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WO2025073902A1 - Optimisation chimique et de séquence d'oligonucléotides antisens pour édition d'arn médiée par adar - Google Patents

Optimisation chimique et de séquence d'oligonucléotides antisens pour édition d'arn médiée par adar Download PDF

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WO2025073902A1
WO2025073902A1 PCT/EP2024/077956 EP2024077956W WO2025073902A1 WO 2025073902 A1 WO2025073902 A1 WO 2025073902A1 EP 2024077956 W EP2024077956 W EP 2024077956W WO 2025073902 A1 WO2025073902 A1 WO 2025073902A1
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modified
oligonucleotide
nucleotides
sequence
mismatch
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Inventor
Thorsten Stafforst
Laura Sophia PFEIFFER
Jin Billy Li
Inga JARMOSKAITE
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Eberhard Karls Universitaet Tuebingen
Leland Stanford Junior University
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Eberhard Karls Universitaet Tuebingen
Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present invention relates to the field of site-directed RNA editing, whereby an RNA sequence is targeted by an antisense oligonucleotide (ASO) for RNA editing of a genetic mutation (“compensatory editing” for repair of disease- causing G-to-A mutations) or for editing of an RNA derived from a wildtype allele to introduce de-novo A-to-G variants (“beneficial editing”).
  • ASO antisense oligonucleotide
  • RNA editing is a natural process through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule in a site- specific way. Unlike DNA editing, the advantage of site-directed RNA editing is that it allows modification of the genetic information in a more precise and efficient manner. Contrary to DNA, RNA is generally quickly degraded and any errors introduced by off-target modifications will be washed out rather than permanently introduced into the modified DNA of a subject. RNA editing may also be less likely to cause an immune reaction since it is an editing mechanism naturally found in humans. Moreover, RNA editing might provide a more natural response than introducing an external, engineered gene.
  • oligonucleotide therapeutics have been developed to silence, restore or modify the expression of disease-causing or disease-associated genes in, e.g., cancer and (other) genetic disorders.
  • Such therapeutics include, e.g., antisense oligonucleotides (ASOs), small interfering RNA (siRNA) and microRNA (miRNA) that interfere with coding and noncoding RNAs in a sequence specific manner.
  • ASOs antisense oligonucleotides
  • siRNA small interfering RNA
  • miRNA microRNA
  • SDRE Site-Directed RNA Editing
  • ADAR1 and hADAR2 are expressed in most tissues and encode active deaminases.
  • Human ADAR3 (hADAR3) has been described to only be expressed in the central nervous system and reportedly has no deaminase activity in vitro. Accordingly, the ADAR may be hADAR1, hADAR2 or hADAR3, or any variant thereof.
  • the ability of ADARs to alter the sequence of RNAs has also been used to artificially target RNAs in vitro in cells for RNA editing. Endogenous ADAR proteins can be directed to edit RNA targets of interest by providing short target specific antisense oligonucleotides (ASOs). However, the optimal design of ADAR recruiting ASOs remains to be determined.
  • ASOs are generally short single-stranded synthetic RNA or DNA molecules, which use Watson-Crick base pairing to bind sequence specifically to the target RNA. They can be broadly classified into 1 st (Gen 1), 2 nd (Gen 2), and 3 rd (Gen 3) generation ASOs.
  • Gen 1 ASOs were initially employed to inhibit translation of Rous sarcoma virus ribosomal RNA (Stephenson and Zamecnik, 1978). They are characterised in having a modified backbone, wherein the nucleotide linkages are modified by sulphur, methyl or amine groups to generate phosphorothioates (PS), methyl- phosphonates, and phosphoramidates, respectively.
  • Gen 2 ASOs show increased nuclease stability and affinity for their RNA targets, which has translated to improved potency and therapeutic index in the clinic.
  • Gen 2 ASOs are typically modified using PS backbone modification and additionally carry alkyl modifications at the 2’ position of the ribose.
  • Such 2’-sugar modifications may include 2’-O-methyl (2’-OMe), 2’-fluoro (2’-F), 2’-O-methoxyethyl (2’-MOE) modifications.
