ANTISENSE OLIGONUCLEOTIDES FOR THE TREATMENT OF HURLER SYNDROME CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE [0001] This PCT application claims the priority benefit of U.S. Provisional Application No.63/622,296, filed on January 18, 2024, which is herein incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] The content of the electronically submitted ST.26 sequence listing in xml format (name 0070WO01ORD_20250115xmL.xml; size: 1,064,699 bytes; and date of creation: Janury 16, 2025) filed with the application is incorporated herein by reference in its entirety. TECHNICAL FIELD [0003] The present disclosure relates to the field of medicine. More particularly, it relates to the field of diseases caused by genetic defects such as, for example, Mucopolysaccharidosis type 1 (MPS 1), of which the most severe form is Hurler syndrome. The disclosure involves the use of RNA editing technology in targeting (pre-) mRNA to deaminate target adenosines present in the target RNA and convert them to inosines using antisense oligonucleotides (ASOs) that enable RNA editing when hybridized to the target (pre-) mRNA. BACKGROUND [0004] MPS 1 is an inherited metabolic disease characterized by a malfunction of the α- L-iduronidase enzyme (hereinafter abbreviated to “Iduronidase”), encoded by the IDUA gene, leading to the storage of glycosaminoglycans (GAGs) dermatan sulfate and heparan sulfate as oligosaccharides in the lysosomes. The IDUA gene has over 100 known mutations in individuals with the disease. Because of the variety of possible genotypes,
there is a broad phenotypical spectrum with the presence or absence of neurological impairment. The classical and most severe form is known as Hurler syndrome (also referred to as MPS-IH), the intermediate form is known as Hurler-Scheie, and the most attenuated form is known as Scheie syndrome. Individuals having Hurler syndrome exhibit progressive developmental delay, corneal clouding, airway obstruction, cardiac disease, enlargement of organs, severe joint restrictions, and have an untreated life expectancy of 10 years. Individuals with MPS 1 require a coordinated team of specialists to address symptoms as they become apparent. [0005] Currently, there are two clinically available treatments for MPS 1: (1) enzyme replacement therapy with Laronidase and (2) hematopoietic stem cell transplantation (HSCT). Laronidase is a human recombinant enzyme intravenously administered into the bloodstream and endocytosed into cells to catabolize accumulated glycosaminoglycans (GAGs) in lysosomes. Laronidase has limitations, for instance related to the blood brain barrier leading to continued progression of neurological disease, lifelong re- administration, and high costs. HSCT, when successful, is an effective approach, but has a high morbidity and mortality related to the procedure, needs to employ preparative chemotherapy, and has the potential of graft failure. Unfortunately, neither treatment fully eliminates the clinical manifestations of the disease, and individuals with MPS 1 may still require intervention through surgery or symptom-specific specialists. [0006] Gene therapy (including gene editing) is a third approach, which has also been explored as a potential therapeutic for this disease, applying retroviral vectors, lentiviral vectors, and adeno-associated virus (AAV) vectors. Yet another therapy that is being explored makes use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing tools for gene therapy purposes because of its targeting accuracy when mediating gene integration. Two mouse studies using CRISPR/Cas9 to integrate the Idua cDNA with liposomal complexes had varied success. In the first study, the liposomal complexes containing vectors with CRISPR/Cas9 and the Idua cDNA were injected into the superficial temporal vein and were retained in the lung, heart, and liver. There was significant Iduronidase activity in serum and reduction of GAGs in several organ systems but not in the brain (Schuh et al.2018, J. Control. Release 288:23-33). The second study with these complexes reported that treated mice showed improved facial bone structure and some recovery of respiratory function but limited overall improvement of cardiovascular pathologies (Schuh et al.2020, Gene Ther.27:74-84).
[0007] It is generally held that current clinical treatments for MPS 1 are inadequate, resulting in a residual disease burden, disability, and early death in treated individuals. Preclinical gene therapy studies for MPS 1 have demonstrated that replacement of Iduronidase activity is therapeutic but struggled with the requirement for long-term Iduronidase restoration in both the systemic and CNS compartments, as well as some limitations due to safety concerns with certain vector-based approaches. However, it remains to be seen how successful gene editing may be in achieving the necessary Iduronidase concentrations to produce a widespread therapeutic effect in the brain as well as in the harder-to-treat parts of the body such as heart valves, spinal meninges, and cartilage. [0008] The incidence of Hurler syndrome is 1:100,000 in Western society, and one of the most common mutations is the guanosine to adenosine mutation in codon 402 of the human IDUA coding sequence, in which the TGG triplet (coding for tryptophan) is mutated into TAG, which then determines a premature stop in the transcript. The mutant variant is also referred to as c.G1205A, G1205A, or c.1205G>A for the coding sequence, and generally referred to as W402X, p.W402X, p.W402*, or p.Trp402Ter for the protein variant (see NCBI ref variant NM_000203.5), and accounts for 40% of the patients. It is position 1205 in the human IDUA mRNA (see SEQ ID NO: 135) that is mutated from a guanosine to an adenosine, which is the middle nucleotide of the codon for tryptophan at position 402. [0009] Notably, the mutation from a guanosine to an adenosine allows for another therapeutic approach known as “RNA editing”, in which an ASO (also referred to as an AON, or when used for RNA editing as an RNA editing oligonucleotide (EON)) targets and hybridizes to the area in the (pre-) mRNA that includes the mutated nucleotide. The oligonucleotide, in concert with an Adenosine Deaminase acting on RNA (ADAR) enzyme that is either administered to the cell but preferably is endogenously present in the cell, causes the deamination of the target adenosine to inosine (A-to-I), which is subsequently read as a guanosine by the translation machinery. In the case of Hurler syndrome, caused by the c.1205G>A mutation, this reversion will result in the production of a wild-type Iduronidase protein. [0010] RNA editing in its basic form is a natural process through which eukaryotic cells alter the sequence of their RNA molecules, often in a site-specific and precise way, thereby increasing the repertoire of genome encoded RNAs by several orders of
magnitude. RNA editing enzymes have been described for eukaryotic species throughout the animal and plant kingdoms, and these processes play an important role in managing cellular homeostasis in metazoans from the simplest life forms (such as Caenorhabditis elegans) to humans. Examples of RNA editing are A-to-I conversions mentioned above, and cytidine (C) to uridine (U) conversions. The most extensively studied RNA editing system is the ADAR enzyme. [0011] ADAR (e.g., ADAR1; ADAR2) is a multi-domain protein, comprising – depending on the enzyme in question – 2 to 3 double-stranded RNA recognition domains and a catalytic domain. The recognition domain recognizes a specific double-stranded RNA (dsRNA) sequence and/or conformation, whereas the catalytic domain converts an adenosine to inosine in a nearby, predefined position in the target RNA, by deamination of the nucleobase. As indicated above, inosine is read as guanosine by the translational machinery of the cell, meaning that, if an edited adenosine is in a coding region of an mRNA or pre-mRNA, it can recode the protein sequence. The c.1205G>A (W402X) mutation is not a natural target for ADAR. [0012] Early attempts to force ADAR into targeted editing required the use of genetically engineered ADAR that was covalently bound to a 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 interesting, it is unlikely that such a system could be used clinically due to the practical difficulty in delivering bulky modified ADAR proteins into the patient’s cells and to the uncertainty around immunogenic responses to administering such proteins to a patient. 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) wherein the longer oligonucleotides (34-mer and 52-mer) could promote editing of the target RNA by endogenous ADAR because of the double-stranded nature of the target RNA and the hybridizing oligonucleotide. However, the oligonucleotides only appeared to function in cell extracts or in amphibian (Xenopus) oocytes by microinjection and suffered from severe lack of specificity; nearly all adenosines in the target RNA strand that was complementary to the ASO were edited. [0013] More recent developments have established that careful design of ASOs can be used to successfully redirect ADAR that is naturally present within a cell to edit the c.1205G>A (W402X) mutation. As described below, early attempts performed using a
biochemical assay consisting of purified reagents led to significant levels of RNA being edited. However, to be clinically useful, the ASOs must be able to survive the in vivo environment inside and outside the target cell after administration and still bring about clinically relevant levels of editing. Criteria relevant to the overall levels of editing achieved clinically include that the ASOs should be suitable resistant to degradation by nuclease activity, should hybridize with suitable strength with the target (pre-) mRNA, should attract and bind the ADAR with suitable strength, should be able to effect editing quickly before being displaced from the target (pre-) mRNA by splicing events within the cell, and should be able to survive long enough to edit new (pre-) mRNA as it is transcribed within the cell. Work remains to be done to identify ASOs that have a suitable balance of these properties to ultimately bring about clinically relevant levels of editing. Exemplary disclosures to date in this area include, but are not limited to, the following. [0014] Intl. Patent Application Publication No. WO 2017/220751 represents an early example of using ASOs to redirect natural ADAR in a mouse model of Hurler syndrome. It discloses one study that was performed by transfecting mRNA and ASOs into an immortalized mouse embryonic fibroblast (MEF) cell, culturing for 48 hours, lysing the cells, isolating the supernatant, and performing an Iduronidase enzyme activity assay on the supernatant. It was identified that specific placement of mismatches between the ASO and the target RNA could lead to improvements in enzyme activity, representing an increase in mRNA editing. [0015] Intl. Patent Application Publication No. WO 2018/041973 discloses an investigation into the use of chemical modifications in ASOs that target mouse Idua mRNA. A wide range of ASOs comprising sugar and/or base modifications in the Central Triplet of nucleotides directly opposite the target adenosine underwent stability and activity assays. The use of DNA nucleotides, 2’-fluoro (2’-F) modifications and phosphorothioate (PS) linkages within the Central Triplet were shown to be beneficial. [0016] Intl. Patent Application Publication No. WO 2019/158475 discloses the use of in silico modelling of ASO/IDUA/ADAR complexes to predict nucleotide sites on the ASO that may tolerate 2’-O-methoxyethyl (2’-MOE) modifications without disrupting the complex. Several ASOs were made according to the prediction and tested against IDUA (pre-)mRNA.
[0017] Intl. Patent Application Publication No. WO 2019/219581 discloses the use of in silico modelling of ASO/IDUA/ADAR complexes to predict which ASO linkages may tolerate PS modifications without disrupting the complex. [0018] Intl. Patent Application Publication No. WO 2020/165077 discloses use of in silico modelling of ASO/IDUA/ADAR complexes to predict which ASO linkages may tolerate phosphonoacetate modifications without disrupting the complex, and to predict which ASO nucleotides may tolerate Unlocked Nucleic Acid (UNA) modifications without disrupting the complex. [0019] Intl. Patent Application Publication No. WO 2020/201406 discloses the use of ASOs comprising methylphosphonate (MP) linkage modifications. An optimized ASO (IDUA268) was found to edit up to 1.4% of RNA in a mouse embryonic fibroblast cell assay (see figure 8 and related description therein). [0020] Intl. Patent Application Publication No. WO 2020/252376 discloses the use of cytidine analogs in ASOs. Optimized sequences having C analogs, such as Benner’s base gave editing of up to 12% in primary mouse liver fibroblast cells (see figure 10 and related description therein). [0021] RNA editing of the c.1205G>A mutation in human IDUA using a 111 nt long ADAR-recruiting oligonucleotide has been disclosed as well (Qu et al.2019, Nat. Biotechnol.37:1059-1069), though some promiscuous editing was observed. [0022] Despite the above disclosures, it remains difficult to predict if there is an ASO sequence, with or without modifications, that can give a balance of stability and activity that leads to higher activity in cells. Such an ASO could find utility in the further development of therapeutically useful medicaments in the treatment of Hurler syndrome. BRIEF SUMMARY [0023] The present disclosure relates to an RNA editing antisense oligonucleotide (ASO) forming a double-stranded complex with a human IDUA RNA molecule, wherein the RNA molecule comprises a target adenosine at position 1205 in 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 in length, wherein the ASO comprises the nucleotide sequence: [0024] 5’-…m5Ce^Zd*Ad^Gm^Af*Ge…-3’,
[0025] wherein: i) m5Ce is a 5-methyl-cytidine comprising a 2’-O-methoxyethyl (2’-MOE) ribose substitution; ii) Zd is a deoxyribonucleoside that is directly opposite the target adenosine and comprises a Benner’s base; iii) Ad is a deoxyadenosine; iv) Gm is a guanosine nucleoside comprising a 2’-O-methyl (2’-OMe) ribose substitution; v) Af is an adenosine nucleoside comprising a 2’-fluoro (2’-F) ribose substitution; vi) Ge is a guanosine comprising a 2’-MOE ribose substitution; vii) ^ denotes a phosphodiester (PO) internucleoside linkage; viii) * denotes a phosphorothioate (PS) internucleoside linkage; and ix) ^ denotes a methylphosphonate (MP) internucleoside linkage, [0026] wherein the nucleoside position numbering is such that Z is nucleoside position 0, and the internucleoside linkage numbering is such that the linkage 5’ from Z is linkage number 0, wherein the nucleoside positions and the linkage positions in the ASO are positively (+) incremented toward the 5’ end and negatively (-) incremented towards the 3’ end. [0027] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0028] 5’-…(X)14*m5Ce^Zd*Ad^Gm^Af*Ge*(Y)9…-3’ [0029] wherein X and Y are nucleotides comprising an adenine, guanine, thymine, uracil, hypoxanthine, or cytosine base, or a derivative or analog of any of such nucleobase moiety, as discussed infra. [0030] In one embodiment, the ASO as disclosed herein consists of the nucleotide sequence: [0031] 5’-…(X)14*m5Ce^Zd*Ad^Gm^Af*Ge*(Y)9…-3’, [0032] wherein X and Y are nucleotides comprising an adenine, guanine, thymine, uracil, hypoxanthine, or cytosine base, or a derivative or analog of any such nucleobase moiety, as discussed infra.
