN, and A-78GN. The results showed... Figure 17As previously observed in in vitro cell transfection assays, the GalNAc version of A-50 (A-50GN) performed inferiorly to the GalNAc-free version (A-50). Notably, when using naked uptake in these GM00798 fibroblasts, A-50 without the GalNAc moiety outperformed ASO carrying the GalNAc moiety (data not shown), indicating that simple in vitro cell entry is easier in the absence of GalNAc. However, this does not reflect the in vivo situation. Clearly, the GalNAc moiety is more beneficial in the in vivo environment when specific targeting of the liver is required. In naked uptake experiments using patient fibroblasts, A-121 (SEQ ID NO: 116) and A-127 (SEQ ID NO: 122) performed well and were superior to A-50GN (data not shown). Furthermore, several novel GalNAc-modified ASOs outperformed A-50GN in transfection assays, particularly A-118 (SEQ ID NO: 113, as described above), as well as A-107 (SEQ ID NO: 102), A-125 (SEQ ID NO: 120), A-128 (SEQ ID NO: 123), A-129 (SEQ ID NO: 124), A-130 (SEQ ID NO: 125), and A-131 (SEQ ID NO: 126). Interestingly, the increased stability of A-118 over A-50, as described above (where the Cm > Gm substitution at position +10 is clearly significant), also contributed to the increased editing efficiency observed in these cells, reflecting previous findings in... Figure 15 The situation observed by A-50 and A-78.

[0314] A-118 and other optimized versions of A-50 will be further tested to determine their in vivo RNA editing efficiency. Notably, ASO A-128, A-129, A-130, and A-131 all contain three deoxynucleotides in the central triplet with an orphan nucleotide in the middle, which further contributes positively to the RNA editing effect on the target adenosine.

[0315] Example 8. Study on specific A-to-I editing of mouse Idua target RNA using multiple ASOs in vivo.

[0316] As a further study of the in vivo studies outlined in Examples 3, 4, and 5, a subsequent longer-duration study was conducted in the same W392X Hurler mouse model to investigate the editing efficacy, restoration of iduronate activity, and reduction of GAG accumulation for up to 20 weeks after ASO administration. Tissues studied included liver, kidney, spleen, heart, lung, quadriceps, as well as plasma and urine. For this purpose, ASO mIdua-85GN (SEQ ID NO: 55), mIdua-75GN (SEQ ID NO: 49), and mIdua-89GN (SEQ ID NO: 58) were compared to their GalNAc-free versions (SEQ ID NO: 128, 127, and 129). Study settings are described in [link to study setup]. Figure 18 Table 1 summarizes the different names used for the three ASOs in this study.

[0317] Table 1. ASO reference names and manufacturing (RM) codes for targeting human IDUA and mouse Idua transcripts, as used in the accompanying examples and figures. GN refers to the GalNAc portion. In some embodiments, such as formulas (A), (B), and (C), the GalNAc structure is as disclosed herein in Formula I and in International Patent Application Publication No. WO2022 / 271806.

[0318]

[0319] All administrations were subcutaneous (SC), except for group 18, where the LNP formulation was administered intravenously (IV). All regimens were administered once weekly, except for group 18, where the LNP formulation was administered only once. Administration was performed only weekly at T=0 and thereafter, for a maximum of 8 administrations. Thus, for example, group 7 received doses only at weeks 0, 1, 2, 3, 4, 5, 6, and 7, while group 5 received doses at weeks 0, 1, 2, and 3. Group 1 served as a wild-type control and received no substance. Samples were also collected from group 1 mice as shown. Groups 2 through 18 were strains of the W392X Hurler mouse model, while group 1 was a wild-type C57BL / 6j strain. Group 2 received only the carrier solution, without any ASO. Autopsy was performed at the last urine / blood sample, and no ASO was administered prior to autopsy. The amount of ASO in groups 11 through 17 was adjusted (to approximately 26 mg / kg) due to the lack of the GalNAc moiety in the compound and chemical diversity. Scr1-GN ASO as Figure 4 As shown. Following the autopsy, tissue, urine, and plasma samples were processed as described above. To determine RNA editing, ddPCR was performed on RNA isolated from different tissues using the method described above. Iduuronidase activity measurements and assessments of reduced GAG accumulation were also performed as described above.