  • these Gen 2 ASOs tend to be less toxic than PS-modified ASOs and have a slightly higher affinity for their target.
  • Gen 3 ASOs tend to be even more heterogenous as they include a large number of chemical modifications that aim to further improve binding-affinity, stability, and pharmacokinetics (Quemener et al., 2019).
  • the diversity of chemical modifications, together with the sequence of the ASO offers considerable flexibility as relates to the therapeutic approach.
  • ASOs can be used to degrade target mRNA, decrease protein levels, modify or correct splicing events, modulate RNA translation or target pathological coding or non-coding RNAs (Quemener et al., 2019).
  • their sequences are generally complementary or at least partially complementary to the target RNA.
  • site- directed mutagenesis i.e., “A-to-I” RNA editing
  • the ASO targeting domain contains a mismatch opposite the targeted adenosine.
  • ASO-based therapies have been gaining more traction over the past years for use in the treatment of different genetic disorders.
  • Oligonucleotide constructs for site-directed RNA editing are described in WO 2016/097212 and WO 2017/010556, which utilise endogenous cellular pathways, i.e., endogenous ADAR, to edit endogenous RNA.
  • ASOs can be chemically modified to improve their properties. For instance, ASOs can be modified to protect them against nucleases and to increase their effectiveness.
  • PS linkages can be found in two stereoisomers, Rp and Sp, and it is known from the art, that Rp and Sp linkages can influence properties such as, e.g., thermal stability, binding affinity, pharmacologic properties, etc., of the ASO.
  • Rp and Sp linkages can influence properties such as, e.g., thermal stability, binding affinity, pharmacologic properties, etc., of the ASO.
  • ASOs are typically very rich in 2’-F-modifications within the 5’ half, which are generally present as blocks of 2’-F-modifications and uniform block of 2’-O-Methyl- modifications within the 3’ terminus on either side of the CBT. That precise, site- specific RNA editing can be achieved by endogenous ADARs has previously been shown by Merkle et al. (2019). They were able to demonstrate that chemically optimized ASOs can be used to recruit endogenous ADARs to edit endogenous transcripts in a simple and programmable way with almost no off-target editing.
  • RNA-editing oligonucleotides comprising a first and second domain, wherein the first domain comprises one or more 2’-F modifications and the second domain comprises one or more sugars that do not have a 2'-F modification.
  • WO 2022/099159 relates to oligonucleotides with a first and second domain, wherein the domains comprise specific percentages of 2’-F modifications and aliphatic substitutions.
  • WO 2018/041973 relates to single-stranded RNA-editing oligonucleotides for the deamination of a target adenosine by an ADAR enzyme whereby the central base triplet (CBT) of three sequential nucleotides comprises a sugar modification and/or a base modification. It was found that deoxyribose at all three positions of the CBT is well tolerated and provides substantial stabilization against nuclease digestion.
  • ASO optimisation for A-to-I editing has led not only to the identification of a CBT but also to a more thorough investigation of the region immediate 5’ and 3’ to the CBT.
  • WO 2021/243023 describes modifications 3’ to the CBT (at position +2 of an oligonucleotide comprising the structure [Am]-X 1 -X 2 -X 3 -X 4 -[Bn], wherein X 4 corresponds to the +2 position). It was found that editing the +2 position can affect the editing rate of the target. Improved editing was observed with a 2’-F modification at the +2 position.
  • PCT/EP2017/065467 describes that additional mismatches in the formed dsRNA, caused by nucleotides in the oligonucleotide that do not form perfect base pairs with the target RNA are tolerable but not essential for specific targeted editing of the target RNA sequence.
  • a method for site-directed A-to-I editing of a target nucleic acid comprising providing to a cell or subject an oligonucleotide according to the invention, or composition according to the invention.
  • a method of treating a disease or condition associated with a point mutation in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide according to the invention, or a composition according to the invention.
  • Linkages may be continuous (consecutive; several in a consecutive order) or discontinuous (interrupted).
  • discontinuous or “interrupted” means that there are not more than, e.g., 7, 8, 9, 10 or more consecutive internucleoside linkage modifications of the same modification.
  • the naturally occurring PO linkages are replaced by modified internucleoside linkages.