[0033] The present disclosure relates to an ASO capable of forming a double-stranded complex with a section of a human IDUA transcript molecule in a human cell, wherein the double-stranded complex can recruit an endogenous (= naturally present) ADAR enzyme present in the cell, wherein the section comprises a target adenosine for deamination by the ADAR enzyme, and wherein the ASO comprises the nucleotide sequence as disclosed herein. [0034] In one embodiment, the ASO as disclosed herein comprises: i) a nucleoside at position +2 that makes a wobble base pair with the RNA molecule; ii) a 2’-F or 2’-OMe ribose substitution at nucleoside position +2, +3, +4, +5, +6, +7, +8, +10, -5, -6, -7, -8, -9, -10, -11, and/or -13; iii) a 2’-OMe substitution at nucleoside position +9, and/or -12; iv) a 2’-OMe or 2’-MOE substitution at nucleoside position +11, +12, +13, +14, and/or +15; v) a PS linkage at linkage position +1, +2, +3, +4, +5, +6, +7, +8, +9, -5, -6, -7, -9, -10, -11, and/or -12; vi) a PS or PNdmi linkage at linkage position -8, and/or -13; vii) a PS or PO linkage at linkage position +10, +11, +12, and/or +13; and/or viii) a PS, a PNdmi, or a PO linkage at linkage position +14. [0035] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0036] 5'-UGCGACACUUCGGU*m5Ce^Zd*Ad^Gm^Af*Ge*CUGCUCCUC-3’ (SEQ ID NO: 136). [0037] In one embodiment, the ASO as disclosed herein comprises at least one nucleotide that comprises one or more additional non-naturally occurring chemical modifications in the ribose, linkage, or base moiety, and wherein the one or more additional ribose modifications is selected from the group consisting of deoxyribose (DNA), Unlocked Nucleic Acid (UNA), and 2’-F. [0038] In one embodiment, the one or more additional modifications is a linkage modification selected from the group consisting of PS, 3'-methylenephosphonate, 5'- methylenephosphonate, 3'-phosphoroamidate and 2'-5’-PO. [0039] In one embodiment, one or more nucleosides in the ASO outside the m5Ce^Zd*Ad^Gm^Af*Ge motif comprise an additional modification that is a mono- or di-substitution at the 2', 3' and/or 5' position of the sugar, selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl (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; -dimethylamino oxyethoxy; and -dimethylaminoethoxyethoxy. [0040] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0041] 5’- C~G~A~C*A*C*U*U*C*G*G*U*m5Ce^Zd*Ad^Gm^Af*Ge*C*U*G*C*U*C*C*U~C -3’ (SEQ ID NO: 213), wherein: i) C, G, A, and U are nucleotides comprising a cytosine, guanine, adenine, or uracil nucleobase, respectively, wherein the uracil nucleobase is optionally a 5- methyluracil (thymine) nucleobase; ii) the nucleotides at position +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, - 7, -8, -9, -10, -11, and -13 comprise, each independently, a 2’-OMe, a 2’-MOE, or a 2’-F ribose substitution; iii) the nucleotides at position +9 and -12 comprise a 2’-OMe ribose substitution; and iv) denotes an internucleoside linkage comprising a PS linkage, a PNdmi linkage, or a PO linkage. [0042] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0043] 5’- C=G=A=C*A*C*U*U*C*G*G*U*m5Ce^Zd*Ad^Gm^Af*Ge*C*U*G*C*U*C*C*U% C-3’ (SEQ ID NO: 214), wherein: i) C, G, A, and U are nucleotides comprising a cytosine, guanine, adenine, or uracil nucleobase, respectively, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) the nucleotides at position +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10, and -11 comprise, each independently, a 2’-MOE, or a 2’-F ribose substitution; iii) the nucleotides at position +10, +11, +12, and +13 comprise, each independently, a 2’-OMe or a 2’-MOE ribose substitution; iv) the nucleotides at position +9, -12, and -13 comprise a 2’-OMe ribose substitution; v) “=” denotes an internucleoside linkage comprising a PO linkage or a PS linkage; and
vi) “%” denotes an internucleoside linkage comprising a PS or PNdmi linkage. [0044] In one embodiment, the ASO as disclosed herein comprises or consists of the nucleotides sequence and chemical modifications 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. [0045] In one embodiment, the ASO as disclosed herein comprises a delivery moiety. A preferred delivery moiety is an 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:
Formula I wherein the ASO is conjugated to connection point E of Formula I, optionally via a linker, as disclosed herein. [0046] In one embodiment, the ASO as disclosed herein comprises or consists of the nucleotide sequence and chemical modifications of any one 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 moiety, which may be any GalNAc moiety known to the person skilled in the art, and is preferably a GalNAc moiety as disclosed herein (such as Formula I which is also referred as “L004”), optionally conjugated to the ASO via a linker, which may be any suitable linker known to the person skilled in the art, and preferably is a linker as disclosed herein. The GalNAc moiety may be conjugated to the ASO at its 5’ terminus and/or at its 3’ terminus and is preferably
conjugated to its 3’ terminus. The GalNAc moiety is preferably a tri-antennary GalNAc moiety. [0047] In one embodiment, the ASO comprises or consists of the 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. [0048] In one embodiment, the ASO comprises or consists of the nucleotide sequence and chemical modifications of any one of SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. These ASOs comprise a GalNAc moiety, which may be any GalNAc moiety known to the person skilled in the art, and is preferably a GalNAc moiety as disclosed herein (such as Formula I which is also referred as “L004”), optionally conjugated to the ASO via a linker, which may be any suitable linker known to the person skilled in the art, and preferably is a linker as disclosed herein. The GalNAc moiety may be conjugated to the ASO at its 5’ terminus and/or at its 3’ terminus and is preferably conjugated to its 3’ terminus. The GalNAc moiety is preferably a tri-antennary GalNAc moiety. [0049] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence and chemical modifications of SEQ ID NO: 16. In a preferred embodiment, the ASO as disclosed herein consists of the nucleotide sequence and chemical modifications of SEQ ID NO: 16. In one embodiment the ASO according to SEQ ID NO: 16 is conjugated to a delivery moiety. In one embodiment, the conjugation is at its 3’ terminus. In one embodiment, the conjugation is at its 3’ terminus, and via a linker. In some embodiments the delivery moiety is a GalNAc, e.g. without limitation a GalNAc of Formula I (see above). [0050] In one embodiment, the ASO as disclosed herein comprises or consists of the structure of Formula (A) as disclosed in Fig.27. [0051] In one embodiment, the ASO as disclosed herein comprises or consists of the structure of Formula (B) as disclosed herein and in Fig.28. [0052] In one embodiment, the ASO as disclosed herein comprises or consists of the structure of Formula (C) as disclosed in Fig.29. [0053] SEQ ID NO:1 in Fig.1A is a section of the human IDUA transcript comprising the c.1205G>A mutation. The ASOs of the present disclosure can form a double-stranded complex with this section of the human IDUA transcript (which may be pre-mRNA or mRNA) and through the recruitment of an endogenous (also referred to as ‘naturally
occurring’ or ‘naturally present’) enzyme with deaminating activity (such as ADAR2) that recognizes and binds the double-stranded complex, the adenosine at position 1205 (in SEQ ID NO: 135) is deaminated into an inosine. [0054] The disclosure also relates to a pharmaceutical composition comprising an ASO as disclosed herein, and a pharmaceutically acceptable carrier. [0055] The disclosure also relates to an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein, for use in the treatment of mucopolysaccharidosis type 1 (MPS 1), preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene. [0056] The disclosure also relates to a use of an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein, in the manufacture of a medicament for the treatment of MPS 1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene. [0057] The disclosure also relates to a method for the treatment of MPS 1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene, comprising administering to a patient in need thereof a therapeutically effective amount of an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein. In one embodiment, the step of administering is via a subcutaneous delivery. In one embodiment, the step of administering is to the central nervous system, optionally via an intrathecal delivery. In one embodiment, the step of administering is via an intrathecal delivery. [0058] The disclosure also relates to a method for the deamination of a target adenosine present in a target RNA molecule in a cell, wherein the target RNA molecule is a human IDUA pre-mRNA or mRNA, or a part 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 cell with an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein under conditions suitable for: a) uptake of the ASO by the cell; b) annealing of the ASO to the target RNA molecule; and c) deamination of the target adenosine in the target RNA molecule to an inosine by a naturally present mammalian ADAR enzyme present in the cell; and ii) optionally processing the cellular pre-mRNA or mRNA to determine the presence of an inosine at the position of the target adenosine in the target RNA molecule. In one embodiment, the optional step (ii) comprises: a) determining the sequence of the target RNA molecule; b) assessing the presence of a functional, elongated, full length
and/or wild type Iduronidase protein; or c) using a functional read-out, wherein the target RNA after the deamination encodes a functional, full length, elongated and/or wild type Iduronidase protein. BRIEF DESCRIPTION OF THE DRAWINGS [0059] The advantages, effects, features, and objects other than those set forth above will become more readily apparent when consideration is given to the detailed description below. Such detailed description refers to the following drawing(s), where: [0060] Fig.1A shows a section of the sequence of the target human IDUA RNA molecule bearing the c.1205G>A mutation causing Hurler syndrome (top sequence; 3’ to 5’; target adenosine A shown in bold font; SEQ ID NO: 1). Below SEQ ID NO: 1 sequences of ASOs (5’ to 3’) that are disclosed herein are shown. 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-methyl-cytidine; Zd (orphan nucleotide), in bold face, is a deoxynucleotide (deoxycytidine analog) carrying a Benner’s base; Ad is deoxyadenosine; Te is 2’-MOE modified thymidine (identical to a 5- methyluridine with a 2’-MOE substitution, herein and elsewhere also denoted as m5Ue); “!” refers to a PNdmi linkage; “^” refers to a MP linkage; “*” refers to a PS linkage. All other internucleoside linkages are PO linkages. The basic sequence of the ASOs shown in SEQ ID NO: 2, 5 to 37, 67 to 72, 86 to 98, and 99 to 113, without the chemical modifications to the sugar or linkage is 5’- TGCGACACUUCGGUCZAGAGCUGCUCCUC-3’ (SEQ ID NO:64) in which the underlined T can be referred to as either a T or a U as indicated above depending on the modification, and wherein the Z in bold face is a nucleotide carrying a Benner’s base. The basic sequence of the ASO shown as A-26, without the chemical modifications to the sugar or linkage is 5’- CGACACUUCGGUCZAGAGCUGCUCCUC-3’ (SEQ ID NO:65) in which the Z in bold face is a nucleotide carrying a Benner’s base. The basic sequence of the ASO shown as A-32, without the chemical modifications to the sugar or linkage is 5’- CTCGACACUUCGGUCZAGAGCUGCUCCUC-3’ (SEQ ID NO:66) in which the underlined T can be referred to as either a T or a U as indicated above
depending on the modification, and wherein the Z in bold face is a nucleotide carrying a Benner’s base. Fig.1B shows the same set of ASOs as provided in Fig.1A, except with the 3’ attachment of GN, which is a delivery moiety, preferably an N-acetylgalactosamine (“GalNAc” or “GN”) moiety, more preferably a tri-antennary GalNAc moiety (for example without limitation as disclosed in Intl. Patent Application Publication No. WO 2022/271806). Other non-limiting embodiments of GN are described in Int. Patent Application Publication 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. Fig.1C shows ASOs mIdua-85-A to -M that are mouse Idua targeting ASOs, which correspond to A-50 (targeting human IDUA) carrying a GalNAc moiety and comprising chirally pure PS linkages (denoted here
that can be either Rp or Sp) as disclosed in Intl. Patent Application No. WO 2019/219581. ASOs mIdua-85-A to -M correspond to the depicted A-50 GN-A to M respectively, that target the human IDUA transcript. Fig.1D shows SEQ ID NO: 135, which represents the full mRNA sequence (5’ to 3’) of the human IDUA gene with the W402X UAG stop codon (underlined) comprising the c.1205G>A mutation with a capital A in bold. Fig.1E shows the sequences of ASOs A-50-A to -M that have the same ASO sequence and modification as A-50 GN-A to -M (see Fig.1C), but without the GalNAc (GN) moiety. [0061] Figs.2A, 2B, 2C, and 2D show the results of an in vitro biochemical editing assay that assesses the percentage editing of target human IDUA in the presence of several ASOs specified in Fig.1A. [0062] Figs.3A, 3B, and 3C show the results of cell-based assays that assess the percentage editing of target human IDUA in a live cellular environment caused by the ASOs specified in Fig.1A recruiting ADAR that is naturally present in the cell. [0063] Fig.4 shows a section of the mouse Idua transcript (SEQ ID NO: 134; 3’ to 5’) resembling the area of the human IDUA transcript with the Hurler syndrome causing G>A mutation shown in Fig.1A and Fig.1D, here also shown in bold face. Below the target sequence the sequences of the ASOs are shown (from 5’ to 3’) used in the in vivo experiments in mice. The chemical modifications are as described for Fig.1A. GN represents a 3’-bound tri-antennary GalNAc moiety as described in Int. Patent Application Publication No. WO 2022/271806. Scr1 is a scrambled oligonucleotide that serves as a negative control for editing.
[0064] Fig.5A shows the percentage editing in the liver of W392X mice after administration of the fourteen ASOs with a GalNAc moiety, and the scrambled ASO (with GN) as shown in Fig.4. The numbers on the X-axis indicate the numbering of the ASOs in Fig.4. The percentage was calculated and relative to wild type levels set at 100%. Fig.5B shows the restoration of Iduronidase activity in the same experiment (at day 14), in comparison to wild type mice, showing an increase in activity up to about 4.5% of wild type activity in comparison to the residual activity observed in the mutant mice (represented by the Scr control), more or less giving the same overall result as shown in Fig.5A with ASOs mIdua-75GN, mIdua-89GN, mIdua-88GN, mIdua-85GN, and mIdua-87GN performing best. Fig.5C shows the restoration of Iduronidase activity in the plasma of the same mice, showing a similar overall pattern as shown in Fig.5A and Fig.5B. [0065] Fig.6 shows the administration scheme for an in vivo study using mIdua-75 and mIdua-85 ASOs (all conjugated to a GalNAc (GN) moiety) in different amounts with different regimens. [0066] Fig.7A shows the average percentage editing in the mice of Group 1 (see Fig.6). Fig.7B shows the average Iduronidase enzymatic activity in this same group over the course of 4 weeks. The right y-axis displays the percentage activity in comparison to wildtype (100%). [0067] Fig.8A shows the average percentage editing after 4 weeks in mice of Group 2 (dosing 10 mg/kg) and Group 3 (dosing 30 mg/kg). EON 11 refers to mIdua-85GN, see Table 1. Fig.8B shows the average Iduronidase enzymatic activity in these same groups over the course of 4 weeks. The right y-axis displays the percentage activity in comparison to wildtype (100%). [0068] Fig.9A shows the average percentage editing after 4 weeks in mice of Group 3 (receiving 30 mg/kg mIdua-85GN (EON 11)) and Group 4 (receiving 30 mg/kg mIdua- 75GN (EON 6)). Fig.9B shows the average Iduronidase enzymatic activity in these same groups over the course of 4 weeks. The right y-axis displays the percentage activity in comparison to wildtype (100%). [0069] Fig.10A shows the average percentage editing after 4 weeks in mice of Group 2 (receiving 10 mg/kg mIdua-85GN (EON 11) twice weekly) and Group 5 (receiving 10 mg/kg mIdua-85GN (EON 11) once per week). Fig.10B shows the average Iduronidase
enzymatic activity in these same groups over the course of 4 weeks. The right y-axis displays the percentage activity in comparison to wildtype (100%). [0070] Fig.11A shows the average percentage editing in mice of Group 2 (necropsy at 4 weeks after twice weekly dosing), Group 6 (necropsy at 8 weeks after twice weekly dosing) and Group 7 (necropsy at 8 weeks after twice weekly dosing in the first two weeks, followed by one dose per two weeks in week 3 to 8) using 10 mg/kg mIdua-85GN (EON 11) at each dosing. Fig.11B shows the average Iduronidase enzymatic activity in these same groups at the end of the regimen. The right y-axis displays the percentage activity in comparison to wildtype (100%). [0071] Fig.12A shows the GAG levels in liver in all 7 groups of mice at necropsy. NT is a control of non-treated mutant mice; WT is the GAG level observed in wildtype mice and WT (NT) is the level GAG observed in non-treated wildtype mice. Fig.12B shows the GAG levels in urine at the end of the regimen in all groups in µg/mg tissue.8 (WT) represents the GAG levels observed in the urine of a group of wildtype mice. NT shows the GAG levels in non-treated mutant mice. [0072] Fig.13 shows the percentage editing of the human IDUA target adenosine in human iPSC-derived hepatocytes after incubation of 2 days (12 left bars, squares), 5 days (12 middle bars, triangles), and 7 days (12 right bars; dots) with six ASOs as indicated, either bound to a GalNAc moiety (GN) or not. Sequences and modifications of the indicated ASOs used are provided in Fig.1A. [0073] Fig.14A shows the HPLC chromatograms of A-50GN and Fig.14B A-78GN before and after treatment in a biochemical stability assay using a variety of nucleases. The peaks in the top chromatograms show the control samples that have been treated using the non-activated biological stability assay and the bottom chromatogram of both figures show the chromatograms of the same samples after treatment in a biochemical stability assay. The peak with retention time 9.10 minutes in the top chromatogram in Fig.14A is A-50GN. The bottom chromatogram of Fig.14A shows the same peak at retention time 9.12 minutes, however with much more degradation products after treatment. Fig.14B shows the same order in chromatograms with the same treatment, however this experiment was performed using A-78GN. This indicates that this molecule is much less prone to degradation compared to A-50GN.
[0074] Fig.15 shows the percentage of stability in a bar diagram of A-50 GN (here A- 50), A-78 GN (here A-78), mIdua-85 and mIdua-88, based on the HPLC chromatograms generated after the stability assay. [0075] Fig.16A shows a set of ASOs based on A-50 GN (SEQ ID NO: 69) in which the linkage modifications are varied to determine the stability of the ASOs based on the presence of PO linkages and PS linkages at different positions. Different positions of A- 50 were also replaced by PNdmi linkages to determine the stability introduced by such linkage modifications. The chemical modifications are as in Fig.1A. “^” refers to a PO linkage; “#” refers to a PNms linkage; Cd is deoxycytidine. L004 is the same GalNAc moiety (GN) as described in Fig.4 and may be replaced by any preferred GalNAc moiety as disclosed herein. Fig.16B shows the same set of ASOs as in Fig.16A but depicted with an ‘-X’ notation and all lacking a GalNAc moiety at the 3’ terminus. [0076] Fig.17 shows the percentage editing in primary human W402X fibroblasts (GM00798), determined by ddPCR, after transfection of the ASOs as specified. 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. A mock transfection served as a negative control. The specific chemical modifications and sequences of the ASOs are provided in Fig.16. [0077] Fig.18 shows the administration scheme for an in vivo study using a mIdua-85, mIdua-75, and mIdua-89 ASOs without and conjugated to a GalNAc (GN) moiety; see also Fig.6. The dose of the ASO’s lacking the GalNAc moiety is adjusted in mg/kg, as indicated. Control group 1 did not receive any administrations, but urine/blood sampling was performed 1 week before the start of the study (-1), at weeks 2, 4, and 6, and finally at necropsy (8 weeks). Group 2 only received the carrier solution. Group 3 received a scrambled control ASO conjugated to a GalNAc moiety. Group 18 received a mIdua-85 ASO formulated in a lipid nanoparticle (LNP). The administration in Group 2 to 17 was subcutaneously (SC), while Group 18 received the LNP formulation intravenously (IV). Group 1 were wildtype C57BL/6j mice, and Groups 1 to 18 were Idua-W392X model mice. [0078] Fig.19A shows the editing percentage of mouse Idua target transcripts, determined in the mouse liver at necropsy after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100%
(although not being edited as such), and the two other negative controls at 8 weeks. Fig. 19B shows the results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety). Fig.19C shows the average Idua enzymatic activity in the liver, for the same GalNAc Groups as shown in Fig.19A at necropsy. Fig.19D shows the average Idua enzymatic activity in the liver, for the same Groups without GalNAc (here ‘naked’) as shown in Fig.19B at necropsy. Fig. 19E shows the GAG levels in the liver for the same GalNAc Groups as shown in Fig. 19A at necropsy. The low level of GAG accumulation in wildtype mice (Group 1) is shown with an open bar. The right axis shows the percentage reduction in GAG levels in comparison to the scrambled control. Fig.19F shows the GAG levels in the liver for the same Groups with GalNAc (here ‘naked’) as shown in Fig.19B. [0079] Fig.20A shows the editing percentage of mouse Idua target transcripts, determined in the mouse kidney at necropsy after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100% (although not being edited as such), and the two other negative controls at 8 weeks. Fig.20B shows the results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety). Fig.20C shows the average Idua enzymatic activity in the kidney, for the same GalNAc Groups as shown in Fig.20A at necropsy. Fig.20D shows the average Idua enzymatic activity in the kidney, for the same Groups without GalNAc (here ‘naked’) as shown in Fig.20B at necropsy. Fig.20E shows the GAG levels in the kidney for the same GalNAc Groups as shown in Fig.20A at necropsy. The low level of GAG accumulation in wildtype mice (Group 1) is shown with an open bar. The right axis shows the percentage reduction in GAG levels in comparison to the scrambled control. Fig.20F shows the GAG levels in the kidney for the same Groups with GalNAc (here ‘naked’) as shown in Fig.20B. [0080] Fig.21A shows the editing percentage of mouse Idua target transcripts, determined in the mouse spleen at necropsy after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100% (although not being edited as such), and the two other negative controls at 8 weeks.
