Single-stranded RNA-editing oligonucleotides
The invention relates to antisense oligonucleotides that are capable of bringing about specific editing of a target nucleotide (adenosine) in a target RNA in a eukaryotic cell, wherein said oligonucleotide does not, in itself, form an intramolecular hairpin or stem-loop structure, and wherein said oligonucleotide comprises a cytidine (a non-complementary nucleotide) or a uridine in position opposite to the target adenosine to be edited in the target RNA region.
1. An antisense oligonucleotide (AON) capable of forming a double stranded complex with a target RNA in a cell for the deamination of a target adenosine present in the target RNA by an ADAR enzyme present in the cell, wherein:
(a) the AON is complementary to a target RNA region comprising the target adenosine, and the AON comprises one or more mismatches, wobbles and/or bulges with the complementary target RNA region;
(b) the AON comprises one or more nucleotides with one or more sugar modifications, provided that the nucleotide opposite the target adenosine comprises a ribose with a 2′-OH group, or a deoxyribose with a 2′-H group;
(c) the AON does not comprise a portion that is capable of forming an intramolecular stem-loop structure capable of binding an ADAR enzyme;
(d) the AON does not include a 5′-terminal O6-benzylguanine modification;
(e) the AON does not include a 5′-terminal amino modification; and
(f) the AON is not covalently linked to a SNAP-tag domain.
2. An AON capable of forming a double stranded complex with a target RNA in a cell for the deamination of a target adenosine present in the target RNA by an ADAR enzyme present in the cell, wherein:
(a) the AON is complementary to a target RNA region comprising the target adenosine, and the AON comprises one or more mismatches, wobbles and/or bulges with the complementary target RNA region;
(b) the AON comprises one or more nucleotides with one or more sugar modifications, provided that the nucleotide opposite the target adenosine comprises a ribose with a 2′-OH group, or a deoxyribose with a 2′-H group;
(c) the AON does not comprise a portion that is capable of forming an intramolecular stem-loop structure capable of binding an ADAR enzyme; and
(d) the AON is not a 17-mer or a 20-mer.
3. An AON capable of forming a double stranded complex with a target RNA in a cell for the deamination of a target adenosine present in the target RNA by an ADAR enzyme present in the cell, wherein:
(a) the AON is complementary to a target RNA region comprising the target adenosine, and the AON comprises one or more mismatches, wobbles and/or bulges with the complementary target RNA region;
(b) the AON comprises one or more nucleotides with one or more sugar modifications, provided that the nucleotide opposite the target adenosine comprises a ribose with a 2′-OH group, or a deoxyribose with a 2′-H group;
(c) the AON does not comprise a portion that is capable of forming an intramolecular stem-loop structure capable of binding an ADAR enzyme; and
(d) the AON is longer than 17 nucleotides, or shorter than 14 nucleotides.
4. An AON capable of forming a double stranded complex with a target RNA in a cell for the deamination of a target adenosine present in the target RNA by an ADAR enzyme present in the cell, wherein:
(a) the AON is complementary to a target RNA region comprising the target adenosine;
(b) the AON comprises one or more nucleotides with one or more sugar modifications, provided that the nucleotide opposite the target adenosine comprises a ribose with a 2′-OH group, or a deoxyribose with a 2′-H group;
(c) the AON does not comprise a portion that is capable of forming an intramolecular stem-loop structure that is capable of binding an ADAR enzyme;
(d) the AON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches, wobbles and/or bulges with the complementary target RNA region.
5. The AON of claim 1 , wherein the nucleotide opposite the target adenosine is a cytidine, a deoxycytidine, a uridine, or a deoxyuridine.
6. The AON of claim 1 , wherein the nucleotide directly 5′ and/or 3′ from the nucleotide opposite the target adenosine comprises a ribose with a 2′-OH group, or a deoxyribose with a 2′-H group.
7. The AON of claim 6 , wherein all other nucleotides in the AON comprise a 2′-O-alkyl group.
8. The AON of claim 1 , comprising at least one phosphorothioate linkage.
9. The AON of claim 8 , wherein the 2, 3, 4, 5, or 6 terminal nucleotides of the 5′ and 3′ terminus of the AON are linked with a phosphorothioate linkage.
10. The AON of claim 9 , wherein the 5 terminal nucleotides of the 5′ and 3′ terminus of the AON are linked with phosphorothioate linkages.
11. The AON of claim 1 , wherein the AON is longer than 10, 11, 12, 13, 14, 15, 16 or 17 nucleotides.
12. The AON of claim 1 , wherein the AON is shorter than 100 nucleotides.
13. The AON of claim 1 , wherein the AON comprises 18 to 70 nucleotides.
14. A pharmaceutical composition comprising the AON of claim 1 and a pharmaceutically acceptable carrier.
15. A method of treating or preventing a genetic disorder in a subject in need thereof, the method comprising administering the AON of claim 1 to the subject.
16. A method of deaminating at least one target adenosine present in a target RNA in a cell, the method comprising:
(i) contacting a cell with the AON of claim 1 thereby to permit the AON to enter the cell and an ADAR enzyme comprising a natural dsRNA binding domain to deaminate the target adenosine in the target RNA to an inosine; and
(ii) optionally identifying the presence of the inosine in the targeted RNA.
17. The method of claim 16 , wherein step (ii) comprises:
(a) sequencing the targeted RNA sequence;
(b) assessing the presence of a functional, elongated, full length and/or wild type protein when the target adenosine is located in a UGA or UAG stop codon, which is edited to a UGG codon through the deamination;
(c) assessing the presence of a functional, elongated, full length and/or wild type protein when two target adenosines are located in a UAA stop codon, which is edited to a UGG codon through the deamination of both target adenosines;
(d) assessing whether splicing of the pre-mRNA was altered by the deamination; or (e) using a functional read-out, wherein the target RNA after the deamination encodes a functional, full length, elongated and/or wild type protein.
18. The AON of claim 1 , wherein the target RNA sequence encodes CFTR, CEP290, alphal-antitrypsin (A1AT), LRRK2, BDNF, or wherein the target RNA is encoded by the IDUA gene.
19. The AON of claim 7 , wherein all other nucleotides in the AON comprise a 2′-O-methyl group.
20. The method of claim 15 , wherein the genetic disorder is selected from the group consisting of: Cystic fibrosis, Hurler Syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, Amyotrophic lateral sclerosis, Asthma, ß-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, Chronic Obstructive Pulmonary Disease (COPD), Distal Spinal Muscular Atrophy (DSMA), Duchenne/Becker muscular dystrophy, Dystrophic Epidermolysis bullosa, Epidermylosis bullosa, Fabry disease, Factor V Leiden associated disorders, Familial Adenomatous, Polyposis, Galactosemia, Gaucher's Disease, Glucose-6-phosphate dehydrogenase, Haemophilia, Hereditary Hematochromatosis, Hunter Syndrome, Huntington's disease, Inflammatory Bowel Disease (IBD), Inherited polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, Mucopolysaccharidosis, Muscular Dystrophy, Myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-esol related cancer, Peutz-Jeghers Syndrome, Phenylketonuria, Pompe's disease, Primary Ciliary Disease, Prothrombin mutation related disorders, such as the Prothrombin G20210A mutation, Pulmonary Hypertension, Retinitis Pigmentosa, Sandhoff Disease, Severe Combined Immune Deficiency Syndrome (SCID), Sickle Cell Anemia, Spinal Muscular Atrophy, Stargardt's Disease, Tay-Sachs Disease, Usher syndrome, X-linked immunodeficiency, and cancer.