[0320] Figure 19A and Figure 19B The percentage of editing in liver samples with and without GalNAc is shown separately at different time points. In mice administered mIdua-85GN, the highest percentage of approximately 5% was reached after 4 weeks, maintained until about 8 weeks, and then slowly declined to approximately 2% after 20 weeks. The percentage of editing provided by ASO without GalNAc was even lower. Interestingly, idurolactinase activity in the liver after mIdua-85GN administration peaked at approximately 21% at 8 weeks and remained there until 16 weeks (see [link to article]). Figure 19C Surprisingly, similar levels were observed in mouse livers treated with ASO that did not contain GalNAc (see...). Figure 19D In these mice (group 16), mIdua-75 also performed relatively well. Regarding GAG reduction, mIdua-85GN was superior to mIdua-75GN and mIdua-89GN, reducing GAG by 65% ​​compared to the disordered control and maintaining it for up to 20 weeks (see [link to relevant documentation]). Figure 19E This effect was also observed in the "naked" version, although not as high as that seen in ASO containing GalNAc (see...). Figure 19F These results clearly demonstrate that while the initial editing effect of mIdua-85GN ASO appears to be relatively low (approximately 5%), it significantly increases iduronate activity, which correlates well with the percentage of editing (R=0.46) and is maintained for an extended period, subsequently leading to a significant reduction in GAG accumulation. This suggests that the inventors have been able to provide in vivo proof of concept that RNA editing produces therapeutically relevant effects in this Hurler mouse model.

[0321] The same measurements were performed on the kidneys from the same mice described above. Regarding editing, there were no significant differences between the GalNAc-containing ASO and the "naked" version (respectively). Figure 20A and Figure 20B Although mIdua-85GN (again) was significantly superior to the other two ASOs. Regarding enzyme activity ( Figure 20C and Figure 20D The same applies to GAG reduction; after 20 weeks of using mIdua-85GN, GAG was reduced by approximately 35% (see...). Figure 20E and Figure 20F ).

[0322] For spleen samples, only edits were measured, and mIdua-85GN and mIdua-85 were also superior to mIdua-75 and mIdua-89 ASO, reaching approximately 0.7% after 4 to 8 weeks (see [link to original text]). Figure 21A and Figure 21B ).

[0323] In the lungs, editing is moderate, reaching a maximum of approximately 2% (see...). Figure 22A and Figure 22B However, the enzyme activity reached approximately 4% (see...). Figure 22C The GAG ​​reduction was still quite significant; compared to the out-of-order control, the non-GalNAc version using mIdua-85 achieved almost a 55% reduction after 20 weeks (see [link to documentation]). Figure 22D ).

[0324] Similar editing results were observed in heart tissue samples from treated mice (see...). Figure 23A and Figure 23B In the early stages (using mIdua-85GN for 4 weeks), approximately 4% enzyme activity was also observed (see...). Figure 23C ), and a relatively diverse range of GAG reduction modes, achieving a reduction of almost 80% after 20 weeks of use with mIdua-85GN (see Figure 23D ).

[0325] Regarding the quadriceps, a similar pattern was observed, with an edit of approximately 1-2% (see...). Figure 24A and Figure 24B At 8 weeks, enzyme activity was approximately 4% (see...). Figure 24C ).

[0326] like Figure 18 As stated, blood and urine samples were collected at different time points and during autopsies. Figure 25 The results show the enzyme activity of different groups of blood samples (plasma from the heart) during the autopsy phase. Consistent with the above, mIdua-85GN performed best, with enzyme activity of approximately 3 to 4% at 4 to 8 weeks compared to samples set as 100% wild-type.

[0327] GAG reduction was also measured in urine samples collected at different time points and during autopsies. Figure 26A and Figure 26B The reduction in GAG at autopsy was shown in ASOs containing GalNAc and those without GalNAc. Although there appeared to be no effect at the earliest 4-week time point, significant reductions in GAG levels were observed from 8 to 20 weeks with all three different ASOs, with mIdua-85GN showing the best performance. While levels considered wild-type (hollow bars) showed a reduction of approximately 60% compared to negative PBS and disordered controls, mice treated with mIdua-85GN showed a reduction of approximately 45% at 12 weeks and maintained the effect at 20 weeks with a reduction of approximately 31 to 33%, again demonstrating that the reduction in GAG after repeated treatment with ASOs was very significant, with mIdua-85GN showing the best performance.

[0328] like Figure 26B As shown, the ASO version without GalNAc provided similar results in these urine samples. Figure 26CThe time-series effect of mIdua-85GN on GAG reduction from T=0 to T=20 weeks is shown compared to a randomized control set to 100% (= no reduction). A clear trend emerged from week 4 and maintained a prolonged period of strong reduction until week 20 when the last batch of mice was sacrificed. This clearly demonstrates that mIdua-85GN ASO is capable of editing the target transcript in this Hurler mouse model. It should be understood that the percentage of editing may be considered moderate, but this moderate editing translates into a very strong effect on enzyme activity, subsequently leading to a very significant and treatment-related reduction in GAG in the urine of these animals.

[0329] Example 9. In vivo intraventricular (ICV) administration of mouse Idua-specific ASO for specific A-to-I editing of mouse Idua target RNA (ICV Study 1).

[0330] This study investigated whether mouse Idua editing could be achieved by administering mouse-Idua-specific ASO to the intraventricular cavity (ICV) of the Hurler Idua-W392X mouse model. ICV injection allows ASO to be widely distributed in different parts of the brain, such as the administered ventricle and cerebrospinal fluid (CSF). ICV injection is commonly used to administer drugs and bypass the blood-brain barrier (BBB), for example, to treat long-term and / or chronic pain, and to administer chemotherapy drugs for glioma. The ASO used in this study was mIdua-71, which has the following nucleotide sequence and chemical composition:

[0331]

[0332] The chemical modifications are shown in Figure 1. Figure 4 As shown in Figure 16. Note that the only difference between mIdua-71 and mIdua-85 (SEQ ID NO: 128) is that the internucleotide bond at position -8 is a PS bond instead of a PNdmi bond. The study design is shown in Table 2.