Fig.21B shows the results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety). [0081] Fig.22A shows the editing percentage of mouse Idua target transcripts, determined in the mouse lungs at necropsy after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100% (although not being edited as such), and the two other negative controls at 8 weeks. Fig.22B shows the results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety). Fig.22C shows the average Idua enzymatic activity in the lungs, for the same GalNAc and ‘naked (no GalNAc) Groups as shown in Fig.22A and Fig.22B at necropsy, together. Fig.22D shows the GAG levels in the lungs for the same Groups as shown in Fig.22C at necropsy. The low level of GAG accumulation in wildtype mice (Group 1) is shown with an open bar. The right axis shows the percentage reduction in GAG levels in comparison to the scrambled control. [0082] Fig.23A shows the editing percentage of mouse Idua target transcripts, determined in the mouse heart at necropsy after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100% (although not being edited as such), and the two other negative controls at 8 weeks. Fig.23B shows the results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety). Fig.23C shows the average Idua enzymatic activity in the heart, for the same GalNAc and ‘naked (no GalNAc) Groups as shown in Fig.23A and Fig.23B at necropsy, together. Fig.23D shows the GAG levels in the heart for the same Groups as shown in Fig.23C at necropsy. The low level of GAG accumulation in wildtype mice (Group 1) is shown with an open bar. The right axis shows the percentage reduction in GAG levels in comparison to the scrambled control. [0083] Fig.24A shows the editing percentage of mouse Idua target transcripts, determined in the mouse quadriceps at necropsy after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20
weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100% (although not being edited as such), and the two other negative controls at 8 weeks. Fig.24B shows the results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety). Fig.24C shows the average Idua enzymatic activity in the quadriceps, for the same GalNAc and ‘naked (no GalNAc) Groups as shown in Fig.24A and Fig.24B at necropsy, together. [0084] Fig.25 shows the average Idua enzymatic activity in the mouse plasma, after administration of mIdua-85GN at 4 weeks (Group 5), 8 weeks (Group 4), 12 weeks (Group 6), 16 weeks (Group 7), and 20 weeks (Group 8), and after administration of mIdua-75GN and mIdua-89GN at 8 weeks (Groups 9 and 10, respectively), in comparison to the wildtype situation (Group 1) set at 100% (although not being edited as such), and the two other negative controls at 8 weeks. The results in the arm without the GalNAc being present (referred to as ‘naked’ which here is only in the context of lacking a GalNAc moiety) is also shown. [0085] Fig.26A shows the GAG levels in the urine samples at necropsy of the indicated groups treated with GalNAc containing ASOs. Fig.26B shows the same but for the ASOs lacking a GalNAc moiety. The wildtype levels (open bars) represent the GAG levels compared to the scrambled control of which the average level was set at 100%. RM4870 = mIdua-85GN, RM5306 = mIdua-75GN, RM4772 = mIdua-89GN, RM4347 = mIdua- 85, RM4337 = mIdua-75, and RM4351 = mIdua-89. Fig.26C shows the GAG levels in a time course upon mIdua-85GN administration from start (T=0) to 20 weeks. Levels are compared to scrambled levels (not shown) that was set at 100%. [0086] Fig.27 shows the structure of ASO A-36 GN (SEQ ID NO: 67; Formula (A)). [0087] Fig.28 shows the structure of ASO A-50 GN (SEQ ID NO: 69; Formula (B)). [0088] Fig.29 shows the structure of ASO A-94 GN (SEQ ID NO: 72; Formula (C)). DETAILED DESCRIPTION [0089] The present disclosure relates to ASOs (also herein and elsewhere referred to as AONs or EONs) and their use in the treatment of disease, particularly MPS 1, more particularly Hurler syndrome. It should be noted that the oligonucleotides herein do not edit the RNA on their own, despite the name that is sometimes used. The
oligonucleotides, when attached to their target region present an environment in which an endogenous ADAR enzyme (preferably ADAR2) can bind to the double-stranded RNA complex and specifically deaminate the target A to I, only because the oligonucleotide herein is hybridized/bound to its target sequence. The ASOs herein target a specific A in the human IDUA pre-mRNA or mRNA and cause the deamination of these to I, read as G in translation. The disclosure is applicable for instance, but not limited to, the editing of the W402X mutation in the human IDUA transcript, reverting the premature stop codon back to a codon encoding tryptophan. Repairing (or deaminating) the A to an I in the W402X mutation will result in translation of a wild-type protein. [0090] An ASO herein does not comprise an ADAR-recruitment portion as described in Intl. Patent Application Publication No. WO 2016/097212. The ASOs herein also do not comprise a portion that can form an intramolecular stem-loop or hair-pin structure that is not complementary to the target sequence, such as found in the oligonucleotides used in CRISPR/Cas9 systems (e.g., Intl. Patent Application Publication No. WO 2019/005884). The ASOs herein are not (covalently) linked to an enzyme that can deaminate a target nucleotide before it enters a target cell but can form a complex with such an enzymatic entity (preferably ADAR) present in a target cell (hence, recruiting, interacting, and complexing with an endogenous ADAR enzyme). The ASOs herein are shorter than such oligonucleotides from the art, which makes them cheaper to produce, easier to use (likely because of increased and more efficient cell entry and trafficking) and easier to manufacture. Intl. Patent Application Nos. WO 2017/220751 and WO 2018/041973 disclose ASOs that are complementary to a target RNA for deaminating a target A present in a target RNA sequence to which the ASO is complementary, but also lacked a recruitment portion while still being capable of harnessing ADAR enzymes present in the cell to edit the target A. The present disclosure makes use of that knowledge and aims to solve the problem of targeting Hurler syndrome mutations, preferably the c.1205G>A (W402X) mutation. [0091] As explained above, the disclosure relates to ASOs forming a double-stranded complex with a human IDUA RNA molecule, wherein the RNA molecule comprises a target adenosine at position 1205 in SEQ ID NO: 135, which represents the 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 nucleotide sequence: 5’-…m5Ce^Zd*Ad^Gm^Af*Ge…-3’, [0092] wherein: i) m5Ce is a 5-methyl-cytidine comprising a 2’-O-methoxyethyl (2’-MOE) ribose substitution; ii) Zd is a deoxyribonucleoside that is directly opposite the target adenosine and comprises a Benner’s base; iii) Ad is a deoxyadenosine; iv) Gm is a guanosine nucleoside comprising a 2’-O-methyl (2’-OMe) ribose substitution; v) Af is an adenosine nucleoside comprising a 2’-fluoro (2’-F) ribose substitution; vi) Ge is a guanosine comprising a 2’-MOE ribose substitution; vii) ^ denotes a phosphodiester (PO) internucleoside linkage; viii) * denotes a phosphorothioate (PS) internucleoside linkage; and ix) ^ denotes a methylphosphonate (MP) internucleoside linkage, wherein the nucleoside position numbering is such that Z is nucleoside position 0, and the internucleoside linkage numbering is such that the linkage 5’ from Z is linkage number 0, wherein the nucleoside positions and the linkage positions in the ASO are positively (+) incremented toward the 5’ end and negatively (-) incremented towards the 3’ end. [0093] It has surprisingly been found that ASOs having this “m5Ce^Zd*Ad^Gm^Af*Ge” motif in the sequence show improved editing activity. [0094] The term ‘Central Triplet’ used herein refers to the three nucleotides opposite the target A in the target RNA, wherein the middle nucleotide in the Central Triplet is directly opposite the target A. The Central Triplet does not have to be in the center of the ASO, as it may be located closer to the 3’ end, or alternatively towards the 5’ end, of the ASO, whatever is preferred for a certain target. The term ‘Central’ in this aspect has therefore more the meaning of the triplet that is in the center of catalytic activity when it comes to chemical modifications and targeting the target A. It should also be noted that the ASOs are sometimes depicted from 3’ to 5’, especially when the target sequence is shown from 5’ to 3’. However, whenever herein the order of nucleotides within the ASO
is discussed, it is always from 5’ to 3’ of the ASO. The position can also be expressed in terms of a particular nucleotide within the ASO while still adhering to the 5’ to 3’ directionality, in which case other nucleotides 5’ of the said nucleotide are marked as positive positions and those 3’ of it as negative positions. [0095] In the present disclosure, the Central Triplet comprises the motif “c-Z*A”, where Z is a Benner’s base (also referred to as “2′-deoxy Benner’s base Z” or “dZ”; see, Yang et al. (2006) Nucl Acid Res 34:6095-6101). A Benner’s base is a cytidine analog. Benner’s dZ nucleotide (6-amino-5-nitro-3-yl-2(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone) has been shown to provide a hydrogen bonding pattern optimal for interaction with a glutamate residue present on the ADAR flipping loop, leading to increased deamination rates in vitro (Doherty et al. (2021) J. Am. Chem. Soc.143:6865-6876). The structure of the Benner’s base is as follows, in which the connection at the lower part is to position 1 of the ribose sugar ring:
[0096] In the disclosure, the nucleosides making up the ASO are numbered by position, wherein the nucleoside position numbering is such that Z, which is the nucleoside directly opposite the target A in the target RNA molecule is nucleoside position 0 (the orphan nucleotide), and the internucleoside linkage numbering is such that the linkage 5' from Z is linkage number 0, wherein the nucleoside positions and the linkage positions in the ASO are positively (+) incremented toward the 5’ end and negatively (-) incremented towards the 3' end. [0097] Disclosed herein is an ASO, wherein the ASO comprises or consists of the nucleotide sequence: 5’-…(X)14*m5Ce^Zd*Ad^Gm^Af*Ge*(Y)9…-3’, wherein X and Y are nucleotides comprising an adenine, guanine, thymine, uracil, hypoxanthine, or cytosine base. [0098] In some embodiments, the ASO further comprises at least one modified internucleoside linkage and at least one nucleotide comprising a modified sugar moiety. [0099] In one embodiment, the ASO as disclosed herein comprises:
(i) a nucleoside at position +2 that makes a wobble base pair with the RNA molecule; (ii) a 2’-F or 2’-OMe ribose substitution at nucleoside position +2, +3, +4, +5, +6, +7, +8, +10, -5, -6, -7, -8, -9, -10, -11, and/or -13; (iii) a 2’-OMe substitution at nucleoside position +9, and/or -12; (iv) a 2’-OMe or 2’-MOE substitution at nucleoside position +11, +12, +13, +14, and/or +15; (v) a PS linkage at linkage position +1, +2, +3, +4, +5, +6, +7, +8, +9, -5, -6, - 7, -9, -10, -11, and/or -12; (vi) a PS or PNdmi linkage at linkage position -8, and/or -13; (vii) a PS or PO linkage at linkage position +10, +11, +12, and/or +13; and/or a PS, a PNdmi, or a PO linkage at linkage position +14. [0100] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0101] 5'-UGCGACACUUCGGU*m5Ce^Zd*Ad^Gm^Af*Ge*CUGCUCCUC-3’ (SEQ ID NO: 136). [0102] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0103] 5’- C~G~A~C*A*C*U*U*C*G*G*U*m5Ce^Zd*Ad^Gm^Af*Ge*C*U*G*C*U*C*C*U~C -3’ (SEQ ID NO: 213), wherein: i) C, G, A, and U are nucleotides comprising a cytosine, guanine, adenine, or uracil nucleobase, respectively, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) the nucleotides at position +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, -7, -8, -9, -10, -11, and -13 comprise, each independently, a 2’-OMe, a 2’-MOE, or a 2’-F ribose substitution; iii) the nucleotides at position +9 and -12 comprise a 2’-OMe ribose substitution; and iv) “~” denotes an internucleoside linkage comprising a PS linkage, a PNdmi linkage, or a PO linkage. [0104] In one embodiment, the ASO as disclosed herein comprises the nucleotide sequence: [0105] 5’- C=G=A=C*A*C*U*U*C*G*G*U*m5Ce^Zd*Ad^Gm^Af*Ge*C*U*G*C*U*C*C*U%
C-3’ (SEQ ID NO: 214), wherein: i) C, G, A, and U are nucleotides comprising a cytosine, guanine, adenine, or uracil nucleobase, respectively, wherein the uracil nucleobase is optionally a 5-methyluracil (thymine) nucleobase; ii) the nucleotides at position +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10, and -11 comprise, each independently, a 2’-MOE, or a 2’-F ribose substitution; iii) the nucleotides at position +10, +11, +12, and +13 comprise, each independently, a 2’-OMe or a 2’-MOE ribose substitution; iv) the nucleotides at position +9, -12, and -13 comprise a 2’-OMe ribose substitution; v) “=” denotes an internucleoside linkage comprising a PO linkage or a PS linkage; and vi) “%” denotes an internucleoside linkage comprising a PS or PNdmi linkage. [0106] The disclosure relates to an ASO targeting the human IDUA transcript, or a part thereof, wherein the ASO comprises the chemical modifications and/or the nucleotide sequence 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. [0107] In one embodiment the ASO as 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. [0108] In one embodiment, the ASO as disclosed herein comprises a delivery moiety. Preferably, the delivery moiety is an N-Acetylgalactosamine (GalNAc) moiety. Preferred GalNAc moieties are Tri-antennary GalNAc moieties as shown in Formula I, II, III, and VI (below). Preferred mono-antennary GalNAc moieties are as shown in Formula IV and V (below). [0109] Formula I, includes connection point E (see WO2022/271806):
[0110] Formula II, includes a linker and a connection point F which conjugates to the ASO (see WO2014179620):
. [0111] Formula III(a) and III(b), includes a linker and a connection point F which conjugates to the ASO (see WO2009/073809):
[0113] Formula III(b). [0114] Formula IV:
. [0115] Formula V:
. [0116] Formula VI (See WO2011104169), wherein the squiggly line indicates a connection point of Formula VI, optionally via linker and/or a spacer, to the ASO:
. [0117] When the GalNAc moiety of Formula I is applied, the GalNAc moiety is preferably conjugated to the ASO at its 3’ terminus, via connection point E of Formula I, optionally via a linker and/or a spacer, for example as depicted in Formula VII (below). [0118] Formula VII:
. [0119] When the GalNAc moiety of Formula II is applied, the GalNAc moiety is preferably conjugated to the ASO at its 5’ terminus, via connection point F of Formula II, optionally via a linker and/or a spacer. [0120] The disclosure relates to an ASO targeting the human IDUA transcript, or a part thereof, wherein the ASO comprises the chemical modifications and/or the nucleotide sequence 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 as 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. [0121] The disclosure relates to an ASO targeting the human IDUA transcript, or a part thereof, wherein the ASO comprises or consists of the sequence and modifications of SEQ ID NO: 16. Preferably, the ASO comprising or consisting of the sequence and modifications of SEQ ID NO: 16 further comprises a delivery moiety, preferably wherein the delivery moiety is a GalNAc moiety, more preferably a tri-antennary GalNAc moiety, even more preferably the GalNAc moiety of Formula I (above). [0122] The disclosure relates to an ASO targeting the human IDUA transcript, or a part thereof, as disclosed herein, wherein the ASO comprises or consists of the structure of Formula (B), see also Fig. 28:
[0124] The disclosure relates to an ASO targeting the human IDUA transcript, or a part thereof, as disclosed herein, wherein the ASO comprises the structure of SEQ ID NO: 64, wherein the ASO is chemically modified by a selection of chemical modifications to the ribose, linkage or nucleobase moiety as taught herein, and preferably wherein the ASO comprises a GalNAc moiety at the 3’ terminus. [0125] In one embodiment, the disclosure relates to an ASO as provided in structural Formula (A), see Fig.27. [0126] In one embodiment, the disclosure relates to an ASO as provided in structural Formula (B), see Fig.28. [0127] In one embodiment, the disclosure relates to an ASO as provided in structural Formula (C), see Fig.29. [0128] In an embodiment, the section of the human IDUA transcript molecule comprises the sequence of SEQ ID NO: 1, wherein the target adenosine represents the c.1205G>A mutation. [0129] In an embodiment, the human IDUA transcript molecule is according to the sequence of SEQ ID NO: 135, wherein the adenosine at position 1205 represents the c.1205G>A mutation. It is to be understood that the sequence of SEQ ID NO: 135 is the mutant human IDUA mRNA sequence, causing Hurler syndrome. [0130] In an embodiment, at least one nucleotide in the ASO as disclosed herein comprises one or more additional non-naturally occurring chemical modifications in the ribose, linkage, or base moiety, wherein the one or more additional ribose modifications is selected from the group consisting of deoxyribose (DNA), Unlocked Nucleic Acid (UNA), and 2’-F. [0131] In an embodiment, the one or more additional modifications is a linkage modification selected from the group consisting of PS, 3'-methylenephosphonate, 5'- methylenephosphonate, 3'-phosphoroamidate and 2'-5’-PO. [0132] In an embodiment, one or more nucleosides in the ASO outside the m5Ce- Zd*Ad^Gm-Af*g motif comprise an additional modification that is a mono- or di- substitution at the 2', 3' and/or 5' position of the sugar, selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; -O- , S-, or N-alkyl (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; -dimethylamino oxyethoxy; and -dimethylaminoethoxyethoxy. [0133] Disclosed herein is also a pharmaceutical composition comprising an ASO as disclosed herein, and a pharmaceutically acceptable carrier. [0134] Disclosed herein is also an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein, for use in the treatment of mucopolysaccharidosis type 1 (MPS 1), preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene. [0135] Disclosed herein is also a use of an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein, in the manufacture of a medicament for the treatment of MPS 1, preferably Hurler syndrome caused by a c.1205G>A mutation in the human IDUA gene. [0136] Disclosed is also a method for the treatment of MPS 1, preferably Hurler syndrome caused by the c.1205G>A mutation in the human IDUA gene, comprising administering to a patient in need thereof a therapeutically effective amount of an ASO as disclosed or a pharmaceutical composition as disclosed. In a preferred embodiment, the step of administering is via a subcutaneous delivery. In another preferred embodiment, the step of administering is to the central nervous system, optionally via an intrathecal delivery. In one embodiment, the step of administering is via an intrathecal delivery. [0137] Disclosed is also method for the deamination of a target adenosine present in a target RNA molecule in a cell, wherein the target RNA molecule is a human IDUA pre-mRNA or mRNA, or a part 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 cell with an ASO as disclosed herein, or a pharmaceutical composition as disclosed herein under conditions suitable for: a) uptake of the ASO by the cell; b) annealing of the ASO to the target RNA molecule; and c) deamination of the target adenosine in the target RNA molecule to an inosine by a naturally present mammalian ADAR enzyme present in the cell; and ii) optionally processing the cellular pre-mRNA or mRNA to determine the presence of an inosine at the position of the target adenosine in the target RNA molecule. The optional step comprises: a) determining the sequence of the target RNA molecule; b) assessing the presence of a functional, elongated, full length and/or wild type Iduronidase protein; or c) using a functional read-out, wherein the target RNA after the deamination encodes a
functional, full length, elongated and/or wild type Iduronidase protein. Preferably, the target adenosine is the c.1205G>A mutation in the human IDUA gene. [0138] Disclosed herein is an ASO consisting of the sequence: 5’-C~G~A~C*A*C*U*U*C*G*G*U*m5Ce^Zd*Ad^Gm^Af*Ge*C*U*G*C*U*C*C*U~C- 3’ (SEQ ID NO: 213), wherein: i) the chemical modifications are as provided for in Fig.1, Fig.4, and Fig. 16; ii) C, G, A, and U are nucleotides comprising a cytosine, guanine, adenine, or uracil nucleobase, respectively, wherein uracil may also be a 5- methyluracil (or thymine) nucleobase; iii) the nucleotides at position +2, +3, +4, +5, +6, +7, +8, +10, +11, +12, +13, -5, -6, -7, -8, -9, -10, -11, and -13 comprise, each independently, a 2’- OMe, a 2’-MOE, or a 2’-F ribose substitution; iv) the nucleotides at position +9 and -12 comprise a 2’-OMe ribose substitution; and v)
denotes an internucleoside linkage comprising a PS linkage, a PNdmi linkage, or a PO linkage. [0139] Disclosed herein is an ASO consisting of the sequence: 5’-C=G=A=C*A*C*U*U*C*G*G*U*m5Ce^Zd*Ad^Gm^Af*Ge*C*U*G*C*U*C*C*U%C-3’ (SEQ ID NO: 214), wherein: i) the chemical modifications are as provided for in Fig.1, Fig.4, and Fig. 16; ii) C, G, A, and U are nucleotides comprising a cytosine, guanine, adenine, or uracil nucleobase, respectively, wherein uracil may also be a 5- methyluracil (or thymine) nucleobase; iii) the nucleotides at position +2, +3, +4, +5, +6, +7, +8, -5, -6, -7, -8, -9, -10, and -11 comprise, each independently, a 2’-MOE, or a 2’-F ribose substitution; iv) the nucleotides at position +10, +11, +12, and +13 comprise, each independently, a 2’-OMe or a 2’-MOE ribose substitution;