  • the linkage is a non-natural internucleoside linkage.
  • stereopure or “stereorandom” refers to chemically modified oligonucleotides.
  • the term “stereopure” refers to oligonucleotides that are chirally pure (or “stereochemically pure”).
  • the term “stereorandom” refers to racemic (or “stereorandom”, “non-chirally controlled”) oligonucleotides.
  • the oligonucleotides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom internucleoside linkages (mixture of Rp and Sp linkage phosphorus at the internucleoside linkage, e.g., from traditional non-chirally controlled oligonucleotide synthesis).
  • an internucleoside linkage is a phosphorothioate (PS) linkage.
  • an internucleoside linkage is a stereorandom PS linkage. In one embodiment, an internucleoside linkage is a chirally controlled PS linkage. In one embodiment, an internucleoside linkage is not chirally controlled. In one embodiment, an internucleoside linkage is not a chirally controlled PS linkage.
  • the term “antisense oligonucleotide” or “ASO” refers to a strand of nucleotide analogue that hybridizes with the complementary (target) RNA in a sequence-specific manner. This may be via Watson-Crick base pairing. The ASO may be chemically modified. The ASO may be sequence modified.
  • RNA refers to an RNA, which is subject to the editing process, and “targeted” by the respective ASO(s) or composition(s) of the invention.
  • off-target or “off-targeting” refers to non-specific and/or unintended genetic modification(s) of the target. Off-target editing may include unintended point mutations, deletions, insertions, inversions, and translocations. For instance, off-target editing may arise from the promiscuous reactivity of the deaminase enzymes.
  • a modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.).
  • a modified sugar is an LNA sugar.
  • LNA locked nucleic acid
  • LNAs locked nucleic acids
  • BNA bridged nucleic acid
  • a sugar modification is 2’-OMe, 2'-O-methoxy-ethyl (2’-MOE), 2’-F, 5’-vinyl, or S-constrained ethyl (S-cEt).
  • a 2’-modification is a C2-stereoisomer of 2’-F-ribose.
  • a 2’-modification is a 2’-O-alkyl modification.
  • the 2’-O-alkyl modification is a 2’-O-methyl-, 2’-O-ethyl-, 2’-O-propyl-, or 2'-MOE modification.
  • a 2’-modification is 2'-OMe.
  • a 2'-modification is 2'-MOE.
  • a 2'-modification is 2'-OR, wherein R is substituted C1-10 aliphatic.
  • the expression “a derivate thereof” refers to a corresponding nucleotide(s) or oligonucleotide(s) that has been chemically derived from said nucleotide or oligonucleotide(s).
  • the term “complementary” or “substantially complementary” refer to nucleic acid sequences, which, due to their complementary nucleotides, are capable of specific intermolecular base-pairing.
  • the oligonucleotide may comprise a nucleic acid sequence complementary to a target sequence, e.g., SERPINA1, or any other target sequence.
  • the ASO may be self-complementary.
  • beneficial editing refers to the editing of a target sequence (or base) derived from a wildtype allele (not a mutated allele) in order to, e.g., modulate the function of a wildtype protein in a useful way to prevent or treat a disease.
  • beneficial editing may include sites, such as STAT1 Y701, that are not causes for genetic diseases but rather represent wildtype protein sites.
  • RNA editing refers to the modification of RNA nucleotides to change and correct one or more detrimental or unfavourable changes in the RNA sequence when compared to wildtype, e.g., a compensatory A-to-I change could help to functionally compensate for an otherwise non-editable mutation to ameliorate a disease phenotype.
  • adenosine deaminase(s) or “adenosine deaminase(s) acting on RNA” [ADAR(s)] refers to any (poly)peptide, protein or protein domain or fragment thereof capable of catalysing the hydrolytic deamination of adenosine to inosine.
  • ADAR(s) adenosine deaminase acting on RNA
  • ADAR(s) refers to any (poly)peptide, protein or protein domain or fragment thereof capable of catalysing the hydrolytic deamination of adenosine to inosine.
  • the term thus not only refers to full-length and wild type ADARs but also to a functional fragment or a functional variant of an ADAR.