[0333] Table 2. Study design for editing mouse Idua transcripts following ICV injection of mIdua-71 ASO in the Hurler model Idua-W392X. aCSF samples were obtained from untreated animals as negative controls (na = not applicable). Autopsies were performed at 2 weeks (6 animals) and 4 weeks (6 animals) post-treatment.

[0334]

[0335] In addition to editing, the distribution of ASO in the brain following ICV injection was investigated. After sacrifice, the brain was dissected and divided into two hemispheres. One hemisphere was used for FISH staining, and the other hemisphere was dissected to represent the frontal cortex (further divided into two parts), hippocampus, cerebellum, and midbrain. A portion of the frontal cortex was used for protein analysis (iduronuclease activity assay), while the other hemisphere, hippocampus, cerebellum, and midbrain were used for ddPCR assays to determine editing. RNA isolation, cDNA preparation, and ddPCR assays for the mIdua target sequence were performed as described above. FISH staining on brain slices indicated that mIdua-71 ASO was well distributed in the brain following ICV administration (data not shown).

[0336] Table 3 shows the results of ddPCR in the frontal cortex, hippocampus, cerebellum and midbrain of four groups of mice (see Table 2), with aCSF samples from untreated mice (groups 1 and 3) serving as controls.

[0337] Table 3. Percentage of edits in the frontal cortex, hippocampus, cerebellum and midbrain of W392X mice after ASO treatment, as determined by ddPCR, according to Table 2.

[0338]

[0339] In mice (group 4) treated with 250 μg mIdua-71 for 4 weeks, an average editing level of approximately 25% was observed in the frontal cortex. Similar levels were observed in the hippocampus, with slightly lower levels in the cerebellum and midbrain. In group 2, across all tissues, the animals achieved an average editing level at 4 weeks higher than at 2 weeks. No editing was observed in untreated animals, with one exception: an outlier in one animal in group 1 at 2 weeks (untreated). When comparing brain slices from mice treated with 250 μg of mIdua-71 to those from mice treated with 100 μg of ASO, there was no significant difference in the percentage of editing (data not shown).

[0340] As described above, iduronate activity was also measured in the same mice. Table 4 shows the results of iduronate activity measurements in mice treated with 250 μgmIdua-71 (groups 2 and 4) compared with untreated animals (aCSF; groups 1 and 3), as shown in Table 2.

[0341] Table 4. Iduuronidase activity (nmol / 24h / mg) in the frontal cortex, hippocampus, cerebellum and midbrain of W392X mice after ASO treatment according to Table 2.

[0342]

[0343] Protein activity generally followed the editing results in these mice, with the highest iduronate activity observed in the frontal cortex after 4 weeks of treatment (mean 12 nmol / 24hr / mg). Activity levels were slightly lower in the hippocampus, cerebellum, and midbrain, but significantly higher than the background levels observed in untreated animals, all of which showed a basal activity of approximately 1–2 nmol / 24hr / mg.

[0344] Example 10. Study on specific A-to-I editing of mouse Idua target RNA by administering mouse Idua-specific ASO with ICV in vivo.

[0345] ICV Study #2: This study investigated whether Idua editing could be achieved in the Hurler Idua-W392X mouse model after administration of 250 μg mIdua-85 (SEQ ID NO: 128; RM4347) and 250 μg or 400 μg mIdua-85GN (SEQ ID NO: 55; RM4870) (both mouse-Idua-specific ASOs). Editing was measured in the frontal cortex, hippocampus, cerebellum and midbrain (as in Example 9), as well as the brainstem and spinal cord (cervical, thoracic, and lumbar regions).

[0346] Although ICV injection enables ASO to be widely distributed throughout the CNS, much of the payload delivered via this route of administration eventually escapes into the peripheral circulation. Therefore, in addition to CNS tissues, this study also evaluated editing, idurolactamase activity, and GAG accumulation in peripheral tissues such as the liver. Furthermore, this study compared the activities of unconjugated GalNAc ASO and GalNAc-conjugated ASO during ICV administration. The study design is shown in Table 5.

[0347] Table 5. Study design for editing mouse Idua transcripts in the Hurler model Idua-W392X after ICV injection of mIdua-85 and mIdua-85GN ASO. aCSF samples (group 1) were obtained from untreated animals as negative controls (na = not applicable). Mice were sacrificed 4 weeks after treatment. GN is the GalNAc structure disclosed in International Patent Application Publication No. WO2022 / 271806, Chinese Formula I.

[0348]

[0349] Table 6 shows the edited results obtained by ddPCR in different brain tissues 4 weeks after treatment with the specified ASO according to Table 5.