v) the nucleotides at position +9, -12, and -13 comprise a 2’-OMe ribose substitution; vi) “=” denotes an internucleoside linkage comprising a PO linkage or a PS linkage; and vii) “%” denotes an internucleoside linkage comprising a PS or PNdmi linkage. [0140] Disclosed herein is also an ASO as disclosed herein, wherein at least one nucleotide comprises one or more additional non-naturally occurring chemical modifications in the ribose sugar moiety, linkage moiety, or base moiety, with the proviso that Z does not comprise a 2’-OMe ribose substitution. [0141] In one embodiment the ASO according to the present disclosure comprises or consists of a nucleotide sequence, including the specified chemical modifications, selected from any of the human IDUA c.1205G>A targeting ASOs depicted in Fig.1A, Fig.1B, Fig.1C, Fig.1E, Fig.16A and Fig.16B. [0142] In one embodiment the ASO according to the disclosure comprises or consists of a nucleotide sequence, including the specified chemical modifications, selected from the group consisting of: SEQ ID NO: 16, 34, 35, 10, 8, 13, 14, 7, 37, 36, 33, 25, 6, 21, and 18, representing ASOs 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, with their respective chemical modifications. [0143] In one embodiment the ASO according to the disclosure comprises or consists of a sequence, including the specified chemical modifications, selected from the group consisting of: 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 ASOs A-50 GN, A-104, A-94 GN, A-38 GN, A-93 GN, A-36 GN, A-78 GN, A-105, A-106, A- 107, A-108, A-109, A-110, A-111, A-112, A-113, A-114, A-115, A-116, A-117, A-118, A-121, A-125, A-128, A-129, A-130, and A-131, respectively, with their respective chemical modifications. [0144] In one embodiment the ASO according to the disclosure comprises or consists of a sequence, including the specified chemical modifications, selected from the group consisting of: SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98 representing ASOs A-50 GN-A to A-50 GN-M, respectively, with their respective chemical modifications (see Fig.1C). The disclosure provides variants of the A-50 GN
ASO (SEQ ID NO: 69) as recited in Fig.1B, comprising one or more additional non- naturally occurring chemical modifications in the sugar, (chirally pure) linkage, or base moiety, with the proviso that the one or more additional chemical modifications are in a different category of sugar, linkage or base moiety modification to any modifications already described. [0145] In one preferred embodiment the ASO according to the disclosure comprises or consists of a sequence, including the specified chemical modifications, of SEQ ID NO: 113, which comprises a 2’-OMe modified guanosine residue (Gm) at position +10 instead of a 2’-OMe modified cytosine residue (Cm) at position +10 in A-50 (SEQ ID NO: 16). [0146] In one preferred embodiment the ASO according to the disclosure comprises or consists of a sequence, including the specified chemical modifications, of SEQ ID NO: 194, which comprises a 2’-OMe modified guanosine residue (Gm) at position +10 instead of a 2’-OMe modified cytosine residue (Cm) at position +10 in A-50 GN (SEQ ID NO: 69). [0147] The skilled person knows that an oligonucleotide, such as an RNA oligonucleotide, generally consists of repeating monomers. Such a monomer is most often a nucleotide or a nucleotide analogue. The most common naturally occurring nucleotides in RNA are A, C, G, and U. These consist of a pentose sugar, a ribose, a 5’-linked phosphate group which is linked via a phosphate ester, and a 1’-linked base. The sugar connects the base and the phosphate and is therefore often referred to as the “scaffold” of the nucleotide. A modification in the pentose sugar is therefore often referred to as a “scaffold modification”. For severe modifications, the original pentose sugar might be replaced in its entirety by another moiety that similarly connects the base and the phosphate. It is therefore understood that while a pentose sugar is often a scaffold, a scaffold is not necessarily a pentose sugar. [0148] A base, sometimes called a nucleobase, is generally adenine, cytosine, guanine, thymine or uracil, or a derivative thereof. C, T, and U are pyrimidine bases, and are generally linked to the scaffold through their 1-nitrogen. A and G are purine bases and are generally linked to the scaffold through their 9-nitrogen. [0149] A nucleotide is generally connected to neighboring nucleotides through condensation of its 5’-phosphate moiety to the 3’-hydroxyl moiety of the neighboring nucleotide monomer. Similarly, its 3’-hydroxyl moiety is generally connected to the 5’- phosphate of a neighboring nucleotide monomer. This forms PO bonds. The POs and the
scaffold form an alternating copolymer. The bases are grafted on this copolymer, namely to the scaffold moieties. Because of this characteristic, the alternating copolymer formed by linked monomers of an oligonucleotide is often called the “backbone” of the oligonucleotide. Because PO bonds connect neighboring monomers together, they are often referred to as “backbone linkages”. It is understood that when a phosphate group is modified so that it is instead an analogous moiety such as a PS, such a moiety is still referred to as the backbone linkage of the monomer. This is referred to as a “backbone linkage modification”. In general terms, the backbone of an oligonucleotide comprises alternating scaffolds and backbone linkages. [0150] In one aspect, the nucleobase in an ASO herein is A, C, G, T, or U. In another aspect, the nucleobase is a modified form of A, C, G, or U. In another aspect, the modified nucleobase is hypoxanthine (the nucleobase in inosine), pseudouracil, pseudocytosine, 1-methylpseudouracil, orotic acid, agmatidine, lysidine, 2-thiouracil, 2- thiothymine, 5-halouracil, 5-halomethyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-formyluracil, 5- aminomethylcytosine, 5-formylcytosine, 5-hydroxymethylcytosine, 7-deazaguanine, 7- deazaadenine, 7-deaza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, 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 derivatives thereof; and degenerate or universal bases, like 2,6- difluorotoluene, or absent like abasic sites (e.g.1-deoxyribose, 1,2-dideoxyribose, 1- deoxy-2-O-methylribose, azaribose). The terms ‘adenine’, ‘guanine’, ‘cytosine’, ‘thymine’, ‘uracil’ and ‘hypoxanthine’ as used herein refer to the nucleobases as such. The terms ‘adenosine’, ‘guanosine’, ‘cytidine’, ‘thymidine’, ‘uridine’ and ‘inosine’ refer to the nucleobases linked to the (deoxy)ribosyl sugar. [0151] The term ‘nucleoside’ refers to the nucleobase linked to the (deoxy)ribosyl sugar, without phosphate groups. A ‘nucleotide’ is composed of a nucleoside and one or more phosphate groups. The term ‘nucleotide’ thus refers to the respective nucleobase- (deoxy)ribosyl-phospholinker, as well as any chemical modifications of the ribose moiety or the phospho group. Thus, the term would include a nucleotide including a locked ribosyl moiety (comprising a 2’-4’ bridge, comprising a methylene group or any other group), a UNA, a threose nucleic acid (TNA), a nucleotide including a linker comprising
a PO, phosphonoacetate, phosphotriester, PS, phosphoro(di)thioate, MP, methyl thiophosphonate, phosphoramidate linkages, and the like. Sometimes the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine/uridine, inosine, and hypoxanthine, are used interchangeably to refer to the corresponding nucleobase on the one hand, and the nucleoside or nucleotide on the other. Thymine (T) is also known as 5-methyluracil (m5U) and is a uracil (U) derivative; thymine, 5-methyluracil and uracil can be interchanged throughout the document text. Likewise, thymidine is also known as 5-methyluridine and is a uridine derivative; thymidine, 5-methyluridine and uridine can be interchanged throughout the document text. Sometimes the terms nucleobase, nucleoside and nucleotide are used interchangeably, unless the context clearly requires differently, for instance when a nucleoside is linked to a neighbouring nucleoside and the linkage between these nucleosides is modified. As stated herein, a nucleotide is a nucleoside plus one or more phosphate groups. The terms ‘ribonucleoside’ and ‘deoxyribonucleoside’, or ‘ribose’ and ‘deoxyribose’ are as used in the art. [0152] Whenever reference is made to an oligonucleotide, oligo, ON, ASO, oligonucleotide composition, antisense oligonucleotide, AON, (RNA) editing oligonucleotide, EON, and RNA (antisense) oligonucleotide, both oligoribonucleotides and deoxyoligoribonucleotides are meant unless the context dictates otherwise. Potentially the oligonucleotide may completely lack RNA or DNA nucleotides (as they appear in nature) and may consist completely of modified nucleotides. Whenever reference is made to an ‘oligoribonucleotide’ it may comprise the bases A, G, C, U or I. Whenever reference is made to a ‘deoxyoligoribonucleotide’ it may comprise the bases A, G, C, T or I. However, an ASO of the present disclosure may comprise a mix of ribonucleosides and deoxyribonucleosides. When a deoxyribonucleoside is used, hence without a modification at the 2’ position of the sugar, the nucleotide is often abbreviated to dA, dC, dG or T in which the ‘d’ represents the deoxy nature of the nucleoside, while a ribonucleoside that is either normal RNA or modified at the 2’ position is often abbreviated without the ‘d’ and often abbreviated with their respective modifications and as explained herein. [0153] Whenever reference is made to nucleotides in the ASO, such as cytosine, 5- methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5- hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine are included; when
reference is made to adenine, N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine and 7-methyladenine are included; when reference is made to uracil, dihydrouracil, iso-uracil, N3-glycosylated uracil, pseudo-uracil, 5-methyluracil, N1-methylpseudouracil, 4- thiouracil and 5-hydroxymethyluracil are included; when reference is made to guanine, 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7- trimethylguanosine and N2,7-dimethylguanosine are included. Whenever reference is made to nucleosides or nucleotides, ribofuranose derivatives, such as 2’-deoxy, 2’- hydroxy, and 2’-O–substituted variants, such as 2’-O-methyl, are included, as well as other modifications, including 2’-4’ bridged variants. Whenever reference is made to oligonucleotides, linkages between two mononucleotides may be PO linkages as well as modifications thereof, including, phosphonoacetate, phosphotriester, PS, phosphoro(di)thioate, MP, phosphoramidate linkers, phosphoryl guanidine, thiophosphoryl guanidine, sulfono phosphoramidate and the like. [0154] In one aspect, an ASO herein comprises a 2’-substituted PS monomer, preferably a 2’-substituted PS RNA monomer, a 2’-substituted phosphate RNA monomer, or comprises 2’-substituted mixed phosphate/PS monomers. It is noted that DNA is considered as an RNA derivative in respect of 2’ substitution. An ASO of the present disclosure comprises at least one 2’-substituted RNA monomer connected through or linked by a PS or phosphate backbone linkage, or a mixture thereof. The 2’-substituted RNA preferably is 2’-F, 2’-H (DNA), 2’-O-Methyl or 2’-O-(2-methoxyethyl). The 2’-O- Methyl is often abbreviated to “2’-OMe” and the 2’-O-(2-methoxyethyl) moiety is often abbreviated to “2’-MOE”. More preferably, the 2’-substituted RNA monomer in the ASO of the present disclosure is a 2’-OMe monomer, except for the monomer opposite the target adenosine, as further outlined herein, which should not carry a 2’-OMe substitution. In a preferred aspect of this aspect is provided an ASO according to the 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 mixtures thereof. Preferably, the monomer opposite the target adenosine is a 2’-H monomer (DNA) but may also be a monomer that allows deamination of the target adenosine, other 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 within the ASO in combination.
[0155] Throughout the disclosure, a 2’-OMe monomer within an ASO herein may be replaced by a 2’-OMe PS RNA, a 2’-OMe phosphate RNA or a 2’-OMe phosphate/PS RNA. Throughout the disclosure, a 2’-MOE monomer may be replaced by a 2’-MOE PS RNA, a 2’-MOE phosphate RNA or a 2’-MOE phosphate/PS RNA. Throughout the disclosure, an oligonucleotide consisting of 2’-OMe RNA monomers linked by or connected through PS, phosphate or mixed phosphate/PS backbone linkages may be replaced by an oligonucleotide consisting of 2’-OMe PS RNA, 2’-OMe phosphate RNA or 2’-OMe phosphate/PS RNA. Throughout the disclosure, an oligonucleotide consisting of 2’-MOE RNA monomers linked by or connected through PS, phosphate or mixed phosphate/PS backbone linkages may be replaced by an oligonucleotide consisting of 2’- MOE PS RNA, 2’-MOE phosphate RNA or 2’-MOE phosphate/PS RNA. [0156] In addition to the specific preferred chemical modifications at certain positions in compounds herein, the compounds herein further may comprise or consist of one or more (additional) modifications to the nucleobase, scaffold and/or backbone linkage, which may or may not be present in the same monomer, for instance at the 3’ and/or 5’ position. A scaffold modification indicates the presence of a modified version of the ribosyl moiety as naturally occurring in RNA (i.e., the pentose moiety), such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2’-modified sugars, 4’-modified sugar, 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-propargyl, 2’-O-allyl, 2’-O-(2-aminopropyl), 2’-O- (2-(dimethylamino)propyl), 2’-O-(2-amino)ethyl, 2’-O-(2-(dimethylamino)ethyl); 2’- deoxy (DNA); 2’-O-(haloalkyl)methyl 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] (MOCE), 2’-O-[2-N-methylcarbamoyl)ethyl] (MCE), 2’-O-[2- (N,N-dimethylcarbamoyl)ethyl] (DCME); 2’-halo e.g.2’-F, FANA; 2'-O-[2- (methylamino)-2-oxoethyl] (NMA); a bicyclic or bridged nucleic acid (BNA) scaffold modification such as a conformationally restricted nucleotide (CRN) monomer, a locked nucleic acid (LNA) monomer, a xylo-LNA monomer, an α-LNA monomer, an α-L-LNA monomer, a β-D-LNA monomer, a 2’-amino-LNA monomer, a 2’-(alkylamino)-LNA monomer, a 2’-(acylamino)-LNA monomer, a 2’-N-substituted 2’-amino-LNA monomer, a 2’-thio-LNA monomer, a (2’-O,4’-C) constrained ethyl (cEt) BNA monomer, a (2’-
O,4’-C) constrained methoxyethyl (cMOE) BNA monomer, a 2’,4’-BNANC(NH) monomer, a 2’,4’-BNANC(NMe) monomer, a 2’,4’-BNANC(NBn) monomer, an ethylene- bridged nucleic acid (ENA) monomer, a carba-LNA (cLNA) monomer, a 3,4-dihydro- 2H-pyran nucleic acid (DpNA) monomer, a 2’-C-bridged bicyclic nucleotide (CBBN) monomer, an oxo-CBBN monomer, a heterocyclic-bridged BNA monomer (such as triazolyl or tetrazolyl-linked), an amido-bridged BNA monomer (such as AmNA), an urea-bridged BNA monomer, a sulfonamide-bridged BNA monomer, a bicyclic carbocyclic nucleotide monomer, a TriNA monomer, an α-L-TriNA monomer, a bicyclo DNA (bcDNA) monomer, an F-bcDNA monomer, a tricyclo DNA (tcDNA) monomer, an F-tcDNA monomer, an alpha anomeric bicyclo DNA (abcDNA) monomer, an oxetane nucleotide monomer, a locked PMO monomer derived from 2’-amino LNA, a guanidine- bridged nucleic acid (GuNA) monomer, a spirocyclopropylene-bridged nucleic acid (scpBNA) monomer, and derivatives thereof; cyclohexenyl nucleic acid (CeNA) monomer, altriol nucleic acid (ANA) monomer, hexitol nucleic acid (HNA) monomer, fluorinated HNA (F-HNA) monomer, pyranosyl-RNA (p-RNA) monomer, 3’- deoxypyranosyl DNA (p-DNA), UNA; an inverted version of any of the monomers above. All these modifications are known to the person skilled in the art. [0157] A “backbone modification” indicates the presence of a modified version of the ribosyl moiety (“scaffold modification”), as indicated above, and/or the presence of a modified version of the PO as naturally occurring in RNA (“backbone linkage modification”). Examples of internucleoside linkage modifications are PS, chirally pure PS, Rp PS, Sp PS, phosphorodithioate (PS2), phosphonoacetate (PACE), thophosphonoacetate, phosphonacetamide (PACA), thiophosphonacetamide, PS prodrug, S-alkylated PS, H-phosphonate, MP, methylphosphonothioate, methylphosphate, methylphosphorothioate, ethylphosphate, ethylphosphorothioate, boranophosphate, boranophosphorothioate, methylboranophosphate, methylboranophosphorothioate, methylboranophosphonate, methylboranophosphonothioate, phosphoryl guanidine (PGO), methylsulfonyl phosphoroamidate, phosphoramidite, phosphonamidite, N3’^P5’ phosphoramidate, N3’^P5’ thiophosphoramidate, phosphorodiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido (TANA); and their derivatives. An ASO, as disclosed herein may also comprise a PNdmi linkage linking the most terminal nucleoside at the 5’ and/or 3’ end, and the one before last nucleoside at each of these
ends, respectively. A PNdmi linkage as preferably used in the ASOs has the structure of the following formula:
[0158] An ASO, as disclosed herein may also comprise a linkage moiety selected from a PS, phosphonoacetate, phosphorodithioate, methyl phosphonate (MP), sulfonylphosphoramidate, or a PNdmi internucleotide linkage. [0159] An ASO as disclosed herein may also comprise one or more linkage modifications according to the structure of formula:
wherein: X = O or S; and R = an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a C1-C6 alkoxy, a substituted C1-C6 alkoxy, a C1-C20 alkyl, a substituted C1-C20 alkyl, a C1-C6 alkenyl, a C1-C6 substituted alkenyl, a C1-C6 alkynyl, a substituted C1-C6 alkynyl, or a conjugate group. In a preferred embodiment, X = O and R = methyl and the linkage modification is referred to as mesyl phosphoramidate, MsPA or PNms. [0160] In one aspect, the ASO as disclosed herein comprises at least one MP internucleoside linkage according to the structure of the following formula:
[0161] As was noted in the art, a preferred position for an MP linkage in an ASO is linkage position -2, thereby connecting the nucleoside at position -1 with the nucleoside at position -2. In one aspect, this position, in an ASO as disclosed herein, comprises a PNms linkage modification as disclosed above, instead of an MP linkage. Intl. Patent Application Publication No. WO 2020/201406 discloses tuse of MP linkage modifications at certain positions surrounding the orphan nucleotide in the first nucleic acid strand. Although the presence of MP linkages is compatible with RNA editing by human ADAR enzymes, introducing MP linkages during the manufacturing of oligonucleotides is challenging in view of additional manufacturing (purification) steps in the coupling and decoupling process. In one aspect, the ASO does not comprise an MP linkage. [0162] ASOs herein do not include a 5’-terminal O6-benzylguanosine or a 5’-terminal amino modification and are not covalently linked to a SNAP-tag domain (an engineered O6-alkylguanosine-DNA-alkyl transferase). In one embodiment, an ASO herein comprises 0, 1, 2 or 3 wobble base pairs with the target sequence, and/or 1, 2, 3, 4, 5, 6, 7, or 8 mismatching base pairs with the target RNA sequence. The target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the ASO comprising the Benner’s base that is directly opposite the target adenosine. An ASO of the present disclosure does not include a boxB RNA hairpin sequence. An ASO according to the present disclosure can utilise endogenous cellular pathways and naturally available ADAR enzymes to specifically edit a target adenosine in a target RNA sequence. An ASO of the disclosure is capable of recruiting ADAR and complex with it and then allow the deamination of a (single) specific target adenosine nucleotide in a target RNA sequence. Ideally, only one adenosine is deaminated. An ASO of the disclosure, when complexed to ADAR, preferably deaminates a single target adenosine. [0163] Analysis of natural targets of ADAR enzymes has indicated that these generally include mismatches between the two strands that form the RNA helix edited by ADAR1 or 2. It has been suggested that these mismatches enhance the specificity of the editing
reaction (Stefl et al. (2006) Structure 14:345-355; and Tian et al. (2011) Nucleic Acids Res.39:5669-5681). Characterization of optimal patterns of paired/mismatched nucleotides between the ASOs and the target RNA also appears crucial for development of efficient ADAR-based ASO therapy. [0164] An ASO herein makes use of specific nucleotide modifications at predefined spots to ensure stability as well as proper ADAR binding and activity as indicated above. These changes may vary and may include modifications in the backbone of the ASO, in the sugar moiety of the nucleotides as well as in the nucleobases or the PO linkages, as outlined in detail above. They may also be variably distributed throughout the sequence of the ASO. Specific modifications may be needed to support interactions of different amino acid residues within the RNA-binding domains of ADAR enzymes, as well as those in the deaminase domain. For example, PS linkages between nucleotides or 2’-OMe or 2’-MOE modifications may be tolerated in some parts of the ASO, while in other parts they should be avoided so as not to disrupt crucial interactions of the enzyme with the phosphate and 2’-OH groups. Specific nucleotide modifications may also be necessary to enhance the editing activity on substrate RNAs where the target sequence is not optimal for ADAR editing. [0165] Previous work has established that certain sequence contexts are more amenable to editing. For example, the target sequence 5’-UAG-3’ (with the target A in the middle) contains the most preferred nearest-neighbor nucleotides for ADAR2, whereas a 5’-CAA- 3’ target sequence is disfavored (Schneider et al. (2014) Nucleic Acids Res.42:e87). The structural analysis of ADAR2 deaminase domain hints at the possibility of enhancing editing by careful selection of the nucleotides that are opposite to the target trinucleotide. For example, the 5’-CAA-3’ target sequence, paired to a 3’-GCU-5’ sequence on the opposing strand (with the A-C mismatch formed in the middle), is disfavored because the guanosine base sterically clashes with an amino acid side chain of ADAR2. [0166] An ASO herein comprises (when not expressed through a viral vector) one or more nucleotides with one or more sugar modifications. Thereby, a single nucleotide of the ASO can have one, or more than one sugar modification. Within the ASO, one or more nucleotide(s) can have such sugar modification(s). It is also an aspect of the disclosure that the nucleotide within the ASO herein that is opposite to the target A that needs to be edited does not contain a 2’-OMe or a 2’-MOE modification. Often the nucleotides that are directly 3’ and 5’ of this nucleotide (the ‘neighbouring nucleotides’)