  • the term “effective amount” in the context of administering a therapy to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s).
  • the term “in combination” in the context of the administration of two or more therapies to a subject refers to the use of more than one therapy (e.g., more than one prophylactic agent and/or therapeutic agent). The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. For instance, one or more ASOs may be used in combination.
  • the terms “prevent”, “preventing” and “prevention” refer to the inhibition of the development or onset of a disease or symptoms thereof.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • the terms “subject” or “patient” are used interchangeable and relate to an animal (e.g., mammals) that may need administration of the compound of the invention in the field of human or veterinary medicine.
  • the subject is a human.
  • the subject may be administered the oligonucleotide of the invention for beneficial editing.
  • the subject may be administered the oligonucleotide of the invention for compensatory editing.
  • pharmaceutically acceptable means approved by a regulatory agency.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the formulation should suit the mode of administration.
  • oligonucleotides of the invention may comprise different types of chemical modifications, e.g., 2’-sugar modifications and internucleoside linkage modifications, along with nucleotide sequence modifications to introduce mismatches relative to the target RNA. Oligonucleotides according to the claims promote increased target editing yields.
  • the oligonucleotides according to the claimed invention may have a mixture of different modifications at the 2’-position of the sugar residue but preferably not stretches of more than 6 nucleotides with the same 2’-modification.
  • Such oligonucleotides may show enhanced editing, specifically when the mismatches are at position N -7 , at position N -9 or at positions N- 9 and N +2 and wherein mismatches at N -7 or N -9 are placed in the context of a G or C 3’ to the mismatch and an A or U 5’ to the mismatch.
  • these features confer high levels of lysosomal stability and RNA editing efficacy of the oligonucleotides.
  • the oligonucleotides of the invention are modified and designed accordingly.
  • the core oligonucleotide comprises the sequence: 5’- a N b c d +4 N+3 N+2 N+1 e N f 0 N g -1 N h -2 N i -3 N j -4 N k -5 N l -6 N m -7 N n -8 N-9 -3’ ; and contains a specific patterns of 2’-modification and internucleoside linkages, as well as nucleotide sequence modifications, which contribute to the advantageous properties of the oligonucleotides.
  • the oligonucleotide is suitable for A-to-I editing and comprises: (I) a nucleic acid sequence of 25 to 70 nucleotides (nt) that is an antisense sequence substantially complementary to the target nucleic acid, (II) a central base triplet (CBT) (5’- N+1 e N0 f N-1 g -3’) with the central nucleotide (N0) directly opposite the target adenosine to be edited when the antisense oligonucleotide is hybridised to the target nucleic acid, and (III) the following core sequence: 5’- a N +4 b N +3 c N +2 d N +1 e N 0 f N -1 g N -2 h N -3 i N -4 j N -5 k N -6 l N -7 m N -8 n N -9 -3’ ; and wherein: (a) at least two of the three nucleot
  • the mismatch is at position -1 (N-1).
  • the target sequence is SNCA.
  • the target sequence is SNCA A53T.
  • the oligonucleotides of the invention do benefit from having a base level of internucleoside linkage modifications, and oligonucleotides having at least 15% modification are preferred as this is beneficial to achieve good RNA editing.
  • the linkage modification content is at least 15 %; and linkages h and i are not phosphorothioate (PS) linkages.
  • nucleotides can tolerate high percentages of 2’- modifications without detrimental loss of activity.
  • 20- 100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100% , 80-100%, or 90-100% of nucleotides are deoxyribonucleosides (DNA) or 2’-modified.
  • 20- 100% of nucleotides are DNA or 2’-modified.
  • 50-100% of nucleotides are DNA or 2’-modified nucleotides.
  • nucleotides 100% of nucleotides are DNA or 2’-modified nucleotides. In one embodiment, 30-95%, 40- 95%, 40-90%, 50-95%, 50-90%, 60-95% or 60-90% of nucleotides are DNA or 2’- modified nucleotides. In one embodiment the above percentages are satisfied with only 2’-modified nucleotides and no DNA.
  • the oligonucleotides comprise modifications at the 2’-position on nucleotides using different modifying groups. In one embodiment, 20-70% of nucleotides are 2’-F-modified. In one embodiment, 35-65% of nucleotides are 2’-F-modified.