[0350] Table 6. Percentage of editing determined by ddPCR in the frontal cortex, hippocampus, cerebellum, midbrain, brainstem, upper cervical, middle thoracic, and lower lumbar regions of the spinal cord, and liver of W392X mice after ASO treatment, according to the study design in Table 5. Group 1 showed background levels of 0.3 to 0.5 in various tissues.

[0351]

[0352] As can be observed in Table 6, the percentage of editing observed in the liver, whether using unconjugated GalNAc mIdua-85 ASO (Group 2) or conjugated GalNAc mIdua-85 ASO (Groups 3 and 4), was higher than the percentage of editing in the background (Group 1). Consistent with the ICV study shown in Example 9, unconjugated GalNAc mIdua-85 ASO measured a significant percentage of editing in all brain tissues (and spinal cord), and was superior to the GalNAc conjugated version mIdua-85GN. This is not entirely unexpected, as GalNAc is typically used to target hepatocytes, not brain cells.

[0353] In these same groups, idurouronase activity in the cortex, hippocampus, cerebellum, midbrain, brainstem, cervical spinal cord, and liver was measured (as described above). These results are shown in Table 7.

[0354] Table 7. Iduuronase activity (nmol / 24h / mg) in the frontal cortex, hippocampus, cerebellum, midbrain, brainstem, cervical spinal cord, and liver of W392X mice after ASO treatment, according to Table 5.

[0355]

[0356] As observed in the study described in Example 9, Idua activity appeared to be potentially rejuvenated in all examined brain tissues, and this iduronate activity was found to generally follow the editing results in different tissues as determined by ddPCR, as shown in Table 6. Notably, iduronate activity also appeared to be significantly increased in the livers of these mice receiving ASO via ICV administration, although no real difference was observed between mIdua-85 and mIdua-85GN-treated animals. To determine whether GAG accumulation was reduced in the livers of these ICV-treated mice, measurements were also performed according to the protocol described above. The values ​​of these measurements are shown in Table 8.

[0357] Table 8. GAG accumulation in the liver of W392X mice after ASO treatment (μg / mg tissue) according to Table 5.

[0358]

[0359] As shown in Table 8, compared with the negative control (Group 1), treatment with mIdua-85 (Group 2) and mIdua-85GN (Groups 3 and 4) appeared to reduce GAG ​​accumulation in the liver of W392X mice by approximately 30%, although the percentage of editing in the liver appeared relatively moderate as shown in Table 6. This indicates that even after administration of ASO targeting mouse Idua transcripts via ICV, ASO can induce editing in brain tissue near the application site, as well as in the liver, and importantly, produce downstream effects such as the observed reduction in GAG accumulation in the liver.

[0360] ICV Study #3: Another study investigated the administration of GalNAc-free mIdua-85 to W392X mice via ICV. This study included analyzing the effects of 250 μg of unconjugated mIdua-85 (GalNAc-free) ICV administration compared to untreated mice (control group). Autopsies were performed on day 28 (4 weeks) post-treatment. Each group (control group and 250 μg mIdua-85 group) contained 5 mice. Editing was measured in the frontal cortex, hippocampus, cerebellum, midbrain, brainstem, striatum, liver, and quadriceps femoris muscle using ddPCR according to the protocol described above. The results of the editing assays are shown in Table 9.

[0361] Table 9. Based on the study design in Table 5, the percentage of edits in the frontal cortex, hippocampus, cerebellum, brainstem, midbrain, striatum, liver, and quadriceps femoris of W392X mice after 4 weeks of treatment with 250 μg mIdua-85, as determined by ddPCR. Untreated (NT) mice (n=5) served as negative controls.

[0362]

[0363] Consistent with observations in earlier ICV study #1 (which compared mIdua-85 and mIdua-85GN), significant editing was observed in all brain tissues of all five treated animals after ICV injection of 250 μg ASO, with editing levels exceeding 20%. Editing was also significantly higher in the liver and quadriceps femoris muscles of mIdua-85 treated animals than in untreated animals.

[0364] Similarly, in these animals, iduronate activity was measured in tissues studied for RNA editing using the methods described in the two earlier ICV studies mentioned above. The results are shown in Table 10.

[0365] Table 10. Iduuronase activity (nmol / 24h / mg) in the frontal cortex, hippocampus, cerebellum, brainstem, midbrain, striatum, liver and quadriceps femoris of W392X mice after 4 weeks of treatment with 250 μg mIdua-85, with untreated (NT) mice (n=5) as negative controls.

[0366]

[0367] Table 10 shows that iduronate activity was restored in the brain tissue of W392X mice treated with mIdua-85, and significant iduronate activity was also detected in the liver and quadriceps femoris muscle of these ICV-treated animals. No significant iduronate activity was observed in any tissue of untreated animals, indicating that, as previously stated, editing leads to an increase in Idua protein activity, which is observable at least 4 weeks after initial treatment.

[0368] Example 11. Intrathecal delivery of edited nucleotides.