in the ASO also lack such a chemical modification, although not both of the neighbouring nucleotides should not contain a 2’-O-alkyl group (such as a 2’-OMe). Either one, or both neighbouring nucleotides or all three nucleotides of the ‘Central Triplet’ may carry 2’- OH. [0167] As outlined herein, the nucleotides outside the Central Triplet are often 2’-OMe or 2’-MOE modified. However, this is not a strict requirement for the ASOs herein. The use of these 2’ substitutions assure a proper stability of those parts of the ASO, but other modifications may be applied as well. [0168] The disclosure also relates to an ASO comprising the sequence “m5Ce-Zd*Ad” as the Central Triplet, where m5Ce is a 5-methyl-cytidine having a 2’-MOE ribose group, Zd is a Benner’s base (i.e., a cytidine analog, which is located opposite the target A) and Ad is A having a 2’-H ribose group. The linkage between the cytidine and Benner’s base is a PO linkage and the linkage between Benner’s base and the A is a PS linkage. [0169] 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, the disclosure relates to an ASO comprising a Central Triplet of 3 sequential nucleotides, wherein the nucleotide directly opposite the target adenosine is the middle nucleotide of the Central Triplet, and wherein two nucleotides in said Central Triplet are DNA nucleotides to render the ASO more stable and/or more effective in inducing deamination of the target A. In another preferred aspect, the remainder of the ASO consists of RNA nucleotides that preferably (but not necessarily) are substituted at the 2’ position of the sugar, preferably with 2’-OMe, 2’-F, or 2’-MOE modifications. Other ribose modifications that are quite compatible with targeted editing in accordance with the disclosure are LNA and 2’-NH2. Different combinations of sugar modifications may be applied. In another aspect, the ASO herein comprises at least one non-naturally occurring internucleoside linkage modification selected from the group consisting of: PS, 3'-methylenephosphonate (i.e., 3’-O-MP internucleoside linkage), 5’- methylenephosphonate (i.e., 5'-O-MP internucleoside linkage), 3'-phosphoroamidate (i.e., N-3’-phosphoroamidate internucleoside linkage) and 2'-5’-PO (i.e., 2'-5'-PO internucleoside linkage). Especially preferred are PS linkages. [0170] The ASOs as disclosed herein may also be administered in the context of aids that will increase the entry of the ASOs into the target cell and/or its endosomal escape as soon as it is in the cell. Moieties that can be applied for such applications are for example
a set of chemical compounds (generally purified from nature) referred to as “saponins” or “triterpene glycosides”. A preferred saponin that can be used in the methods as disclosed herein is AG1856, disclosed in Intl. Patent Application Publication No. WO 2021/122998 and further described for use with RNA editing producing oligonucleotides in GB Patent Application No.2300865.9 (unpublished). Such saponin may be administered separately from the ASO, or together, and is preferably conjugated to the ASO to for example the 5’ or 3’ terminus, using a suitable linker if needed. [0171] The term ‘comprising’ encompasses ‘including’ as well as ‘consisting of’, for example, a composition ‘comprising X’ may consist exclusively of X or may include something additional (e.g., X + Y). The term ‘about’ in relation to a numerical value x is optional and means, for example, x+10%. The word ‘substantially’ does not exclude ‘completely’ (e.g., a composition that is ‘substantially free from Y’ may be completely free from Y). Where relevant, the word ‘substantially’ may be omitted from the definition of the disclosure. The term ‘downstream’ in relation to a nucleic acid sequence means further along the sequence in the 3' direction; the term ‘upstream’ means the converse. Thus, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand but is downstream of the stop codon in the antisense strand. References to ‘hybridisation’ typically refer to specific hybridisation and exclude non-specific hybridisation. Specific hybridisation can occur under experimental conditions chosen, using techniques well known in the art, to ensure that most stable interactions between probe and target are where the probe and target have at least 70%, preferably at least 80%, more preferably at least 90% sequence complementarity. When an ASO and a target sequence have 100% complementarity it means that there are no mismatches or wobble base pairs. The target A in the target RNA molecule is generally not fully complementary to the orphan nucleotide, which, as outlined herein is often a C or a deoxyribonucleotide carrying a Benner’s base Z, which is a C analog and therefore also not a perfect complementary match with the target A and should be considered a ‘mismatch’. Hence, an ASO as disclosed herein is not 100% complementary to the target sequence. The term ‘mismatch’ is used herein to refer to opposing nucleotides in a double stranded RNA complex which do not form perfect base pairs according to the Watson- Crick base pairing rules. In the historical sense, mismatched nucleotides are G-A, C-A, U-C, A-A, G-G, C-C, U-U pairs. In some embodiments first nucleic acid strands of the present disclosure comprise fewer than four mismatches with the target sequence, for
example 0, 1 or 2 mismatches. ‘Wobble base pairs’ are G-U, I-U, I-A, and I-C base pairs. Although a G:G pairing would be considered a mismatch, that does not necessarily mean that the interaction is unstable, which means that the term ‘mismatch’ may be somewhat outdated based on the current disclosure where a Hoogsteen base-pairing may be seen as a mismatch based on the origin of the nucleotide but still be relatively stable. An isolated G:G pairing in duplex RNA can for instance be quite stable, but still be defined as a mismatch. In one embodiment, an ASO comprises 1, 2, 3, 4, 5, 6, 7, or 8 mismatches, which includes the base pair of the target adenosine with the nucleoside in the ASO comprising the Benner’s base (that is directly opposite the target A). In one embodiment, an ASO comprises 0, 1, 2 or 3 wobble base pairs. [0172] Various chemistries and modifications are known in the field of oligonucleotides that can be readily used in accordance with the disclosure, see above. The regular internucleoside linkages between the nucleosides may be altered by mono- or di-thioation of the PO bonds to yield PS esters or phosphorodithioate esters, respectively. Other modifications of the internucleoside linkages are possible, including amidation and peptide linkers. In one embodiment, an ASO comprises 1, 2, 3, 4 or more PS linkages between the most terminal nucleotides of the ASO (hence, at both the 5’ and 3’ end), which means that in the case of 4 PS linkages, the ultimate 5 nucleotides are linked accordingly. It will be understood by the skilled person that the number of such linkages may vary on each end, depending on a variety of aspects, such as toxicity. [0173] In one embodiment, an ASO comprises a substitution of one of the non-bridging oxygens in the PO linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue comprises PS, phosphonoacetate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate. [0174] Particularly preferred are internucleoside linkages that are modified to contain a PS. Many of these non-naturally occurring modification of the linkage, such as PS are chiral, which means that there are Rp and Sp configurations, known to the person skilled in the art. In a preferred aspect, the chirality of the PS linkages is controlled, which means
that each of the linkages is either in the Rp or in the Sp configuration, whichever is preferred. The choice of an Rp or Sp configuration at a specified linkage position may depend on the target sequence and the efficiency of binding and induction of providing RNA editing. However, if such is not specifically desired, a composition may comprise ASOs as active compounds with both Rp and Sp configurations at a certain specified linkage position. Mixtures of such ASOs are also feasible, wherein certain positions have preferably either one of the configurations, while for other positions such does not matter. Intl. Patent Application Publication No. WO 2019/219581 discloses specific disallowed and tolerated positions of Rp and Sp configurations of PS linkages in which the known structural features of ADAR2 when bound to RNA were modelled in silico using mouse Idua mRNA as the model target sequence. This knowledge was applied to the ASOs of the present disclosure and the sequences of the anticipated ASOs with their chemical modifications including the positions of the tolerated Rp and Sp linkages. These Rp and Sp specificities were applied to a preferred ASO (A-50; SEQ ID NO: 16 and 69 (with GN)) for targeting the human IDUA transcript. These ASOs (with GN) are given as A-50 GN-A to -M in Fig.1C and without GN in Fig.1E. Linkage positions +5, +6, +7, +8, -4, and -5 are PO linkages herein, because Intl. Patent Application Publication No. WO 2019/219581 indicated that neither Rp or Sp PS linkages are tolerated at these positions. Linkages +1, +2, +3, +9, +11, +12, +13, -7, -9, -10, -11, and -12 are stereo random PS linkages (no preference for Sp or Rp configurations), whereas linkages 0, +14, -2, -8, and -13 are not amended in comparison to A-50. [0175] In one embodiment, an ASO as disclosed herein comprises the sequence and chemical modifications (including the stereo-pure linkages) of ASOs 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), as depicted in Fig.1C. [0176] The skilled person knows that a variety of mono- and tri-antennary GalNAc moieties are available in the art. Hence, the GalNAc moiety (GN) with ASO positions (primarily on the 3’ end) as indicated in the accompanying figures herein may also be replaced by an alternative mono- or tri-antennary GalNAc moiety if the (therapeutic) application of the ASO desires it. Preferred GalNAc moieties are given in Formula I, II, III, and IV herein. [0177] In one embodiment, an ASO comprises one or more sugar moieties that are mono- or di-substituted at the 2', 3' and/or 5' position such as: -OH; -F; substituted or
unsubstituted, linear or branched lower (Cl-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by 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; -dimethylamino oxyethoxy; and - dimethylaminoethoxyethoxy. [0178] The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or derivative thereof. A preferred derivatized sugar moiety comprises an LNA, in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2'-O, 4'-C-ethylene-bridged nucleic acid. These substitutions render the nucleotide analogue RNase H and/or nuclease resistant, and increase the affinity for the target RNA. [0179] In one embodiment, a nucleotide analogue 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 -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art. Purine nucleobases and/or pyrimidine nucleobases may be modified to alter their properties, for example by amination or deamination of the heterocyclic rings. The exact chemistries and formats may vary from oligonucleotide construct to oligonucleotide construct and from application to application, and may be worked out in accordance with the wishes and preferences of those of skill in the art. It is believed in the art that 4 or more consecutive DNA nucleotides in an oligonucleotide create so-called ‘gapmers’ that – when annealed to their RNA cognate sequences – induce cleavage of the target RNA by RNase H. According to the disclosure, RNase H cleavage of the target RNA is generally to be avoided as much as possible. [0180] All chemical modifications listed herein that may be used in the ASO of the present disclosure may also be used for a sense strand that is complementary to the ASO, when the ASO and the complementary strand form a so-called heteroduplex RNA editing oligonucleotide (HEON) complex, as described in Intl. Patent Application No. WO 2024/084048, except that the opposite sense strand does not have a so-called ‘orphan nucleotide’, which is the nucleotide in the ASO that is positioned directly opposite the target A in the IDUA transcript. Hence, the modification related to the orphan nucleotide relates only to the ASO, where all other modifications relate to the ASO and any
(protecting) sense oligonucleotide that may be used together with the ASO in a pharmaceutical product. This includes the use of hydrophobic moieties (such as tocopherol and cholesterol) and cell-specific ligands (such as GalNAc moieties), that have also been described in detail in Intl. Patent Application No. WO 2024/084048, which may either be bound to the ASO or its opposite strand, or both. [0181] In some embodiments, the ASO comprises a cell-specific ligand as delivery moiety. In a non-limiting embodiment, the delivery moiety is a GalNAc moiety. In one embodiment the GalNAc moiety is attached to the 3’ terminus of each oligonucleotide. Non-limiting examples of GalNAc moieties are disclosed in the following Int. Patent Application Publication Nos.: WO 2022/271806; WO 2009/073809; WO 2014/025805; WO 2015/006740; WO 2019/053661; WO 2014/179620; WO 2021/261998; U.S. Patent Application Publication No.2019/0256849; WO2011104169; WO2018013525; WO2023034719; WO2023114746; WO2018185241; WO2018185240; WO2019/128611; WO2020093061; WO2021127214; WO2023045995; WO2022076922; WO2022/189861; and WO2016100401. [0182] In one embodiment the GalNAc moiety is according to the structure of Formula I in Int. Patent Application Publication No. WO 2022/271806. This embodiment of the GalNAc moiety is reproduced in Formula I (herein above). [0183] In some embodiments, any one of the ASOs as described herein is conjugated to connection point E of Formula I, optionally via a linker. [0184] In one embodiment the GalNAc moiety is attached to the 3’ terminus of each oligonucleotide and the GalNAc moiety is according to the structure of Formula I in Int. Patent Application Publication No. WO 2022/271806. [0185] Mutagenesis studies of human ADAR2 revealed that a single mutation at residue 488 from glutamate to glutamine (E488Q), gave an increase in the rate constant of deamination by 60-fold when compared to the wild-type enzyme (Kuttan & Bass (2012) Proc Natl Acad Sci USA 109:3295-3304). During the deamination reaction, ADAR flips the edited base out of its RNA duplex, and into the enzyme active site. When ADAR2 edits A in the preferred context (an A:C mismatch) the nucleotide opposite the target A is often referred to as the orphan nucleotide (or ‘orphan cytidine’ as the case may be), as indicated above. The crystal structure of ADAR2 E488Q bound to double-stranded RNA revealed that the glutamine (Gln; Q) side chain at position 488 can donate an H-bond to the N3 position of the orphan cytidine, which leads to the increased catalytic rate of
ADAR2 E488Q. In the wild-type enzyme, wherein a glutamate (or glutamic acid; Glu; E) is present at position 488 instead of a glutamine (Gln) the amide group of the glutamine is absent and is instead a carboxylic acid. To obtain the same contact of the orphan cytidine with the E488Q mutant would then, for the wild-type situation, require protonation for this contact to occur. To make use of endogenously expressed ADAR2 to correct disease relevant mutations, it is essential to maximize the editing efficiency of the wild type ADAR2 enzyme present in the cell. Intl. Patent Application Publication No. WO 2020/252376 discloses use of AONs with modified RNA bases, especially at the position of the orphan nucleotide to mimic the hydrogen-bonding pattern observed by the E488Q ADAR2 mutant. By replacing the nucleotide opposite the target A in the AON with cytidine analogs that serve as H-bond donors at N3, it was envisioned that it would be possible to stabilize the same contact that is believed to provide the increase in catalytic rate for the mutant enzyme. Two cytidine analogs were of particular interest: pseudoisocytidine (also referred to as ‘piC’; Lu et al. (2009) J. Org. Chem. 74:8021- 8030; Burchenal et al. (1976) Cancer Res 36:1520-1523) and Benner’s base Z (also referred to as ‘dZ’ or ‘Zd’; Yang et al. (2006) Nucl Acid Res 34:6095-6101) that were initially selected because they offer hydrogen-bond donation at N3 with minimal perturbation to the shape of the nucleobase. Benner’s base is also referred to as 6-amino- 5-nitro-2(1H)-pyridone. The presence of the cytidine analog in the ASO may exist in addition to modifications to the ribose 2’ group. The ribose 2’ groups in the orphan nucleotide 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., a bridged nucleic acid such as a locked nucleic acid (LNA)), or other 2’ substitutions. The 2’-4’ linkage can be selected from linkers known in the art, such as a methylene linker or constrained ethyl linker. [0186] The orphan nucleotide in the ASO as disclosed herein is preferably a cytidine or analog thereof (such as a nucleotide carrying a Benner’s base) or a uridine or analog thereof (such as iso-uridine). The orphan nucleotide, whether it is a cytidine or analog thereof, or a uridine or analog thereof, preferably comprises a deoxyribose (2’-H; = DNA) but may also comprise a diF modification at the 2’ position of the sugar (see, Intl. Patent Application No. PCT/EP2023/069609; not published). In an embodiment, at least one and in another embodiment both the neighbouring (i.e., directly adjacent) nucleotides flanking the orphan nucleotide do not comprise a 2’-OMe modification. Complete modification wherein all nucleotides of the oligonucleotide hold a 2’-OMe modification (including the
orphan nucleotide), with natural bases, results in a non-functional oligonucleotide as far as RNA editing goes (known in the art), presumably because it hinders the ADAR activity at the targeted position. In general, an adenosine in a target RNA can be protected from editing by providing an opposing nucleotide with a 2'-OMe group (at least when there are no other chemical substitutions or modifications within the nucleotide), or by providing a guanine or adenine as opposing base, as these two nucleobases are also able to reduce editing of the opposing A. [0187] An ASO as disclosed herein is normally longer than 10 nucleotides, preferably more than 11, 12, 13, 14, 15, 16, 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, still more preferably shorter than 50 nucleotides. In a preferred aspect, the ASO herein comprises 18 to 70 nucleotides, more preferably comprises 18 to 60 nucleotides, and even more preferably comprises 18 to 50 nucleotides. Hence, in a particularly preferred aspect, the ASO herein comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In one embodiment, the ASO is 27, 28, 29, or 30 nucleotides in length. [0188] In another preferred aspect, at either end or both termini of an ASO herein, inverted deoxyT or dideoxyT nucleotides may be incorporated. [0189] It is known in the art, that RNA editing entities (such as human ADAR enzymes) edit double-stranded RNA structures with varying specificity, depending on several factors. One important factor is the degree of complementarity of the two strands making up the double-stranded RNA sequence. Perfect complementarity of the two strands usually causes the catalytic domain of hADAR to deaminate adenosines in a non- discriminative manner, reacting more or less with any adenosine it encounters. The specificity of hADAR1 and 2 can be increased by ensuring the presence of a number of mismatches in the double-stranded RNA, which presumably helps to position the double- stranded RNA binding domains in a way that has not been clearly defined yet. Additionally, the deamination reaction itself can be enhanced by providing an ASO that comprises a mismatch opposite the adenosine to be edited. In the disclosure, this is achieved using a nucleoside comprising a Benner’s base. Upon deamination of the adenosine in the target strand, the target strand will obtain an inosine which, for most biochemical processes, is “read” by the cell’s biochemical machinery as a G. Hence, the
skilled person will appreciate that after the A to I conversion, the mismatch has been resolved, because I is perfectly capable of base pairing with the opposite C (or the cytidine analog) in the targeting portion of the oligonucleotide construct herein. However, after deamination, there is clearly no need for the ASO to hybridize to the target sequence. After the mismatch has been resolved due to editing, the substrate is released and the oligonucleotide construct-editing entity complex is released from the target RNA sequence, which then becomes available for downstream biochemical processes, such as splicing and translation. [0190] The desired level of specificity of editing the target RNA sequence may vary from application to application. Following the instructions in the present disclosure, those of skill in the art will be capable of designing the complementary portion of the oligonucleotide according to their needs, and, with some trial and error, obtain the desired result [0191] Pharmaceutical compositions and uses [0192] The disclosure also relates to a pharmaceutical composition comprising an ASO herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier can simply be a saline solution. This can usefully be isotonic or hypotonic, particularly for pulmonary delivery. The pharmaceutical composition may be sterile. The disclosure also provides a delivery device (e.g., a syringe) which includes a pharmaceutical composition herein. [0193] The ASO herein is suitably administrated in aqueous solution, or in suspension, optionally comprising additives, excipients and/or other ingredients, compatible with pharmaceutical use. The pharmaceutical composition may comprise the ASO at concentrations ranging from about 1 ng/ml to about 1 g/ml, preferably from about 10 ng/ml to about 500 mg/ml, more preferably from about 100 ng/ml to about 100 mg/ml. Dosage to a patient may suitably range from between about 1 µg/kg to about 100 mg/kg, preferably from about 10 µg/kg to about 10 mg/kg, more preferably from about 100 µg/kg to about 1 mg/kg. [0194] Administration to a patient may be by inhalation (e.g., through nebulization), intranasally, orally, by injection or infusion, intravenously, subcutaneously, intra- dermally, intra-cranially, intravitreally, intramuscularly, intra-tracheally, intra- peritoneally, intra-rectally, in the central nervous system, e.g intrathecally, and the like.