  • nucleotides 20-60% of nucleotides are 2’-O-methyl (2’- OMe)-modified. In one embodiment, 25-55% of nucleotides are 2’-OMe-modified.
  • the modified oligonucleotides of the invention do not require all of the internucleoside linkages to be modified, provided that a minimum level of internucleoside modification is incorporated and provided that linkages d and e of the core oligonucleotide sequence are modified. In one embodiment, the internucleoside linkage modification content is at least 15 %.
  • the internucleoside linkage modification content is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90%. In one embodiment, no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, or 30% of the linkages are internucleoside linkage modifications.
  • Internucleoside linkage modifications such as PS linkages, tend to have a positive effect on the pharmacokinetics as well as stability, protein binding, and intracellular localization of ASOs. However, at the same time, it is desirable to reduce overall PS content to reduce, e.g., toxicity and non-specific protein binding.
  • (a) no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the linkages outside the CBT are internucleoside linkage modifications; or (b) 15-90% of the linkages are internucleoside linkage modifications, preferably wherein 40-80%, most preferably 45-60%, of the linkages are internucleoside linkage modifications. In one embodiment, no more than 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the linkages outside the CBT are internucleoside linkage modifications.
  • 15-90% of the linkages are internucleoside linkage modifications, preferably wherein 40-80%, most preferably 45-60%, of the linkages are internucleoside linkage modifications.
  • the internucleoside linkage modification content is at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
  • the internucleoside linkage modification content is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20%.
  • the internucleoside linkage modification content is 10-90%, 15-90%, 15-80%, 15-70%, 15-60%, 20-90%, 10-80%, 20-80%, 25-80%, 30-80%, 30-90%, 40-90%, 40-80%, 40-70%, 45-90%, 45-85%, 45-75%, 45- 70%, 45-60% or 45-55%.
  • 15-90% of the linkages are internucleoside linkage modifications.
  • 40-80% of the linkages are internucleoside linkage modifications.
  • 45-60% of the linkages are internucleoside linkage modifications.
  • the internucleoside linkage modification content is 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%.
  • the internucleoside linkage modification content is 30%. In one embodiment, the internucleoside linkage modification content is 15%.
  • Oligonucleotides of different lengths may require a different mixture of particular 2’-modifications and internucleoside linkage modifications in order to provide optimal RNA editing. The shorter the oligonucleotide, the better may be the endosomal escape and the lower the cytotoxicity. Also, shorter oligonucleotides may experience higher specificity. On the other hand, while longer oligonucleotides may bind stronger or faster to their respective RNA target, editing-boosting bulges, mismatches and wobbles may also work better in long oligonucleotides.
  • the oligonucleotides of the invention may be of varying lengths. In some instances, the oligonucleotides may range from about 25-70nt in length, e.g., about 25-39nt or about 40-60nt in length. In one embodiment, the oligonucleotide has a length of 25-60nt. In one embodiment, the oligonucleotide has a length of 40-59nt. In one embodiment, the oligonucleotide has a length of 25-59nt.
  • the oligonucleotide has a length of 40nt. In one embodiment, the oligonucleotide has a length of 42nt. In one embodiment, the oligonucleotide has a length of 59nt. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention. In one embodiment, the oligonucleotide has a length of 42 or 59 nucleotides and wherein outside of the CBT no more than 3 nucleotides are deoxyribonucleotides.
  • the oligonucleotide comprises an internucleoside linkage modification selected from the group consisting of phosphorothioate (PS), 3'- methylenephosphonate, 5'-methylenephosphonate, 3'-phosphoroamidate, 2'- 5'phosphodiester, and phosphoryl guanidine (PN).
  • the internucleoside linkage modification is a PS linkage.
  • the internucleoside linkage modification is a 3'-methylenephosphonate linkage.
  • the internucleoside linkage modification is a 5'-methylenephosphonate linkage.