[0369] This embodiment describes a nonhuman primate (NHP) study in cynomolgus monkeys involving the delivery of an editing guide oligonucleotide via the intrathecal (IT) pathway. The editing guide oligonucleotide (“test article”) was administered to the animals according to the study design shown in Table 11. In this embodiment, the test article was a guide oligonucleotide according to SEQ ID NO: 208, targeting β-actin transcripts. This study demonstrates the editing efficiency and exposure following IT delivery. SEQ ID NO: 208 represents:

[0370]

[0371] The modifications are shown in Figure 1. Figure 4 As shown in Figure 16.

[0372] method

[0373] 1. Animals. Twenty untreated female cynomolgus monkeys had IT catheters implanted in their lumbar region to facilitate the administration of test items.

[0374] 2. Reagents. The test item consists of single-stranded edited oligonucleotides according to SEQ ID NO: 208.

[0375] 3. In vivo study design. Dosage groups and final autopsy time points are shown in Table 11. Test articles were administered via IT catheters at the total doses, volumes, and concentrations shown in Table 11. Toxicity assessments were based on mortality, clinical observations, body weight, qualitative food consumption, neurobehavioral observations, and clinical and anatomical pathology.

[0376] 4. Tissue Collection. Continuous plasma and cerebrospinal fluid (CSF) collection was performed on the day of administration, up to 360 hours post-administration. Terminal tissue collection was performed on the necropsy day as shown in Table 11. Tissue from each animal (if present) was preserved as shown in Table 12.

[0377] 4.1 Histology. All tissues from each monkey designated "E" (Table 12, Autopsy, Organ Weight and Gross Visual Inspection sections) were incubated in 10% neutral buffered formalin (NBF) at room temperature for at least 24 hours but no more than 28 hours. After removal from NBF, the tissues were washed three times with 1x PBS and stored in PBS at 4°C for up to 72 hours until processed into paraffin blocks. After processing into paraffin blocks, the tissues were cut into nominal 5-micrometer thick sections, slides were prepared, and stained with hematoxylin and eosin. For the spinal cord (mid-thoracic and mid-lumbar segments), one transverse section and one oblique transverse section (including the injection site) were taken. For the spinal cord (mid-cervical segment), one transverse section and one parasagittal longitudinal section were taken.

[0378] 4.2 Microscopic examination. All tissues indicated by “E” from all animals (Table 12, Autopsy, Organ Weight and Macroscopic Examination sections) were examined under a microscope. After the initial microscopic evaluation, an independent peer review was conducted.

[0379] 4.3 Cryopreservation of tissues for exploratory analysis. The following samples were collected from each monkey after any of the following tissue samples were collected for microscopic evaluation:

[0380] Brain, left frontal cortex; left temporal cortex; left caudate nucleus; left putamen; left hippocampus; left thalamus; left medulla oblongata; left pons; left substantia nigra; left amygdala; left parietal cortex; left cerebellum; dorsal root ganglion, lumbar vertebrae (targeting L5); dorsal root ganglion, thoracic vertebrae; dorsal root ganglion, cervical vertebrae; heart, apex; kidney, left cortex; liver, left lateral lobe; sciatic nerve, left; spinal cord, cervical; spinal cord, thoracic; spinal cord, lumbar; spleen; superior cervical ganglion, left.

[0381] For each brain region in the left hemisphere (frontal cortex, temporal cortex, caudate nucleus, putamen, hippocampus, thalamus, medulla oblongata, pons, substantia nigra, amygdala, parietal cortex, and cerebellum), collect up to four samples (25 to 100 mg each) in pre-chilled 2 mL RNase-free tubes; rapidly freeze in liquid nitrogen; and place on dry ice until transferred to a freezer maintained at -60 to 80°C. Record the weight of all samples.

[0382] For the spinal cord region (cervical, thoracic, and lumbar), collect up to four samples (25 to 100 mg each) in pre-chilled 2 mL RNase-free tubes; rapidly freeze in liquid nitrogen; and place on dry ice until transferred to a freezer maintained at -60 to 80°C. Record the weight of all samples.

[0383] For dorsal root ganglia (DRGs), four DRGs (two bilateral pairs) were collected from each region (cervical, thoracic, and lumbar), weighed, and the nerve roots were trimmed. Each bilateral pair was placed in a pre-chilled 2 mL RNase-free tube. The entire left superior cervical ganglion was sampled, weighed, and placed in a pre-chilled 2 mL RNase-free tube. Each sample was rapidly frozen in liquid nitrogen and placed on dry ice until transferred to a freezer maintained at -60 to 80°C. The weight of all samples was recorded.

[0384] For liver (left lateral lobe), spleen, heart (apex), left kidney (cortex), and left sciatic nerve samples, collect up to four samples (25 to 100 mg) from each tissue and place them in pre-chilled 2 mL RNase-free tubes; rapidly freeze in liquid nitrogen; and place on dry ice until transferred to a freezer maintained at -60 to 80°C. Record the weight of all samples.