Administration may be in solid form, in the form of a powder, a pill, a gel, an eye-drop, or in any other form compatible with pharmaceutical use in humans. [0195] Methods of treatment & therapeutic uses [0196] As described above, the disclosure provides an ASO for forming a double- stranded complex with a human IDUA RNA molecule in a human cell. Thus, the therapeutic effect is preferably on a human cell in vivo. Of course, the methods may also be carried out in vitro or ex vivo. [0197] The disclosure provides an ASO herein, or pharmaceutical composition herein, for use in the treatment of disease. The disclosure also provides the use of an ASO herein, or pharmaceutical composition herein, in the manufacture of a medicament for the treatment of disease. The disclosure also provides a method for treating a disease in a patient, comprising administering a therapeutically effective amount of an ASO herein or a pharmaceutical composition herein. Preferably the disease is MPS 1. More preferably the disease is Hurler syndrome, which results from a c.1205G>A mutation in the patient’s IDUA gene. The ASO is preferably administered therapeutically, rather than prophylactically. [0198] The therapeutically effective amount of ASO herein to be administered, the dosage and the dosing regimen can vary from cell type to cell type, the disease to be treated, the target population, the mode of administration (e.g., systemic versus local), the severity of disease and the acceptable level of side activity, but these can and should be assessed by trial and error during in vitro research, in pre-clinical and clinical trials. The trials are particularly straightforward when the modified sequence leads to an easily detected phenotypic change. It is possible that higher doses of ASO could compete for binding to an ADAR within a cell, thereby depleting the amount of the entity, which is free to take part in RNA editing, but routine dosing trials will reveal any such effects for a given ASO and a given target. [0199] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of an ASO and/or ASO-GalNAc conjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A “therapeutically effective amount” is an amount of a pharmaceutical composition that is
effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. [0200] One suitable trial technique involves delivering an ASO herein to cell lines, or a test organism and then taking biopsy samples at various time points thereafter. The sequence of the target RNA can be assessed in the biopsy sample and the proportion of cells having the modification can easily be followed. After this trial has been performed once then the knowledge can be retained, and future delivery can be performed without needing to take biopsy samples. A method of the disclosure can thus include a step of identifying the presence of the desired change in the cell’s target RNA sequence, thereby verifying that the target RNA sequence has been modified. This step will typically involve sequencing of the relevant part of the target RNA, or a cDNA copy thereof (or a cDNA copy of a splicing product thereof, in case the target RNA is a pre-mRNA), as discussed above, and the sequence change can thus be easily verified. Alternatively, the change may be assessed on the level of the protein (length, glycosylation, function or the like), or by some functional read-out, such as a(n) (inducible) current, when the protein encoded by the target RNA sequence is an ion channel, for example. [0201] After RNA editing has occurred in a cell, the modified RNA can become diluted over time, for example, due to cell division, limited half-life of the edited RNAs, etc. Thus, in practical therapeutic terms, a method herein may involve repeated delivery of an ASO herein until enough target RNAs have been modified to provide a tangible benefit to the patient and/or to maintain the benefits over time. [0202] Methods for deamination [0203] The disclosure also provides a method for the deamination of at least one specific target adenosine present in a target RNA molecule in a cell, wherein the target RNA molecule is a human IDUA pre-mRNA or mRNA, or a part thereof, the method comprising the steps of: (i) providing the cell with an ASO herein or pharmaceutical composition herein; (ii) allowing uptake by the cell of the ASO; (iii) allowing annealing of the ASO to the target RNA molecule; (iv) allowing a mammalian ADAR enzyme comprising a natural double-stranded RNA binding domain as found in the wild type enzyme to deaminate the target adenosine in the target RNA molecule to an inosine; and optionally (v) identifying the presence of the inosine in the target RNA molecule. Such a method may be carried out in vitro, in vivo, or ex vivo. The identification of step (v) may be by: (a) sequencing the target RNA molecule; (b) assessing the presence of a functional,
elongated, full length and/or wild-type Iduronidase protein; or (c) using a functional read- out, wherein the target RNA after the deamination encodes a functional, full length, elongated and/or wild type Iduronidase protein. [0204] The functional assessment for each of the diseases mentioned herein will generally be according to methods known to the skilled person. A very suitable manner to identify the presence of an inosine after deamination of the target adenosine is of course RT-PCR and sequencing, using methods that are well-known to the person skilled in the art. In one embodiment, the rate of editing may be calculated by comparing the percentage adenosine versus guanosine detected at the target following editing. In one embodiment, the ASO of the disclosure provides editing at a rate of 30%, 35%, 40%, 45%, 50%, 55%, 60% or more, as measured in mammalian cells, such as patient derived fibroblasts. [0205] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Database entries and electronic publications disclosed in the present disclosure are incorporated by reference in their entireties. The version of the database entry or electronic publication incorporated by reference in the present application is the most recent version of the database entry or electronic publication that was publicly available at the time the present application was filed. The database entries corresponding to gene or protein identifiers (e.g., genes or proteins identified by an accession number or database identifier of a public database such as Genbank, Refseq, or Uniprot) disclosed in the present application are incorporated by reference in their entireties. The gene or protein-related incorporated information is not limited to the sequence data contained in the database entry. The information incorporated by reference includes the entire contents of the database entry in the most recent version of the database that was publicly available at the time the present application was filed. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES Example 1. Use of chemically modified oligonucleotides for A-to-I editing of human IDUA target RNA in an in vitro biochemical editing assay. [0206] To obtain the human IDUA target RNA, a PCR was performed using a hIDUA G- block (IDT), which contained the sequence for the T7 promotor and (a part of) the sequence of human IDUA as template using forward primer 5’-CTC GAC GCA AGC CAT AAC AC-3’ (SEQ ID NO: 38) and reverse primer 5’-TGG ACC GAC TGG AAA CGT AG-3’ (SEQ ID NO: 39). The PCR product was then used as template for the in vitro transcription. The MEGAscript T7 transcription kit was used for this reaction. The RNA was purified on a urea gel then extracted in 50 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.1% SDS, 0.3 M NaCl buffer and phenol-chloroform purified. The purified RNA was used as target in the biochemical editing assay. [0207] Guide oligonucleotides A-6, 26, 32, 35, 36, 37, 38, 39, 40, 42, 43, 44, 50 and 55 were annealed to the human IDUA target RNA, which was done in a buffer (5 mM Tris- Cl pH 7.4, 0.5 mM EDTA and 10 mM NaCl) at the ratio 1:3 of target RNA to oligonucleotide (200 nM target RNA and 600 nM oligonucleotide). The samples were heated at 95°C for 3 min and then slowly cooled down to 4°C. Next, the editing reaction was carried out. The annealed oligonucleotide / target RNA was mixed with protease inhibitor (cOmpleteTM, Mini, EDTA-free Protease I, Sigma-Aldrich), RNase inhibitor (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) such that their final concentration was 6 nM oligonucleotide and 2 nM target RNA. The reaction was started by adding purified ADAR2 (GenScript) to a final concentration of 6 nM into the mix and incubated for predetermined time points (0 sec, 30 sec, 1 min, 2 min, 4 min, 8 min, and 16 min) at 37°C. Each reaction was stopped by adding 95 µl of boiling 3 mM EDTA solution. A 6 µl aliquot of the stopped reaction mixture was then used as template for cDNA synthesis using Maxima reverse transcriptase kit (Thermo Fisher) with random hexamer primer (ThermoFisher Scientific). Initial denaturation of RNA was performed in the presence of the primer and dNTPs at 95°C for 5 min, followed by slow cooling to 10°C, after which first strand synthesis was carried out according to the manufacturer’s instructions in a total volume of 20 µl, using
an extension temperature of 62°C. Products were amplified for pyrosequencing analysis by PCR, using the Amplitaq gold 360 DNA Polymerase kit (Applied Biosystems) according to the manufacturer’s instructions, with 5 µl of the cDNA as 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 protocol: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, and a final extension of 72°C for 7 min. [0208] Because inosines base-pair with cytidines during the cDNA synthesis in the reverse transcription reaction, the nucleotides incorporated in the edited positions during PCR will be guanosines. The percentage of guanosine (edited) versus adenosine (unedited) was defined by pyrosequencing. Pyrosequencing of the PCR products and data analysis were performed by the PyroMark Q48 Autoprep instrument (QIAGEN) following the manufacturer’s instructions with 10 µl input of the PCR product and 4 µM of the following sequencing primer: hIDUA-Seq, 5’-GCT GGC GCT GCT GGA T-3’ (SEQ ID NO: 42). The settings specifically defined for this target RNA strand included two sets of sequence information. The first of these defines the sequence for the instrument to analyze, in which the potential for a particular position to contain either an adenosine or a guanosine is indicated by
GAG GAG CAG CTC T A/G GGC CGA AGT GTC GCA GGC CGG GAC C (SEQ ID NO: 43). The dispensation order was defined for this analysis as follows: CGA GAG CAG CTC GTA GCG AGT GTC GCA GAC GCA C (SEQ ID NO: 44). The analysis performed by the instrument provides the results for the selected nucleotide as a percentage of adenosine and guanosine detected in that position, and the extent of A-to-I editing at a chosen position will therefore be measured by the percentage of guanosine in that position. [0209] The results shown in Fig.2A, Fig.2B, Fig.2C, and Fig.2D indicate that all tested guide oligonucleotides can edit the target hIDUA RNA.
Example 2. Use of modified oligonucleotides for specific A-to-I editing of human IDUA target RNA in primary human patient fibroblast. [0210] Next it was investigated whether the modified oligonucleotides of the previous example edit the target adenosine in human IDUA RNA carrying the c.1205G>A mutation in cells. For this, primary human W402X fibroblasts (GM00798) were used. About 75,000 cells per 24 well plate were seeded 24 hrs before transfection, which was performed with 100 nM guide oligonucleotide and Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions (at a ratio 1:2, 1 µg oligonucleotide to 2 µl Lipofectamine 2000). RNA was extracted from cells 48 hrs after transfection 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, with a combination of random hexamer and oligo-dT primers. The cDNA was used as template for digital droplet PCR (ddPCR) with respectively 50 ng RNA input per reaction. The ddPCR assay for absolute quantification of nucleic acid target sequences was performed using BioRad’s QX-200 Droplet Digital PCR system.4 µl of 2x diluted cDNA obtained from the RT cDNA synthesis reaction was used in a total mixture of 21 µl of reaction mix, including the ddPCR Supermix for Probes no dUTP (Bio Rad), a Taqman SNP genotype assay with the following forward and reverse primers combined with the following gene-specific probes: [0211] Forward primer: 5’-CTG TTG CGC AAG CCG GTG-3’ (SEQ ID NO: 45) [0212] Reverse primer: 5’-CCA CCG TGT GGT TGC TGT C-3’ (SEQ ID NO: 46) [0213] Wild type probe (FAM NFQ labeled): 5’-/56-FAM/AG CTC T+G+G +GCC GAA GTG T/3IABkFQ/-3’ (SEQ ID NO: 47) [0214] Mutant probe (HEX NFQ labeled): 5’-/5HEX/AG +CTC T+A+G +GCC GAA GTG T/3IABkFQ/-3’ (SEQ ID NO: 48) [0215] A total volume of 21 µl PCR mix including cDNA was filled in the middle row of a ddPCR cartridge (BioRad) using a multichannel pipette. The replicates were divided by two cartridges. The bottom rows were filled with 70 µl of droplet generation oil for probes (BioRad). After the rubber gasket replacement, droplets were generated in the QX200 droplet generator.42 µl of oil emulsion from the top row of the cartridge was transferred to a 96-wells PCR plate. The PCR plate was sealed with a tin foil for 4 sec at 170°C using the PX1 plate sealer, followed by the following PCR program: 1 cycle of
enzyme activation for 10 min at 95°C, 40 cycles denaturation for 30 sec at 95°C and annealing/extension for 1 min at 63 °C, 1 cycle of enzyme deactivation for 10 min at 98°C, followed by a storage at 8°C. After PCR, the plate was read and analysed with the QX200 droplet reader. The results shown in Fig.3A, Fig.3B, and Fig.3C indicate that all tested oligonucleotides cause editing. Example 3. Use of modified oligonucleotides for specific A-to-I editing of mouse Idua target RNA and restoration of protein activity in vivo. [0216] In a next experiment it was investigated whether A-to-I editing could also occur in the liver of mice with a W392X mutation (a known Hurler mouse model; see e.g., Int. Patent Application Publication No. WO 2018/041973) using oligonucleotides carrying a GalNAc (GN) ligand moiety. Fig.4 shows the names, SEQ ID NO’s and sequences of the fourteen mouse Idua sequence specific ASOs that were initially tested together with a scrambled oligonucleotide (Scr1), also comprising a GalNAc moiety. The GalNAc moiety was attached to the 3’ terminus of each oligonucleotide and was according to the structure of Formula I in Int. Patent Application Publication No. WO 2022/271806. Besides editing efficiency (percentages) it was also investigated whether Iduronidase protein activity could be restored after oligonucleotide treatment. For this, 4 mice (per group; one group per ASO) were subcutaneously injected with 10 mg/kg ASO on day 0, day 4, day 8, and day 12. The animals were sacrificed on day 14. RNA editing was determined in the liver using ddPCR to quantify the percentage of editing of the target RNA, generally as outlined above, with the following set of primers and probes: [0217] Forward primer: 5’- CTCACAGTCATGGGGCTC-3’ (SEQ ID NO: 130) [0218] Reverse primer: 5’- CACTGTATGATTGCTGTCCAAC-3’ (SEQ ID NO: 131) [0219] Wild type probe (FAM NFQ labeled): 5’-/56-FAM/ A+CAACTC+T+GGGCAGAGG+T/3IABkFQ/-3’ (SEQ ID NO: 132) [0220] Mutant probe (HEX NFQ labeled): 5’-/5HEX/ A+CAACTC+T+A+GGCAGAGG+T/3IABkFQ/-3’ (SEQ ID NO: 133) [0221] An Iduronidase activity analysis was performed on liver tissue to quantify the enzyme activity upon treatment with the specific ASOs using an activity assay generally as described in WO2018/041973. The scrambled oligonucleotide (group of 4 animals)
and a non-treated (NT) group of 2 animals served as negative controls, whereas samples of wild-type animals served as positive controls in both assays (set as 100%). [0222] The results are shown in Fig.5A and Fig.5B. In Fig.5A the editing percentages are shown on day 14 and in Fig.5B the Iduronidase activity is shown in the same samples from the same livers of the same mice treated with the guide oligonucleotides as depicted in Fig.5A. Highest editing percentages were determined after treatment with oligonucleotides mIdua-75GN, -89GN, -88GN, -85GN (with one animal showing 8% editing), and -87GN (SEQ ID NO’s: 49, 58, 57, 55, and 56, respectively). The person skilled in the art appreciates that RNA turnover may be very dynamic, may be very rapid and may range from tissue-to-tissue, from cell-to-cell, and from target-to-target RNA, whereas the availability of the guide oligonucleotide that should induce the editing adds another layer of complexity to this assessment. This makes that RNA editing percentages may differ at different points in time, and that a certain percentage at a strict timepoint may not fully translate to the amount of protein translated from the edited RNA versus the amount of protein translated from non-edited RNA, let alone the possibility of degradation differences between edited and non-edited target RNA. Rather than strictly assessing editing percentages, the activity of the Iduronidase protein (absent in W392X mice) reveals an additional pointer to whether the edited RNA translates into a wildtype protein. Fig.5B clearly shows that upon ASO treatment Iduronidase activity could be determined up to almost 6-fold increase (approximately 4.5% activity) compared to levels observed with the scrambled oligonucleotide, again with mIdua-75GN, -89GN, -88GN, - 85GN, and -87GN performing best albeit at slightly different efficiencies. It can be concluded that the best performing guide oligonucleotides – when looked at editing percentages – also provide the highest percentages in protein restoration in vivo. Fig.5C shows the Iduronidase activity in plasma of the same animals on day 14, clearly indicating that the best performing oligonucleotides mentioned above (in respect of RNA editing and Iduronidase activity in the liver) also show the highest levels of Iduronidase activity in plasma, which provides an excellent tool as a biomarker in future assessments.