  • the internucleoside linkage modification is a 3'- phosphoroamidate linkage. In one embodiment, the internucleoside linkage modification is a 2'-5'-phosphodiester linkage. In one embodiment, the internucleoside linkage modification is a PN linkage. In one embodiment, the at least one internucleoside linkage modification is PS. In one embodiment, the oligonucleotide contains a continuous stretch of PS linkages. In one embodiment, the continuous stretch of PS linkages is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more linkages long. In one embodiment, the continuous stretch of PS linkages is 25 linkages long. In one embodiment, the continuous stretch of PS linkages is 6 linkages long.
  • the continuous stretch of PS linkages is 3 linkages long.
  • the positioning of internucleoside linkages within the oligonucleotide plays an important role when determining a balance between high editing yields and a long half-life.
  • the need for a carefully considered placement of backbone modifications reflects the sensitivity of the CBT and bases surrounding the CBT for subtle chemical modifications on the ribose, the nucleobase or the linkage.
  • positions h and i may be chemically modified or unmodified.
  • PS linkages are not placed at positions h and i, which may be detrimental to the editing efficacy of the constructs. Accordingly, in one embodiment, linkage h and i are not chemically modified.
  • linkage h is not chemically modified. In one embodiment, linkage i is not chemically modified. In some embodiments, linkages h and i are phosphate (PO) linkages. In some embodiments, linkages h and i are not phosphorothioate (PS) linkages. In one embodiment, up to three linkages selected from the group consisting of linkages b, c, f, g and j are also PS linkages. It is, however, excluded that all linkages a to j are PS linkages. In especially preferred embodiments the linkage f is a PS linkage. In especially preferred embodiments, linkages a, d and e are PS linkages whereas linkages h and i are PO linkages.
  • N0 is deoxycytidine or FANA-cytidine.
  • N0 is DNA.
  • N0 is deoxycytidine.
  • N0 is FANA- cytidine.
  • Other modifications may include nucleobase replacement by (N) heterocycles or aromatic rings that stack well in the RNA duplex, such as, e.g., a Benner’s base Z (dZ) (and/or analogues) or 8-oxo-adenosine (8-oxo-A).
  • N0 is a Benner’s base.
  • N0 is 8-oxo-adenosine.
  • no more than 6 consecutive nucleotides are 2’-H (DNA) modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-H- modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-H- modified. The 2’-sugar modification may be 2’-ribose. In one embodiment, no more than 6 consecutive nucleotides are 2’-H (DNA) modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-H-modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-H-modified. In one embodiment, no more than 6 consecutive nucleotides are 2’-F-modified.
  • no more than 5 consecutive nucleotides are 2’-F-modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-F-modified. In one embodiment, no more than 6 consecutive nucleotides are 2’-O-alkyl-modified. In one embodiment, no more than 5 consecutive nucleotides are 2’-O-alkyl-modified. In one embodiment, no more than 4 consecutive nucleotides are 2’-O-alkyl-modified, optionally wherein no more than 4 consecutive nucleotides are 2’-OMe-modified.
  • the oligonucleotide comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more continuous PS linkages.
  • each terminus contains 3 consecutive internucleoside linkage modifications.
  • each terminus contains 4, 5, or 6 consecutive internucleoside linkage modifications.
  • each terminus contains no more than 8, 7, 6, 5, 4, or 3 consecutive internucleoside linkage modifications.
  • each terminus contains 3 consecutive internucleoside linkage modifications.
  • the modification is a 3'-methylenephosphonate, 5'-methylenephosphonate, 3'- phosphoroamidate, 2'-5'phosphodiester, or a phosphoryl guanidine (PN) modification.
  • PN phosphoryl guanidine
  • the internucleoside linkage modification is a PS linkage modification. In another embodiment, the internucleoside linkage modification is a 3’- 3’ or 5’-5’ phosphate ester bonds (3′-P-3′ and 5′-P-5′).
  • the oligonucleotides of the invention may have a limited DNA content outside of the CBT. In one embodiment, DNA is located outside the CBT. In one embodiment, DNA is located 3’ and/or 5’ of the CBT. In one embodiment, DNA is located 3’ of the CBT. In one embodiment, DNA is located 5’ of the CBT.