[0385] 1. RNA Isolation. RNA was extracted from exploratory tissue lysates for exploratory pharmacodynamic endpoints (e.g., RNA editing efficiency). Frozen tissue blocks were transferred to 1 ml of cold Trizol (Life Technologies) and immediately homogenized on ice using a Qiagen Ruptor or OMNI Soft Tissue Disruptor (high speed 10–30 seconds, depending on the tissue). The homogenate equivalent to up to 35 mg of tissue weight (25 mg for liver, kidney, and spleen) was diluted to 1 ml at room temperature with Trizol. RNA was extracted by adding 200 µl of chloroform and shaking vigorously. After centrifugation, 450–500 µl of the upper aqueous phase was mixed with an equal volume of 70% ethanol. RNA was isolated from the mixture using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. DNase was applied on the column. RNA was eluted with 30 µl of RNase-free water. RNA concentration and quality were determined using an Agilent Bioanalyzer 4200 and RNAScreenTape (Agilent), and RNA samples were stored at -80°C.

[0386] 2. cDNA Synthesis. 0.5 µg of RNA sample was mixed with 0.5 µl of 100 mM Oligo(dT)18 (Thermo Scientific), 0.5 µl of 100 µM Random Hexamer (Thermo Scientific), 1.0 µl of dNTP mix (Thermo Scientific), and RNase- and DNase-free water to a final volume of 15.5 µl. The solution was incubated at 65°C for 5 min and then cooled at 4°C for 1 min. After mixing with 4 µl of 5x RT buffer and 0.5 µl of Maxima Reverse Transcriptase (Thermo Scientific), the reaction solution was placed in a thermal cycler and run under the following conditions: 25°C for 10 min, 60°C for 30 min, 85°C for 5 min, and 4°C indefinitely. After the reaction, the cDNA sample was diluted 1:80 with RNase- and DNase-free water.

[0387] 3. Digital droplet PCR. Digital droplet PCR (ddPCR) was performed using 1 µl of cDNA sample in a 20 µl ddPCR Multiplex Supermix (4x) (BioRad). ddPCR probes and primers were custom-made by IDT.

[0388] The probe sequence is as follows:

[0389] / 56-FAM / AG GTG A+T+G +GCA TTG CTT TCG T / 3IABkFQ / (SEQ ID NO: 209) and

[0390] / 5HEX / AG +GTG A+T+A +GCA TTG CTT TCG TGT / 3IABkFQ / (SEQ ID NO: 210).

[0391] The primer sequences are as follows:

[0392] 5'-AGT CCT CTC CCG AGT CCA CA-3' (SEQ ID NO: 211) and

[0393] 5'-GGG GCA TGA AGG CTC ATT ATT CAA-3' (SEQ ID NO: 212).

[0394] Use a final concentration of 250 nM (probe). Use a final concentration of 500 nM (primer). Generate droplets on a Bio-Rad Automated Droplet Generator (BioRad). Perform PCR on a C1000 Touch™ Thermal Cycler (BioRad) under the following conditions: 95°C for 10 min, 1 cycle; 95°C for 30 s and 64°C for 1 min, 40 cycles; 98°C for 10 min, 1 cycle; and 4°C indefinitely. After the reaction, analyze the droplets on a QX600 Droplet Reader (BioRad). Acquire and analyze data using BioRad QX manager Standard Edition v.2.20 software. Gated FAM-positive, HEX-positive, and FAM / HEX-positive populations in 2D plots of amplitudes in Channel 1 and Channel 2. Calculate the editing efficiency percentage using the following formula: G(cp / µl) / (G(cp / µl) + A(cp / µl)) x 100%.

[0395] Table 11. NHP study design with different doses, time points, and repeated administrations. Group 1 did not receive the test item but was treated the same as the other animals, serving as a negative control.

[0396]

[0397] Table 12. Organ / tissue collection.

[0398]

[0399] result

[0400] Editing efficiency measured across multiple organizations is shown in Table 13.

[0401] Table 13. Editing efficiency measured across organizations.

[0402]

[0403]

[0404]

[0405]

[0406]

[0407] Exposure analysis. Tissue materials, methods, and data from HPLC / HRMS analysis.

[0408] To analyze the delivery of test articles to tissues and understand exposure-response relationships, tissue samples were collected at time points corresponding to edit measurements, and the exposure to total guide oligonucleotides in the tissues was quantified by HP-LC / MS. Briefly, tissue standards were prepared in control tissue homogenates. To control assay variability, internal standards were added to all standards and samples.

[0409] Tissue samples were homogenized in cell lysis buffer. For total oligonucleotide assays, tissue standards and samples were digested with proteinase K and then loaded onto Oasis Wax micro-elution solid-phase extraction (SPE) plates (Waters Inc, Milford, MA) for separation. The SPE plates were washed with elution buffer and then eluted with elution buffer. The eluent from the SPE plates was dried, reconstituted, and injected into an LC / MS system.