Example 4. Use of mIdua-75GN and mIdua-85GN ASOs for specific A-to-I editing of mouse Idua target RNA and restoration of protein activity in vivo. [0223] Next, guide oligonucleotides mIdua-75GN and mIdua-85GN (see Example 3 and the results in mice with these ASOs in Fig.4) were used in a similar experiment as shown in Example 3. However, dosing and regimens schedules were altered, and sampling was set at different time points. Furthermore, the editing and protein activity assays were performed at 4 and 8 weeks respectively, after sacrifice. All mice were injected with ASOs subcutaneously. Each group contained 4 animals, except for group 1 that contained 20 animals, and wherein 4 animals were sacrificed at 12 hr, 48 hr, 1 week, 2 weeks and at 4 weeks, respectively, after a single dose. The seven groups were set up as shown in Fig 6. [0224] Fig.7A shows the results from group 1 and indicates that the editing levels were between 1 and 2% after 48 hr and at time points 1 and 2 weeks, and that it appears to decline after 4 weeks. Interestingly however, the Iduronidase activity was also checked at these same timepoints, and it appeared that over time, Iduronidase activity increases. From Fig.7B it appears that enzyme activity may even increase after 4 weeks after a single dose at day 0. These experiments show that the protein production (from edited transcripts) and the activity performed by these proteins may follow the editing of the transcript, which may be a very dynamic process with new RNA being transcribed, old RNA being degraded, whereas the ASOs that are available can induce editing when the transcript is free to target. Protein being translated from these edited transcripts may in fact have a much longer half-life and stay around for much longer than each of the edited transcripts in time. Such events need further investigation to clarify whether this is true. [0225] Group 2 and Group 3 only differed in the dose that was administered, with Group 2 receiving 10 mg/kg and Group 3 receiving 30 mg/kg. In both groups administration was twice per week. Figs.8A-8B (in which the ASO mIdua-85GN is referred to as EON 11) show the percentage editing in the liver at necropsy (4 weeks after day 0) and suggests that there is a dose-dependency, wherein percentages editing reached 3.5% in mice administered with 30 mg/kg and 2.5% in mice administered with 10 mg/kg. It is unclear why these percentages are lower than in the previous experiments described in Example 3. Importantly however, Iduronidase activity increased to approximately 13% on average
in the mice receiving 30 mg/kg, and 5% in the mice receiving 10 mg/kg, indicating a strong dose-dependent effect on the protein activity level. [0226] Group 3 and 4 only differed in the ASO that was administered. The ASO mIdua- 75GN is also referred to as EON 6 and mIdua-85GN is also referred to as EON 11. Figs. 9A-9B show that mIdua-85GN (EON 11) outperformed mIdua-75GN (EON 6) in RNA editing percentage as well as in Iduronidase activity, which resembles the findings as shown in Figs.5A-5C. [0227] Group 2 and 5 only differed in the regimen, with Group 2 receiving mIdua-85GN (EON 11) administrations (10 mg/kg) twice weekly for 4 weeks, and Group 5 receiving the same (10 mg/kg per dose) only once a week. Editing percentages and Iduronidase activities did not differ between these groups suggesting that the ASO is available in excess during the experiment. Results are shown in Figs.10A. [0228] Group 2 and 6 differed in the sense that the same dosing regimen (twice weekly with 10 mg/kg) was either 4 weeks until necropsy (Group 2) or 8 weeks until necropsy (Group 6) with mIdua-85GN as the administered ASO. Figs.11A-11B show that editing percentages and Iduronidase activity increased when the administration was prolonged. However, as Figs.11A-11B also show, altering the administration regimen to first twice weekly, followed by an administration biweekly (Group 7) did not provide an improved pattern. The second bar in both Figs.11A and 11B represents Group 2, the third bar in both Figs.11A and 11B represents Group 6, and the fourth bar in both Figs.11A and 11B represents Group 7. Example 5. Use of mIdua-75GN and mIdua-85GN ASOs for specific A-to-I editing of mouse Idua target RNA leading to GAG reduction in vivo. [0229] The samples (liver at sacrifice as well as urine samples taken during the process) from all mice in the 7 groups described in Example 4 were tested for GAG content. The most severe form of MPS 1 is Hurler syndrome. MPS 1 is an inherited metabolic disease characterized by a malfunction of the Iduronidase enzyme, encoded by the IDUA gene, leading to the storage of the GAGs dermatan sulfate and heparan sulfate as oligosaccharides in the lysosomes. Restoration of Iduronidase activity because of RNA editing as disclosed herein should lead to a reduction of GAG content in a variety of tissues, including the liver and circulation (represented by excreted urine). Hence, all mice in all groups described in Example 4 were sampled and samples were tested for
GAG content using the Blyscan GAG assay kit (Blyscan, no. B1000).20 mg to 50 mg liver tissue was homogenized with a pestle and digested in 1 mL papain extraction reagent at 65 °C for 24 hr. The total amount of GAG in each sample was measured according to manufacturer’s protocol. Finally, the measured GAGs were normalized to mg tissue. Urine samples were filtered by centrifugation using the Ultrafree-MC HV Centrifugal Filters (Cat# UFC30HV00, Millipore). The GAG content in urine was measured also by using the Blyscan GAG assay kit (Blyscan, no. B1000). The total amount of GAG in each sample was measured according to manufacturer’s protocol. Finally, the measured GAGs were normalized to the amount of creatinine using creatinine assay kit (Cat# MAK080, Sigma) performed according to manufacturer's protocol. [0230] Figs.12A-12B show that in Groups 2, 3, 4, 5, 6, and 7 there was a significant decrease in GAG content in comparison to non-treated mice (Fig.12A shows the levels in liver and Fig.12B shows the levels in urine) and the levels reached were comparable to the wild-type situation in which GAG levels are close to zero. The single dose administered in Group 1 did not show a reduction (data not shown). [0231] These results together show that RNA editing of the mouse Idua transcript comprising a W392X mutation that is similar to the mutation causing Hurler syndrome in humans results in significant increase in Iduronidase activity on a prolonged period of time, which subsequently appears to result in a significant decrease in GAG levels both in liver and in urine, indicating that the RNA editing and restoration of the protein activity may have a significant impact in a human Hurler syndrome patient when treated with an oligonucleotide as disclosed herein, using an appropriate regimen of administration. Example 6. Stability study. [0232] The efficacy of an ASO to generate RNA editing on a particular target sequence depends on a variety of features, including the ability to interact with the complementary part of the target sequence (and to release again), the ability to recruit endogenous ADAR as a double-stranded complex with the target molecule, and the stability of the ASO in general because it is always prone to degradation by cellular components such as nucleases, especially when it has entered the target cell and travels towards the target molecule.
[0233] To investigate the stability of a GalNAc-conjugated A-50 (A-50GN) that targets the human IDUA transcript in comparison to its counterpart A-78 (also GalNAc- conjugated; A-78GN), which has the same sequence and chemical modifications as A- 50GN but comprises PS linkages instead of PO linkages in the 5’ terminal part (see, Fig. 1B), and the counterpart ASOs used for targeting the mouse Idua target molecule: GalNAc-conjugated mIdua-85GN and mIdua-88GN (Fig.4), a biochemical stability assay was used. For this, 10 µM end concentration of each A-50GN (abbreviated to A-50 in Fig.15), A-78GN (abbreviated to A-78 in Fig.15), mIdua-85GN, and mIdua-88GN was assayed using a mixture of: i) snake venom phosphodiesterase (6.5x10-5 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; Thermo Fisher Scientific; cat# EN0531); and iv) RNAse A (1.3x10-1 µg/µl; Biolabs; cat# M0303L) in a buffer of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 1 mM EDTA and 50% glycerol in a total volume of 50 µL. As a control the same ASOs were diluted in PBS with an end concentration of 10 µM in 50 µL. After 24 hr incubation at 37 °C, the reaction was stopped with 50 µL 8.0 M Guanidine HCl buffer (Thermo Fisher Scientific; cat# 24115) and heated for 2 min at 95 °C. Samples were stored at −20 °C until further analysis. [0234] HPLC analysis was performed using Ion-Pair Reversed Phase (IPRP) UPLC combined with UV detection and mass spectrometry (MS) analysis. IPRP is based on the electrostatic interaction between an oligonucleotide and an ion pairing reagent that is added to the mobile phase, such as tri- or tetra-alkylamine salts, and the hydrophobic interactions between the ion pairing reagent and the non-polar stationary phase. For this method, triethylamine (TEA) and hexafluoro-isopropanol (HFIP) was used as ion-pairing reagent and methanol as organic modifier. UV detection was used at a wavelength of 260 nanometres and MS detection for the identification. Fig.14A shows the chromatograms of A-50GN and Fig.14B shows the chromatograms of A-78GN, clearly indicating the higher stability of A-78GN in comparison to A-50GN, with much more degradation peaks visible in the A-50GN chromatograms. The MS spectra of the peaks in the chromatogram of the degraded A-50GN samples indicate a cleavage at linkage position +10, between the 2’-OMe modified cytidine (Cm) at nucleotide position +10 and the 2’- MOE modified adenosine (Ae) at nucleotide position +11. Fig.15 shows the percentage of stability in a bar diagram relative to the untreated sample, indicating that A-78GN had a stability of 96%, mIdua-85GN of 91%, mIdua-88GN of 88%, and that A-50GN had a
stability of 48%. Without wishing to be bound by theory, the fact that mIdua-85GN is more stable than A-50GN, even though it contains the same chemical modifications in the same positions within the nucleotide sequence as A-50GN, can be explained by the fact that mIdua-85GN contains a different nucleotide sequence with a 2’-OMe modified guanosine at nucleotide position +10 instead of a cytidine. [0235] Stability of an ASO, e.g. GalNAc-conjugated A-50 (A-50GN) can be measured by any other assay. A non-limiting assay includes exposing the ASO to a liver homogenate extract and analyzing the stability of the ASO by HPLC analysis. Example 7. Use of modified oligonucleotides for specific A-to-I editing of human IDUA target RNA in primary human patient fibroblasts. [0236] The skilled person knows that PS linkages improve the stability of ASOs and that PO bonds are more prone to be a target for nucleases. However, it is preferred to use PO bonds instead of PS linkages because it resembles the natural situation better (wherein RNA becomes double-stranded and recruits ADAR for RNA editing) and because of potential toxicity and manufacturing issues. For this reason, it was investigated whether replacing the PO linkages in the 5’ terminal part of A-50 (but not as many as used in A- 78) would improve the stability of A-50 to a level as observed with A-78, especially in respect of the linkage between nucleotide +9 and +10 as discussed above. To study this, and to determine the effect observed at position +10 as outlined above, a further set of ASOs was designed, all with a 3’ attached GalNAc moiety, provided in Fig.16, wherein a variety of positions in the 5’ terminal part of the ASO was either a PS linkage or a PO linkage. The only difference between A-50 GN and A-118 (SEQ ID NO: 113) is that A- 118 comprises a Gm nucleotide (underlined in Fig.16) at position +10, instead of a Cm. This alteration provides the ASO with an increased stability of approximately 35% over the A-50 GN in a stability assay (data not shown), indicating that the cleavage between the Cm at +10 and the Ae at position +11 can be prevented by changing the Cm at position +10 to a Gm. [0237] All ASOs in Fig.16 were tested for RNA editing in primary human W402X fibroblasts (GM00798) followed by RNA isolation and ddPCR analysis as described above in Example 2, in comparison to A-50, A-50GN, and to A-78GN. The results are shown in Fig.17. As observed before in in vitro cell transfection assays, the GalNAc
version of A-50 (A-50GN) underperforms in comparison to the version without the GalNAc (A-50). Notably, when gymnotic uptake is used in these GM00798 fibroblasts, A-50 without the GalNAc moiety outperforms the ASOs carrying the GalNAc moiety (data not shown), indicating that simple cell entry in vitro is easier when the GalNAc is absent. But that does not reflect the situation in vivo. Obviously, the GalNAc moiety is more beneficial in in vivo settings when the liver needs to be targeted specifically. A-121 (SEQ ID NO: 116) and A-127 (SEQ ID NO: 122) performed good and better than A- 50GN in a gymnotic uptake experiments using patient fibroblasts (data not shown). Furthermore, several GalNAc-modified new ASOs outperformed A-50GN in the transfection assay, especially A-118 (SEQ ID NO: 113, discussed above), and further 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 mentioned above, in which apparently the Cm > Gm swap at position +10 is important, also contributes to the increased editing efficiency observed in these cells, reflecting what was observed earlier with A-50 and A-78 in Fig.15. [0238] A-118 and the other optimized versions of A-50 are further tested to determine their RNA editing efficiency in vivo. Notably, ASOs A-128, A-129, A-130, and A-131 all contain three deoxynucleotides in the Central Triplet with the orphan nucleotide in the middle, which further contributes positively to the effect in RNA editing of the target adenosine. Example 8. In vivo study using a variety of ASOs for specific A-to-I editing of mouse Idua target RNA. [0239] Further to the in vivo studies as outlined in Examples 3, 4 and 5, a subsequent longer lasting study was performed in the same W392X Hurler mouse model to study editing efficacy, Iduronidase activity restoration, and reduction of GAG accumulation up to 20 weeks after administration of ASOs. Tissues that were addressed were liver, kidney, spleen, heart, lungs, quadriceps, together with plasma and urine. For this, ASOs mIdua- 85GN (SEQ ID NO: 55), mIdua-75GN (SEQ ID NO: 49) and mIdua-89GN (SEQ ID NO: 58) were compared to their versions without the GalNAc attached (SEQ ID NO: 128, 127,
and 129 respectively). The study setup is provided in Fig.18. An overview of the different names in this study used for the three ASOs is provided in Table 1. [0240] Table 1. ASO referral names, manufacturing (RM) codes, for targeting human IDUA and mouse Idua transcripts, as used in the accompanying examples and drawings. GN refers to a GalNAc moiety. In some embodiments, e.g. Formulae (A), (B), and (C), the GalNAc structure is as disclosed in Formula I as disclosed herein, and Int. Patent Application Publication No. WO2022/271806. a
[0241] All administrations were subcutaneously (SC) except for group 18, wherein the LNP formulation was administered intravenously (IV). All regimens were 1x per week, except for group 18, wherein the LNP formulation was administered only once. Administrations occurred only at T=0 and each week thereafter, with a maximum of 8 administrations. Hence, for example, group 7 received only doses at T=0, 1, 2, 3, 4, 5, 6, and 7 weeks, while group 5 received doses at T=0, 1, 2, and 3 weeks. Group 1 served as a wildtype control, without administering anything. Samples were also drawn from the mice in group 1 as indicated. The strain for groups 2 to 18 was the W392X Hurler mouse model, whereas the strain for group 1 was the wildtype C57BL/6j strain. Group 2 only received the carrier solution, without any ASOs. Necropsy was performed at the last instance of urine/blood sampling, with no administration of ASOs shortly before necropsy. The amount of ASO in groups 11 to 17 was adjusted (to approximately 26 mg/kg) because of the absence of the GalNAc moiety in the compound and the variety in chemistry. The Scr1-GN ASO was as shown in Fig.4. After necropsy, tissues, urine and plasma samples were treated as outlined above, and to determine RNA editing, ddPCR was used as outlined above on RNA isolated from the different tissues. Iduronidase
activity measurement and assessment of reduction in GAG accumulation were also performed as outlined above. [0242] Fig.19A and Fig.19B show the percentage editing in the liver samples at different points in time for the ASOs carrying a GalNac and without a GalNAc, respectively. It appears that the highest percentage of about 5% is reached after 4 weeks in the mice treated with mIdua-85GN, which maintains to about 8 weeks and then slowly declines to approximately 2% after 20 weeks. The ASO’s without the GalNAc provided lower percentages editing. Interestingly, the Iduronidase enzyme activity in liver after administration of mIdua-85GN reaches its peak at 8 weeks with around 21%, which is maintained up to 16 weeks (see Fig.19C). Surprisingly, similar levels were observed in the livers of mice treated with the ASO’s without a GalNAc (see Fig.19D). In these mice (Group 16) mIdua-75 also performed relatively good. In respect of the GAG reduction, mIdua-85GN outperformed mIdua-75GN and mIdua-89GN with a reduction of 65% in comparison to the scrambled control that was maintained up to 20 weeks (see Fig.19E). The effect was also observed in the ‘naked’ versions, albeit not as high as seen with the GalNAc containing ASO’s (see Fig.19F). These results clearly show that the mIdua- 85GN ASO, although the initial editing effect appears to be not extremely high, with a percentage of around 5%, the Iduronidase activity is significantly increased, which correlated well with the editing percentage (R=0,46), and which was maintained for a prolonged period, which subsequently resulted in a significant decrease in GAG accumulation. This shows that the inventors were able to provide in vivo proof of concept that the RNA editing results in a therapeutic relevant effect, using this Hurler mouse model. [0243] The same assays were performed on the kidneys derived from the same mice as described above for the liver samples. There is not a significant difference in effect between the GalNAc containing ASO’s and the ‘naked’ versions, in respect of editing (Fig.20A and Fig.20B, respectively), although mIdua-85GN (again) clearly outperformed the other two ASO’s. The same holds true for the enzymatic activity (Fig. 20C and Fig.20D, respectively) and the GAG reduction which appeared to be around 35% after 20 weeks using mIdua-85GN (see Fig.20E and Fig.20F). [0244] For the spleen samples, only editing was measured and here mIdua-85GN and mIdua-85 outperformed the mIdua-75 and mIdua-89 ASO’s as well, reaching approximately 0.7% after 4 to 8 weeks (see Fig.21A and Fig.21B).
[0245] In the lungs, editing was modest, reaching a maximum of approximately 2% (see Fig.22A and Fig.22B). However, with an enzyme activity reaching approximately 4% (see Fig.22C), the GAG reduction was still quite striking, reaching almost a reduction of 55% in comparison to the scrambled control, and applying the non-GalNAc version of mIdua-85, after 20 weeks (see Fig.22D). [0246] Similar editing results were observed in heart tissue samples of the treated mice (see Fig.23A and Fig.23B), with also around 4% enzyme activity in an early phase (4 weeks with mIdua-85GN, see Fig.23C), and with a relatively varied GAG reduction pattern, with reaching almost 80% reduction observed with mIdua-85GN after 20 weeks (see Fig.23D). [0247] In respect of the quadriceps, a similar pattern was observed, with editing of approximately 1-2% (see Fig.24A and Fig.24B) and with an enzyme activity of approximately 4% at 8 weeks (see Fig.24C). [0248] As outlined in Fig.18, blood and urine samples were drawn at different timepoints and at necropsy. Fig.25 shows the enzyme activity results in the blood samples (plasma derived from cardiac blood) of the different groups at the necropsy stage. In line with the above, mIdua-85GN performed best, with again approximately 3 to 4% enzyme activity at 4 to 8 weeks and compared to the wildtype sample that was set at 100%. [0249] GAG reduction was also measured in urine samples that were taken at different timepoints and at necropsy. Fig.26A and Fig.26B show the GAG reduction at necropsy for the GalNAc containing ASOs and the ASOs without GalNAc, respectively. Whereas there appears to be no effect at the earliest time point of 4 weeks, there is a clear reduction in GAG content from 8 weeks to 20 weeks using all three different ASOs, with mIdua-85GN performing best. While the level that is considered as wildtype (open bars) shows approximately a reduction compared to the negative PBS and scrambled controls of around 60%, mIdua-85GN treated mice showed a reduction of approximately 45% at 12 weeks and maintaining these effects to a reduction of approximately 31 to 33% at 20 weeks, again showing that the GAG reduction is very significant upon repeated treatment with ASOs in which mIdua-85GN performed best. [0250] As shown in Fig.26B, the ASO version without the GalNAc provided a similar result in these urine samples. Fig.26C shows the time course of the effect in GAG reduction in urine samples yielded by mIdua-85GN from T=0 to T=20 weeks compared
to the scrambled control, which was set at 100% (= no reduction). A clear trend starts at 4 weeks and strong reduction is maintained for a prolonged period, up to 20 weeks when the last mice were sacrificed. This is a clear indication that the mIdua-85GN ASO can provide editing of the target transcripts in this Hurler mouse model. It is appreciated that editing percentages may be regarded modest, but this modest editing translates into a very strong effect on enzyme activity and subsequently on a very significant and therapeutic relevant GAG reduction in the urine of these animals. Example 9. In vivo study using intracerebroventricular (ICV) administration of a mouse Idua- specific ASO for specific A-to-I editing of mouse Idua target RNA (ICV study 1). [0251] In this study, it was investigated whether mouse Idua editing could be achieved after intracerebroventricular (ICV) administration of a mouse-Idua specific ASO in the Hurler Idua-W392X mouse model. ICV injection enables one to achieve a broad spread of the ASOs throughout different parts of the brain, such as the cerebral ventricles, where it is administered, and the cerebrospinal fluid (CSF). ICV injection is often used to administer drugs and bypass the blood-brain-barrier (BBB), to administer drugs in the treatment of long-term and/or chronic pain for example, and to administer chemotherapy to treat gliomas. The ASO that was used in this study is mIdua-71 that has the following nucleotide sequence and chemistry: [0252] 5’- Te!TeTeGeAeGm*Am*Cm*Cf*Uf*Cf*Uf*Gf*Uf*m5CeZd*Ad^GmAf*Ge*Uf*Uf*Gf *Uf*Uf *Cf*Um*Cm!Cm-3’ (SEQ ID NO: 215) [0253] wherein the chemical modifications are as provided in Fig.1, Fig.4, and Fig.16. It is noted that mIdua-71 only differs from mIdua-85 (SEQ ID NO: 128) in that the internucleosidic linkage at linkage position -8 is a PS linkage instead of a PNdmi linkage. The study setup is provided in Table 2. [0254] Table 2. Study design for editing a mouse Idua transcript in the Hurler model Idua-W392X after ICV injection with mIdua-71 ASO. The aCSF samples are from non- treated animals and served as negative controls (n.a. = not applicable). Necropsy was performed 2 weeks after treatment (6 animals) and 4 weeks after treatment (6 animals).
[0255] Besides editing, the distribution of ASOs in the brain after ICV injection was studied. After sacrifice, the brains were dissected and split into two hemispheres, wherein one hemisphere was used for FISH staining, whereas the other hemisphere was dissected into the parts representing the frontal cortex (further split in two parts), hippocampus, cerebellum, and midbrain. One part of the frontal cortex was used for protein analysis (Iduronidase activity assay), whereas the other half, the hippocampus, cerebellum, and midbrain were used for a ddPCR assay to determine editing. RNA isolation, cDNA manufacturing and ddPCR assays were performed for the mIdua target sequence as disclosed above. FISH staining on the brain sections indicated that the mIdua-71 ASO was well distributed across the brain after ICV administration (data not shown). [0256] Table 3 shows the results of the ddPCR in the frontal cortex, hippocampus, cerebellum, and midbrain in the four groups of mice (see Table 2), in which the aCSF samples from non-treated mice (Groups 1 and 3) served as controls. [0257] Table 3. Editing percentages as determined by ddPCR in the frontal cortex, hippocampus, cerebellum and midbrain of W392X mice after treatment with an ASO according to Table 2.