  • the nucleotide has a length of 25-30, 25-40, 25-60, 28-60, 28- 55, or 28-45 nucleotides. In one embodiment, the nucleotide has a length of 25-60 and a deoxyribonucleoside content outside the CBT that is 10-40%, more preferably 11-30%, and even more preferably 13-25%. In one embodiment, the oligonucleotide does not contain any unmodified RNA nucleotides. [0083] In one embodiment, at least one of the three nucleotides of the CBT is chemically modified at the 2' position of the sugar moiety, wherein said modification is a 2’-F-modification.
  • the mismatch is a C-to- C mismatch. In one embodiment, the mismatch is a C-C mismatch. In one embodiment, the mismatch is a G-U mismatch. In one embodiment, the mismatch is a C-C mismatch or a G-U mismatch. [0093] In one embodiment, the mismatch is located 3’ and/or 5’ to the nucleotide that is opposite to the target adenosine in the target RNA. The mismatch may comprise a single nucleotide or sequential nucleotides.
  • the CBT has been underlined.
  • the length of the oligonucleotide can be shortened without losing its editing efficacy provided the oligonucleotide comprises specific 2’-sugar and internucleoside linkage modifications.
  • the oligonucleotide may be 40nt long.
  • the oligonucleotide is 40nt long and comprises a C at position N -7 .
  • the oligonucleotide comprises SEQ ID NO: 4.
  • the oligonucleotides comprise a C6-amino-linker at the 5’ terminus. In some embodiments, the oligonucleotides comprise a C6-amino-linker at the 3’ terminus.
  • the modified oligonucleotide comprises a moiety or is conjugated to a moiety that enhances cellular uptake of the oligonucleotide. In one embodiment, the moiety enhancing cellular uptake is a triantennary N-acetyl galactosamine (GalNAc3), which is preferably conjugated to the 3' terminus or to the 5' terminus of the oligonucleotide.
  • the modified oligonucleotides of the invention may be incorporated into compositions. Accordingly, provided herein is a composition containing one or more of the oligonucleotide(s) of the invention. In some embodiments, the compositions are pharmaceutical compositions. In the context of the invention, the term composition and pharmaceutic compositions are used interchangeably. In one embodiment, the composition contains one or more oligonucleotides of the invention. In some embodiments, provided herein is a composition comprising a plurality of oligonucleotides. As used herein, pharmaceutical composition means a substance or a mixture of substances suitable for administering to an individual.
  • the compositions of the invention include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH, and ionic strength, and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol).
  • the material are incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
  • an oligonucleotide according to the invention can be delivered as is, i.e., naked and/or in isolated form to an individual, through an organ, e.g., mucosa of the eye, or directly to a cell.
  • the oligonucleotide of the invention is administered and delivered ‘as is’, also referred to as ‘naked’.
  • the oligonucleotide is dissolved in a solution that is compatible with the delivery method. Such delivery may be in vivo, in vitro or ex vivo.
  • Suitable exemplary excipients or transfection reagents comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINETM 2000 (lnvitrogen), lipofectin TM , or derivatives thereof, and/or viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a target cell.
  • PEI polyethylenimine
  • LipofectAMINETM 2000 lipofectin TM
  • viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a target cell.
  • the oligonucleotide or composition may be administered as a monotherapy or in combination with a further different medicament.
  • the oligonucleotide of the invention may be administered together with a medicament suitable for the treatment or prevention of alpha-1-antitrypsin (A1AT) deficiency or retinitis pigmentosa.
  • A1AT alpha-1-antitrypsin
  • the provided oligonucleotides or compositions are surprisingly effective in target editing.
  • a change is measured by an increase of a desired mRNA and/or protein level compared to a reference sample or condition.
  • a change is measured by an increase in the editing efficacy (%) mediated by the oligonucleotide or composition of the invention.
  • an oligonucleotide or composition containing the same is administered to a mammal, preferably a human.
  • an oligonucleotide or composition containing the same is administered to a naive subject, i.e., a subject that does not have a disease or disorder or has not previously received any oligonucleotide.
  • an oligonucleotide or composition containing the same is administered to a naive subject that is at risk of developing a disease or disorder.
  • an oligonucleotide or composition containing the same is administered to a patient before symptoms manifest or symptoms become severe.