[0410] The total concentration of guided oligonucleotides was measured using a Thermo Orbitrap Exploris 240 mass spectrometer (Thermo Scientific, San Jose, CA) with guided oligonucleotide peaks. The mass spectrometer was operated in negative ion detection mode. All data were processed using Xcalibur version 4.4 (Thermo Scientific, San Jose, CA).

[0411] Table 14 shows measurable exposures in multiple central nervous system regions in animals administered the test article intrathecally. Variations in brain exposure in some groups may indicate incomplete IT administration. Peripheral tissues (liver and kidneys) were also exposed to the test article, indicating that significant amounts of the compound administered with IT reached systemic circulation. Kidney concentrations were consistently higher than those in the liver.

[0412] Table 14. Summary of exposure data.

[0413]

[0414] In summary, the ICV studies outlined in Examples 9 and 10 and the IT studies in Example 11 demonstrate that direct application to the brain (in mice via ICV; in non-human primates and humans via intrathecal (IT) administration) is a feasible method for delivering RNA-edited ASOs to target cells, particularly brain cells. Furthermore, following ICV administration, RNA-edited ASOs can be delivered to peripheral tissues, such as liver and muscle cells, through both conjugated and unconjugated ASOs, i.e., in the absence of GalNAc conjugation.

Claims

1. An RNA editing antisense oligonucleotide (ASO) that forms a double-stranded complex with a human IDUA RNA molecule, wherein the RNA molecule contains a target adenosine at position 1205 of SEQ ID NO: 135, optionally wherein the ASO is 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, wherein the ASO comprises the following nucleotide sequence: ， in: i) m5Ce is a 5-methyl-cytidine containing 2'-O-methoxyethyl (2'-MOE) ribose substitution; ii) Zd is a deoxyribonucleoside that is directly opposite to the target adenosine and contains a Benner base; iii) Ad is deoxyadenosine; iv) Gm is a guanosine nucleoside containing a 2'-O-methyl (2'-OMe) ribose substitution; v)Af is an adenosine nucleoside containing a 2'-fluoro(2'-F) ribose substitution; vi) Ge is a guanosine containing a 2'-MOE ribose substitution; vii) This represents the nucleoside bond between phosphodiester (PO); viii) This indicates the nucleoside bond between phosphate thioesters (PS); and ix) This represents the internucleotide bond between methylphosphonate (MP) nucleotides. The nucleoside position is numbered such that Z is nucleoside position 0, and the inter-nucleoside bond is numbered such that the 5' bond of Z is bond number 0. The nucleoside position and bond position in the ASO increase positively (+) toward the 5' end and negatively (-) toward the 3' end.

2. The ASO according to claim 1, wherein the ASO comprises the following nucleotide sequence: ， Where X and Y are nucleotides containing adenine, guanine, thymine, uracil, hypoxanthine, or cytosine bases.

3. The ASO according to claim 1 or 2, wherein the ASO further comprises at least one modified nucleoside internucleotide bond and at least one nucleotide comprising a modified sugar moiety.

4. The ASO according to any one of claims 1-3, wherein the ASO comprises the following nucleotide sequence: ， Where X and Y are nucleotides containing adenine, guanine, thymine, uracil, hypoxanthine, or cytosine bases.

5. The ASO according to any one of claims 1-4, wherein the ASO comprises: i) The nucleoside at position +2 that forms a wobbling base pair with the RNA molecule; ii) 2'-F or 2'-OMe ribose substitution at nucleoside positions +2, +3, +4, +5, +6, +7, +8, +10, -5, -6, -7, -8, -9, -10, -11 and / or -13; iii) 2'-OMe substitution at nucleoside positions +9 and / or -12; iv) 2'-OMe or 2'-MOE substitution at nucleoside positions +11, +12, +13, +14 and / or +15; v) PS keys at key positions +1, +2, +3, +4, +5, +6, +7, +8, +9, -5, -6, -7, -9, -10, -11 and / or -12; vi) PS or PNdmi keys at key positions -8 and / or -13; vii) The PS or PO keys at key positions +10, +11, +12 and / or +13; and / or viii) PS, PNdmi, or PO key at key position +14.

6. The ASO according to any one of claims 1-5, wherein the ASO comprises a nucleotide sequence .

7. The ASO according to any one of claims 1-6, wherein at least one nucleotide contains one or more additional non-naturally occurring chemical modifications in the ribose, bond, or base portion, and wherein the one or more additional ribose modifications are selected from deoxyribose (DNA), unlocked nucleic acid (UNA), and 2'-F.

8. The ASO according to claim 7, wherein the one or more additional modifications are bond modifications selected from PS, 3'-methylenephosphonate, 5'-methylenephosphonate, 3'-phosphatidyl ester and 2'-5'-PO.