[0258] An average editing level of approximately 25% editing was observed in the frontal cortex of the mice treated with 250 μg mIdua-71 after 4 weeks (Group 4). A similar level was observed in the hippocampus and slightly lower levels in cerebellum and midbrain. In group 2, in all tissues, the animals reached higher average editing levels after 4 weeks than after 2 weeks. No editing was observed in the non-treated animals, with one exception: an outlier in one animal in group 1, after 2 weeks (of no treatment). No significant differences in editing percentages were observed between the brain sections from the 250 μg treated mice when compared to mice that were treated with 100 μg ASO (data not shown). [0259] As indicated above, Iduronidase activity was also determined in the same mice. Table 4 shows the results of the Iduronidase activity assay in the mice treated with 250 μg mIdua-71 (Group 2 and 4) in comparison to the non-treated animals (aCSF; Groups 1 and 3), see Table 2. [0260] Table 4. Iduronidase activity (in nmol/24h/mg) in the frontal cortex, hippocampus, cerebellum and midbrain of W392X mice after treatment with an ASO according to Table 2. 2
6
9 1
[0261] The protein activity generally follows the editing results in these mice with the highest Iduronidase activity (average 12 nmol/24hr/mg) observed in the frontal cortex after 4 weeks of treatment. With slightly lower activity levels in the hippocampus, cerebellum and midbrain, albeit at a significantly higher level than the background observed in the non-treated animals that all displayed a base activity of approximately 1-2 nmol/24hr/mg. Example 10. In vivo studies using ICV administration of a mouse Idua-specific ASO for specific A-to-I editing of mouse Idua target RNA. [0262] ICV study#2: This study investigated whether mouse Idua editing could be achieved after ICV administration of 250 μg mIdua-85 (SEQ ID NO: 128; RM4347) and 250 μg or 400 μg mIdua-85GN (SEQ ID NO: 55; RM4870), that are both mouse-Idua specific ASO’s, in the Hurler Idua-W392X mouse model. Editing was determined in the frontal cortex, hippocampus, cerebellum and midbrain (as in Example 9) and also in the brain stem, and the spinal cord (cervical, thoracic, and lumbar). [0263] While ICV injection enables broad distribution of ASOs through the CNS, much of the payload delivered via this route of administration ultimately escapes into peripheral circulation. Thus, in addition to CNS tissue, this study also assessed peripheral tissue (e.g. liver) for editing, iduronidase activity, and GAG accumulation. Further, this study compared activity of an unconjugated ASO and a ASO conjugated to GalNAc when delivered via ICV administration. The study design is provided in Table 5. [0264] Table 5. Study design for editing a mouse Idua transcript in the Hurler model Idua-W392X after ICV injection with mIdua-85 and mIdua-85GN ASO’s. The aCSF samples (Group 1) are from non-treated animals and served as negative controls (n.a. = not applicable). Mice were sacrificed 4 weeks after treatment. GN is the GalNAc structure as disclosed in Formula I of Int. Patent Application Publication No. WO2022/271806.
[0265] Table 6 shows the editing results obtained after ddPCR in the different brain tissues after 4 weeks upon treatment of the indicated ASO’s in Table 5. [0266] Table 6. Editing percentages as determined by ddPCR in the (frontal) cortex, hippocampus, cerebellum, midbrain, brain stem, the cervical (upper), thoracic (mid) and lumbar (lower) parts of the spinal cord, and the liver of W392X mice after treatment with an ASO according to the study design in Table 5. The value of 0.3 to 0.5 observed in Group 1 in a variety of tissues is background level.
[0267] As can be observed in Table 6, the percentages of editing observed in the liver, either with the unconjugated mIdua-85 ASO (Group 2) or the GalNAc-conjugated mIdua- 85 ASO (Groups 3 and 4), is higher than the editing percentages of the background (Group 1). Consistent with the ICV study shown in Example 9, significant editing percentages were measured in all brain tissues (and in the spinal cord) with the unconjugated mIdua-85 ASO that outperformed the GalNAc-conjugated version mIdua- 85GN. This is not completely unexpected, since GalNAc is generally used to target hepatocytes, not brain cells. [0268] In these same groups, Iduronidase activity was measured (as outlined above) in the cortex, hippocampus, cerebellum, midbrain, brain stem, the cervical part of the spinal cord, and the liver. These results are provided in Table 7. [0269] Table 7. Iduronidase activity (in nmol/24h/mg) in the (frontal) cortex, hippocampus, cerebellum, midbrain, brain stem, the cervical part of the spinal cord, and the liver of W392X mice after treatment with an ASO according to Table 5. 3 9 2 4 4 7 8 7 3 6 5 4
8
1 7 3 5 1 8 6 2 1 4 6 6 9 4 4
[0270] As observed in the study outlined in Example 9, it appeared possible to restore the Idua activity in all brain tissues that were examined, and it was found that this Iduronidase activity in general follows the editing results in the different tissues, as determined through ddPCR and as provided in Table 6. Strikingly, also the Iduronidase activity in the liver of these mice that received the ASO’s through ICV administration, appeared to be significantly increased, although no real difference was observed between the mIdua-85 and mIdua-85GN treated animals. To determine whether GAG accumulation was reduced in the liver of these ICV-treated mice, this was also determined according to the protocols outlined above. The values of these determinations are provided in Table 8. [0271] Table 8. GAG accumulation (in μg/mg tissue) in the liver of W392X mice after treatment with an ASO according to Table 5. 9 2 4 1
[0272] As seen in Table 8, GAG accumulation in the liver appeared to be reduced with approximately 30% in W392X mice after treatment with mIdua-85 (Group 2) and mIdua-
85GN (Groups 3 and 4) in comparison to the negative control (Group 1), even though the editing percentages in the liver, as shown in Table 6, appeared relatively modest. This shows that even after ICV administration of an ASO targeting the mouse Idua transcript, the ASO can bring about editing in the brain tissues that are close to the administration site, but also in the liver, and importantly, yield a downstream effect as observed in the reduced accumulation of GAGs in the liver. [0273] ICV study #3: Another study investigated ICV administration of mIdua-85 without GalNAc conjugation in W392X mice. The study included analyzing the effect of 250 μg of unconjugated mIdua-85 (without GalNAc) that was administered ICV, in comparison to mice that were untreated (control group). Necropsy was on day 28 (4 weeks) after treatment. Each group (control and 250 μg mIdua-85 group) comprised 5 mice. Editing was determined in the frontal cortex, hippocampus, cerebellum, midbrain, brain stem, striatum, liver, and quadriceps, using ddPCR and following the protocols as outlined above. The results of the editing determination are provided in Table 9. [0274] Table 9. Editing percentages as determined by ddPCR in the (frontal) cortex, hippocampus, cerebellum, brain stem, midbrain, striatum, liver, and quadriceps of W392X mice, 4 weeks after treatment with 250 μg mIdua-85 (5 animals) and non-treated (NT) mice (5 animals) that served as a negative control. 2 8 1 3 2 8 3 9 1 1 2 5 2 1 4 4 1 6
6 2 3 8 4 9 4 8 4 9 4 8 2 3 6 3 6 6 2 1 9 9
[0275] In line with what was observed in the earlier ICV study #1, in which mIdua-85 was compared to mIdua-85GN, significant editing was observed in all brain tissues upon ICV injection of 250 μg ASO, in all five treated animals, with editing levels above 20%. Editing was also significantly higher in the liver and quadriceps in the mIdua-85 treated animals in comparison to the non-treated animals. [0276] Also in these animals, the Iduronidase activity was measured, according to methods described in the two earlier ICV studies above, in the tissues that were studied for RNA editing. The results are provided in Table 10. [0277] Table 10. Iduronidase activity (in nmol/24h/mg) in the (frontal) cortex, hippocampus, cerebellum, brain stem, midbrain, striatum, liver, and quadriceps of W392X mice 4 weeks after treatment with 250 μg mIdua-85 (5 animals) and non-treated (NT) mice (5 animals) that served as a negative control. 7 2 6
5 7 5 9 2 9 1 7 4 5 1 9 5 4 9 5 5 4 7 4 7 9 7 8 2 3 8 5 6 6 4 1 9 5 3 9 1
[0278] Table 10 shows that Iduronidase activity was restored in the brain tissues of mIdua-85 treated W392X mice, whereas also significant Iduronidase activity could be detected in the liver and quadriceps of these ICV treated animals. No significant
Iduronidase activity was observed in any of the tissues in the non-treated animals, showing that editing – as observed earlier – is followed by an increased Idua protein activity, which can be observed at least up to 4 weeks after initial treatment. Example 11. Intrathecal delivery of an editing nucleotide. [0279] This example describes a non-human primate (NHP) study delivering an editing guide oligonucleotide via intrathecal (IT) route in cynomolgus monkeys. The animals were administered with an editing guide oligonucleotide (“test article”) according to the study design shown in Table 11. In this example the test article is a guide oligonucleotide according to SEQ ID NO: 208, targeting Beta-actin transcripts. The study shows editing efficiency and exposure after IT delivery. SEQ ID NO: 208 represents: [0280] 5’- Gm!Am*Am*Am*Gm*Cf*Am*Af*Um*Gf*m5Ce*Zd*Ad^Um*Cf*Ae*Cf*Cm *Uf*Cm*Cf*Cm*Cm*Um!Gm-3’ [0281] wherein the modifications are as provided in Fig.1, Fig.4, and Fig.16. [0282] Methods [0283] 1. Animals. Twenty drug-naïve female cynomolgus monkeys had an IT catheter implanted in the lumbar region to facilitate test article administration. [0284] 2. Reagents. Test article consisted of a single-stranded editing oligonucleotide according to SEQ ID NO: 208. [0285] 3. In-Life Study Design. Dose groups and terminal necropsy time points are depicted in Table 11. Test article was administered via IT catheter at total doses, volumes, and concentrations depicted in Table 11. Assessment of toxicity was based on mortality, clinical observations, body weights, qualitative food consumption, neurobehavioral observations, and clinical and anatomic pathology. [0286] 4. Tissue Collection. Serial plasma and cerebrospinal fluid (CSF) collection was performed on dosing days for up to 360 hours post-dose. Terminal tissue collection was performed on the necropsy day indicated in Table 11. Tissues (when present) from each animal were preserved, as indicated in Table 12. [0287] 4.1 Histology. All tissues denoted by “E” in Table 12 (Necropsy, Organ Weights, and Macroscopic Observations section) from each monkey were incubated in 10% neutral buffered formalin (NBF) for at least 24 hrs, but no more than 28 hrs, at RT. Upon
removal from NBF, tissues were rinsed three times with 1x PBS and stored in PBS at 4ºC for up to 72 hrs until processed to paraffin block. Once processed to paraffin block, tissues were sectioned at a nominal 5 micron, and slides were prepared and stained with haematoxylin and eosin. For spinal cord (mid-thoracic and mid-lumbar), one transverse and one transverse oblique section (including the injection site) were taken. For spinal cord (mid cervical) one transverse and one parasagittal longitudinal section was taken. [0288] 4.2 Microscopic Observations. All tissues denoted by “E” in Table 12 (Necropsy, Organ Weights, and Macroscopic Observations section) from all animals were examined microscopically. Following completion of the primary microscopic evaluation, an independent peer review evaluation was performed. [0289] 4.3 Frozen Tissue Collection for Exploratory Analysis. After the collection of any of the following tissue samples for microscopic evaluation, the following samples were collected from each monkey: [0290] Brain, left frontal cortex; Brain left, temporal cortex; Brain, left caudate; Brain, left putamen; Brain, left hippocampus; Brain, left thalamus; Brain, left medulla; Brain left, pons; Brain left, substantia nigra; Brain left, amygdala; Brain left, parietal cortex; Brain left, cerebellum; Dorsal Root Ganglion, lumbar (to target L5); Dorsal Root Ganglion, thoracic; Dorsal Root Ganglion, cervical; Heart, apex; Kidney, left cortex; Liver, left lateral lobe; Sciatic, left; Spinal cord, cervical; Spinal cord, thoracic; Spinal cord, lumbar; Spleen; Superior cervical ganglion, left. [0291] For the left brain hemisphere for each brain region (frontal cortex, temporal cortex, caudate, putamen, hippocampus, thalamus, medulla, pons, substantia nigra, amygdala, parietal cortex, and cerebellum), up to four samples (25 to 100 mg each) were collected and placed in a pre-chilled, 2 mL, RNase-free tube; snap frozen in liquid nitrogen; and placed on dry ice until transferred to a freezer, set to maintain 60 to 80°C. All sample weights were recorded. [0292] For the spinal cord region (cervical, thoracic, and lumbar), up to four samples (25 to 100 mg each) were collected and placed in a pre-chilled, 2 mL, RNase-free tube; snap frozen in liquid nitrogen; and placed on dry ice until transferred to a freezer, set to maintain -60 to 80°C. All sample weights were recorded. [0293] For the dorsal root ganglions (DRG), four DRG (two bilateral pairs) from each region (cervical, thoracic, and lumbar) were collected, weighed, and had nerve roots trimmed out. Each bilateral pair was placed in a pre-chilled, 2 mL, RNase-free tube. The
left superior cervical ganglion was sampled whole, weighed, and placed into a pre- chilled, 2 mL, RNase-free tube. Each sample was snap frozen in liquid nitrogen and placed on dry ice until transferred to a freezer, set to maintain -60 to 80°C. All sample weights were recorded. [0294] For the liver (left lateral lobe), spleen, heart (apex), left kidney (cortex), and left sciatic nerve sample, up to four samples (25 to 100 mg each) of each tissue were collected and placed in a pre-chilled, 2 mL, RNase-free tube; snap frozen in liquid nitrogen; and placed on dry ice until transferred to a freezer, set to maintain -60 to 80°C. All sample weights were recorded. [0295] 1. RNA Isolation. RNA was extracted from exploratory tissue lysates for exploratory pharmacodynamic endpoints (e.g. RNA editing efficiency). Frozen tissue chunks were transferred to 1 ml of cold Trizol (Life Technologies) and immediately homogenized using a Qiagen Ruptor or OMNI Soft Tissue Disruptor on ice (high speed for 10-30 sec, depending on the tissue). Homogenates equivalent to up to 35 mg of tissue weight (25 mg for liver, kidney, and spleen) were diluted with Trizol to a volume of 1 ml at RT. 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. The mixture was subjected to RNA isolation using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. DNase on-column treatment was applied. RNA was eluted with 30 µl of RNase-free water. RNA concentration and quality were determined using an Agilent Bioanalyzer 4200 with RNA ScreenTape (Agilent), and RNA samples were stored at -80°C. [0296] 2. cDNA Synthesis. A 0.5 µg 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 make a final volume of 15.5 µl. The solution was incubated at 65°C for 5 min and 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 thermocycler 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, cDNA samples were diluted with RNase- and DNase-free water at a 1:80 ratio.
[0297] 3. Digital droplet PCR.1 µl of cDNA sample was used to perform digital droplet PCR (ddPCR) with ddPCR Multiplex Supermix (4x) (BioRad) in a reaction volume of 20 µl. ddPCR probes and primers were custom-made by IDT. [0298] The sequences of probes are as follows: [0299] /56-FAM/AG GTG A+T+G +GCA TTG CTT TCG T/3IABkFQ/ (SEQ ID NO: 209) and [0300] /5HEX/AG +GTG A+T+A +GCA TTG CTT TCG TGT /3IABkFQ/ (SEQ ID NO: 210). [0301] The sequences of primers are as follows: [0302] 5’-AGT CCT CTC CCG AGT CCA CA-3’ (SEQ ID NO: 211) and [0303] 5’-GGG GCA TGA AGG CTC ATT ATT CAA-3’ (SEQ ID NO: 212). [0304] A final concentration of 250 nM (probes) was used. A final concentration of 500 nM (primers) was used. Droplets were generated on a Bio-Rad Automated Droplet Generator (BioRad). PCR was performed in a C1000 Touch™ Thermal Cycler (BioRad) using the following conditions: 95°C for 10 min for 1 cycle; 95°C for 30 sec and 64°C for 1 min for 40 cycles; 98°C for 10 min for 1 cycle; and 4°C indefinitely. After the reaction was completed, droplets were analysed on a QX600 Droplet Reader (BioRad). Data was acquired and analysed using BioRad QX manager Standard Edition v.2.20 software. FAM-positive, HEX-positive, and FAM/HEX-positive populations were gated in the 2D plot of channel 1 and channel 2 amplitude. Editing efficiency percentage was calculated using the equation: G(cp/µl) / (G(cp/µl) + A(cp/µl)) x 100%. [0305] Table 11. NHP study design of different doses, timepoints and repeat dosing. Group 1 did not receive the test article, but was treated in line with the other animals, and served as negative control.
[0306] Table 12. Organ/tissue collection.
[0307] Results [0308] The editing efficiencies measured across various tissues are provided in Table 13. [0309] Table 13. Editing efficiency measured across tissues.
[0310] Exposure analyses. Tissue Materials, Methods, and Data from HPLC/HRMS Analysis. [0311] To analyse test article delivery to tissues and understand exposure response relationships, tissue samples were collected at timepoints corresponding to the editing measurements, and the exposure of the total guide oligonucleotide in tissue was quantified by HP-LC/MS. Briefly, tissue standards were prepared in control tissue homogenate. To control assay variability, an internal standard was added to all standards and samples.
[0312] Tissue samples were homogenized in cell lysis buffer. For total guide oligonucleotide measurements, tissue standards and samples were digested with proteinase K prior to being loaded onto an Oasis Wax micro-elution solid phase extraction (SPE) plate (Waters Inc, Milford, MA) for isolation. The SPE plate was washed with wash buffers and then analytes were eluted with elution buffer. Eluants from the SPE plates were dried, reconstituted, and injected onto an LC/MS system. [0313] The total guide oligonucleotide concentrations were measured using a Thermo Orbitrap Exploris 240 (Thermo Scientific, San Jose, CA) mass spectrometer using the guide oligonucleotide peak for quantification. The mass spectrometer was operated in negative ion detection mode. All data were processed using Xcalibur version 4.4 (Thermo Scientific, San Jose, CA). [0314] Table 14 shows that animals dosed intrathecally with test article had measurable exposure in multiple central nervous system regions. The variability in brain exposure in some groups is likely indicative of incomplete IT dosing. Peripheral tissues (liver and kidney) were also exposed to test article, indicating a significant amount of IT dosed compound reaching the systemic circulation. Kidney concentrations were consistently higher than liver. [0315] Table 14. Summary of exposure data. r 1 5 1 8 4 4 1 5 6 1
3 1 3 5
[0316] Taken together, the ICV studies outlined in Example 9 and 10, and the IT study in Example 11 demonstrate that direct administration into the brain (in mice by ICV administration; in non-human primates and humans by intrathecal (IT) administration) is a feasible way of getting an RNA editing ASO at the cell of interest, especially brain cells. Furthermore, upon ICV administration, RNA editing ASO is delivered to peripheral tissues, e.g. the liver and muscle cells, and this is achieved with both conjugated and unconjugated ASO, i.e. in the absence of a GalNAc conjugation.