  • a symptom of a condition, disorder or disease associated with a G-to-A mutation can be any condition, disorder or disease that can benefit from an A- to-I conversion.
  • methods of treating a disease or condition associated with a point mutation in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide according to the invention or composition according to the invention.
  • the genetic disease or genetic disorder is associated with a G-to-A mutation in a subject.
  • an effective amount of the oligonucleotide of the invention or the composition of the invention is administered to the subject.
  • Also provided herein is the use of an oligonucleotide of the invention in therapy.
  • the method is for treating multiple system atrophy, dementia with Lewy bodies and Parkinson’s disease (PD).
  • the methods of editing may be in vitro, in vivo, or ex vivo.
  • the invention provides a method for site-directed A-to-I editing of a target RNA, comprising providing to a cell or subject an oligonucleotide or composition of the invention.
  • the method comprising a step of contacting a target RNA with the oligonucleotide of the invention or the composition of the invention.
  • Construct TMR291 (v16, no mismatch outside the CBT) is a chemically optimized, 59nt long oligonucleotide and served as control.
  • Construct TMR305 (v16m) represents a chemically and sequence optimized version, carrying a G ⁇ C nucleotide change at position -7 (N -7 ). Results are shown in Figure 1.
  • Table 1 SERPINA1 E342K targeting construct sequences including nucleobase and backbone modifications used in Example 1.
  • TMR543/v17m This chemically and sequence optimized construct is referred to as TMR543/v17m.
  • the 40nt targeting constructs, sequences, and modification pattern are listed in Table 2. The results are shown in Figure 2. Editing efficacy (in %) of target RNA was determined as described under Example 1 above. Table 2: SERPINA1 E342K targeting construct sequences including nucleobase and backbone modifications used in Example 2.

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Abstract

L'invention concerne des oligonucléotides antisens modifiés appropriés pour éditer une adénosine dans un acide nucléique cible en inosine (édition A en I), l'oligonucléotide comprenant : (I) une séquence d'acide nucléique de 25 à 70 nucléotides (nt) qui est une séquence antisens sensiblement complémentaire de l'acide nucléique cible, (II) un triplet de bases central (CBT) (5'- N+1 e N0 f N-1 g -3') avec le nucléotide central (N0) directement opposé à l'adénosine cible à éditer lorsque l'oligonucléotide antisens est hybridé à l'acide nucléique cible, et (III) la séquence de cœur suivante : 5'- a N+4 b N+3 c N+2 d N+1 e N0 f N-1 g N-2 h N-3 i N-4 j N-5 k N-6 l N-7 m N-8 n N-9 -3' ; et : (a) au moins deux des trois nucléotides du CBT étant chimiquement modifiés à la position 2'de la fraction sucre, étant des désoxyribonucléosides, ou une combinaison de ceux-ci et d et e étant des modifications de liaison internucléosidiques ; (b) le nucléotide N-2 portant une modification 2'-O-alkyle ; et le nucléotide N-3 portant une modification 2´-fluoro (2'-F) ; (c) au moins 10 % de nucléotides de l'oligonucléotide sont modifiés 2'-F et au moins 10 % de nucléotides de l'oligonucléotide sont modifiés 2'-O-alkyle, pas plus de 6 nucléotides consécutifs ayant la même modification 2' ; et l'oligonucléotide comprenant une ou plusieurs disparités nucléotidiques à l'extérieur du CBT qui est/ne sont pas complémentaires de la position correspondante dans l'élément nucléique cible ; et la disparité étant : à la position -7 (N-7), un G ou C étant directement en 3' de la disparité et un A ou U étant directement en 5' de la disparité ; à la position -9 (N-9), un G ou C étant directement en 3' de la disparité et un A ou U étant directement en 5' de la disparité ; ou à la position -9 (N-9), un G ou C étant directement en 3' de la disparité et un A ou U étant directement en 5' de la disparité, et à la position +2 (N+2).
PCT/EP2024/077956 2023-10-06 2024-10-04 Optimisation chimique et de séquence d'oligonucléotides antisens pour édition d'arn médiée par adar Pending WO2025073902A1 (fr)

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