9. The ASO according to any one of claims 1-8, wherein the ASO contains One or more nucleotides outside the motif contain additional modifications, said modifications being mono- or di-substituted at the 2', 3', and / or 5' positions of the sugar, selected from: -OH; -F; substituted or unsubstituted linear or branched lower (C1-C2) nucleotides. 10 Alkyl, alkenyl, alkynyl, alkylaryl, allyl or aralkyl, which may be interspersed with one or more heteroatoms; -O-, S- or N-alkyl (e.g., -O-methyl); -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 ASO of claim 1, wherein the ASO comprises a nucleotide sequence: (SEQ ID NO: 213), where: i) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) The nucleotides at positions +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, -7, -8, -9, -10, -11, and -13 each independently contain a 2'-OMe, 2'-MOE, or 2'-F ribose substitution; iii) The nucleotides at positions +9 and -12 contain a 2'-OMe ribose substitution; and iv) "~" indicates a nucleoside inter-bond including a PS bond, PNdmi bond, or PO bond.

11. The ASO of claim 1, wherein the ASO comprises the following nucleotide sequence: (SEQ ID NO: 214), where: i) C, G, A and U are nucleotides containing cytosine, guanine, adenine or uracil nucleobases, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) The nucleotides at positions +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10 and -11 each independently contain a 2'-MOE or 2'-F ribose substitution; iii) The nucleotides at positions +10, +11, +12, and +13 each independently contain a 2'-OMe or 2'-MOE ribose substitution; iv) The nucleotides at positions +9, -12, and -13 contain a 2'-OMe ribose substitution; v) "=" indicates a nucleoside internucleotide bond including a PO bond or a PS bond; and vi) "%" indicates a nucleoside inter-bond including a PS bond or a PNdmi bond.

12. The ASO according to any one of claims 1-11, comprising SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 180, 181, 182, 183, 184, 185, 191, 194, or 195.

13. The ASO according to any one of claims 1-11, comprising SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 180, 181, 182, 183, 184, 185, 191, 194 or 195.

14. The ASO according to any one of claims 1-12, comprising a delivery portion.

15. The ASO of claim 14, wherein the delivery portion is N-acetylgalactosamine (GalNAc).

16. The ASO according to claim 15, wherein the GalNAc is of formula I: Formula I The ASO is connected to the connection point E of Formula I, optionally via a connector.

17. The ASO according to any one of claims 14-16, comprising SEQ ID NO: 67, 68, 69, 70, 71, 72, 99, 100, 101, 102, 103, 104, 110, 113, 114, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, or 179.

18. The ASO according to any one of claims 14-16, comprising SEQ ID NO: 67, 68, 69, 70, 71, 72, 99, 100, 101, 102, 103, 104, 110, 113, 114, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 or 179.

19. An RNA editing ASO comprising SEQ ID NO:

16.

20. The ASO of claim 19, wherein the ASO comprises SEQ ID NO:

16.

21. The ASO of claim 19, comprising a delivery portion.

22. The ASO of claim 21, wherein the delivery portion is GalNAc.

23. The ASO according to claim 22, wherein the GalNAc is of formula I: Formula I The ASO is connected to the connection point E of Formula I, optionally via a connector.

24. The ASO of claim 23, wherein the ASO comprises formula (B).

25. The ASO according to claim 23, wherein the ASO is composed of formula (B).

26. A pharmaceutical composition comprising ASO according to any one of claims 1-25, and a pharmaceutically acceptable carrier.

27. The ASO according to any one of claims 1-25 or the pharmaceutical composition according to claim 26 for the treatment of mucopolysaccharidosis type 1 (MPS 1), preferably Hurler syndrome caused by the c.1205G>A mutation in the human IDUA gene.

28. Use of the ASO according to any one of claims 1-25 or the pharmaceutical composition according to claim 26 in the preparation of a medicament for treating MPS 1, preferably Hurler syndrome caused by the c.1205G>A mutation in the human IDUA gene.

29. A method for treating MPS 1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene, comprising administering to a patient in need a therapeutically effective amount of ASO according to any one of claims 1-25 or a pharmaceutical composition according to claim 26.

30. The method of claim 29, wherein the application is by subcutaneous delivery.

31. The method of claim 29, wherein the administration is to the central nervous system, optionally via intrathecal delivery.

32. A method for deamination of a target adenosine present in a target RNA molecule in a cell, wherein the target RNA molecule is human IDUA precursor mRNA or mRNA, or a portion thereof, wherein the target adenosine is the c.1205G>A mutation in SEQ ID NO: 135, the method comprising the following steps: i) Contacting the cells with the ASO according to any one of claims 1-25 or the pharmaceutical composition according to claim 26 under conditions suitable for: - The cells take up the ASO; - The annealing of the ASO with the target RNA molecule; and - By using a naturally occurring mammalian ADAR enzyme present in the cells, the target adenosine in the target RNA molecule is deaminosed to inosine; and ii) Optionally process the cell precursor mRNA or mRNA to determine the presence of inosine at the location of the target adenosine in the target RNA molecule.

33. The method of claim 32, wherein step ii) comprises: - Determine the sequence of the target RNA molecule; - Assess the presence of functional, extended, full-length, and / or wild-type idurolactamase protein; or - Using functional readouts, wherein the deamination of the target RNA encodes functional, full-length, extended, and / or wild-type idurolactamase proteins.

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