JP-2025514327-A5 -

Dates

Publication Date

20260508

Application Date

20230428

Description

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. While specific embodiments have been described in detail so far, embodiments will be better understood by referring to the following examples. These examples are included for illustrative purposes only and are not intended to limit the scope of the embodiments. This disclosure provides, for example, the following: [Section 1] Protected oligonucleotide, (a) A sequence complementary to crRNA, (b) comprising at least one chemically modified nucleotide, The protective oligonucleotide can bind to the crRNA, and The protective oligonucleotide, upon binding, confers nuclease resistance to the crRNA. [Section 2] The protected oligonucleotide according to claim 1, wherein the at least one chemically modified nucleotide includes modifications of a ribose group, a phosphate group, a nucleic acid base, or a combination thereof. [Section 3] The protected oligonucleotide according to claim 2, wherein the modification of the ribose group is independently selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'- O- (2-methoxyethyl) (MOE), 2'-NH₂(2'-amino), 4'-thio, bicyclic nucleotide, locked nucleic acid (LNA), 2'-(S)-constrained ethyl (S-cEt), constraint MOE, and 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'- BNANC ). [Section 4] The protected oligonucleotide according to claim 2 or 3, wherein at least 80% of the ribose group is chemically modified. [Section 5] The protected oligonucleotide according to claim 2 or 3, wherein at least 90% of the ribose group is chemically modified. [Section 6] The protected oligonucleotide according to claim 2 or 3, wherein 100% of the ribose group is chemically modified. [Section 7] The protected oligonucleotide according to any one of claims 1 to 6, wherein the crRNA includes a guide sequence portion and a repeat sequence portion that can hybridize to a target polynucleotide sequence. [Section 8] The protective oligonucleotide according to any one of claims 1 to 7, wherein the protective oligonucleotide binds to the crRNA to form a double helix, and optionally, the protective oligonucleotide binds to a region of the crRNA that is not completely chemically modified. [Section 9] The protected oligonucleotide according to claim 8, wherein the double hemisphere has a melting temperature (Tm) above 37°C along the entire length of the double hemisphere. [Section 10] The protected oligonucleotide according to claim 8, wherein the double helix has a melting temperature (Tm) of less than 37°C over the complementary region including the guide sequence portion. [Section 11] The protective oligonucleotide according to any one of claims 7 to 10, wherein the protective oligonucleotide is dissociated from the crRNA by binding of the tracrRNA to the guide sequence portion of the crRNA. [Section 12] The protective oligonucleotide according to any one of the preceding claims, further comprising at least one site bonded to the protective oligonucleotide. [Section 13] The protective oligonucleotide according to claim 12, wherein the at least one site is attached to at least one of the 5' end and/or 3' end of the protective oligonucleotide. [Section 14] The protective oligonucleotide according to claim 12, wherein at least one of the sites increases cellular uptake of the protective oligonucleotide. [Section 15] The protective oligonucleotide according to claim 12, wherein the at least one site promotes the specific tissue distribution of the protective oligonucleotide. [Section 16] The protected oligonucleotide according to claim 12, wherein the at least one site is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, ganglioside analogs, nucleoside analogs, endogenous cannabinoids, vitamins, receptor ligands, peptides, aptamers, and alkyl chains. [Section 17] The protected oligonucleotide according to claim 12, wherein the at least one site is selected from the group consisting of cholesterol, cholesterol-triethylene glycol (TEGChol), docosahexaenoic acid (DHA), docosanic acid (DCA), lithocholic acid (LA), GalNAc, amphiphilic block copolymer (ABC), hydrophilic block copolymer (HBC), poloxamer, Cy5, and Cy3. [Section 18] The protective oligonucleotide according to claim 12, wherein at least one of the sites is bonded to the protective oligonucleotide via a linker. [Section 19] The protected oligonucleotide according to claim 18, wherein the linker is selected from the group consisting of ethylene glycol chains, alkyl chains, polypeptides, polysaccharides, and block copolymers. [Section 20] The protected oligonucleotide according to claim 18, wherein at least one of the sites is a modified lipid. [Section 21] The protective oligonucleotide according to item 20, wherein the modified lipid is a branched lipid.