Double-stranded RNA molecules for ocular administration

Abstract

The present invention relates to a double-stranded RNA molecule and its pharmaceutical composition, which are conjugated with at least one conjugating moiety and are used for local administration to the eye.The double-stranded RNA molecule is complementary to the target expressed in the eye, for example, completely complementary, and can inhibit the expression of the target expressed in the eye.The double-stranded RNA molecule can be used in the treatment of eye symptoms and diseases.

Description

[Technical Field]

[0001] The present invention relates to double-stranded RNA molecules conjugated to at least one conjugating moiety for topical administration to the eye, and pharmaceutical compositions thereof, and their use in the treatment of ocular conditions and diseases. [Background technology]

[0002] Double-stranded RNA molecules, such as siRNA molecules, can regulate the expression of a target nucleic acid by binding to complementary mRNA after transcription, typically resulting in degradation of the target mRNA and loss of translation, and thereby specifically inhibiting expression of the target nucleic acid.

[0003] siRNA molecules can induce RNA-dependent gene silencing via the RNA-induced silencing complex (RISC) in the cytoplasm of cells, where they interact with Argonaute, the catalytic RISC component.

[0004] The delivery of double-stranded RNA molecules to tissue sites or specific cells can be impaired or hindered by various factors that reduce the stability of double-stranded RNA molecules or prevent double-stranded RNA molecules from being effectively delivered to their target sites.Such factors include but are not limited to: susceptibility to endogenous RNase, short half-life and low stability, recognition by immune system, large size and charge and endosomal trapping.

[0005] One tissue site that can be particularly challenging for delivery is the eye, which can be affected by various symptoms, pathologies and diseases.The delivery of double-stranded RNA molecules to eye can bring about beneficial therapeutic effects by targeting and inhibiting the gene of interest that is expressed in eye, which can be involved in causing pathology.

[0006] There is a need for an effective mechanism for delivering double-stranded RNA molecules to the eye. Summary of the Invention

[0007] The present invention provides a double-stranded RNA molecule for topical administration to the eye, the double-stranded RNA molecule being capable of binding to a target sequence, the double-stranded RNA molecule comprising a first strand having a 5' end and a 3' end and a second strand having a 5' end and a 3' end, the first strand being complementary to the second strand, the first strand comprising a contiguous nucleotide sequence of at least 8 nucleotides in length that is complementary to the target sequence, and the double-stranded RNA molecule being conjugated to at least one conjugate moiety.

[0008] In some embodiments, the double-stranded RNA molecule can be a small interfering RNA (siRNA) molecule. In some embodiments, the double-stranded RNA molecule can be a short hairpin RNA (shRNA) molecule.

[0009] In some embodiments, the double-stranded RNA molecule may be capable of inhibiting expression of a target.

[0010] The double-stranded RNA molecule is conjugated to at least one conjugate moiety.In some embodiments, the double-stranded RNA molecule can be conjugated to at least two or at least three conjugate moieties.In some embodiments, the double-stranded RNA molecule can be conjugated to two conjugate moieties.In some embodiments, the double-stranded RNA molecule can be conjugated to three conjugate moieties.

[0011] In some embodiments, one conjugate moiety or multiple conjugate moieties may be covalently attached to the double-stranded RNA molecule.

[0012] In some embodiments, the conjugate moiety can be a fatty acid molecule or a cholesterol molecule.

[0013] In embodiments where more than one conjugate moiety is present, the conjugate moiety may be a combination of one or more fatty acid molecules and one or more cholesterol molecules, or may be a combination of two or more fatty acid molecules, or may be a combination of two or more cholesterol molecules. In embodiments where more than one conjugate moiety is present, each conjugate moiety is independently selected such that the conjugate moieties attached to the double-stranded RNA molecule may or may not be the same.

[0014] In some embodiments, the conjugate moiety can be a fatty acid molecule.

[0015] In some embodiments, the fatty acid molecule is selected from the list consisting of C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39 and C40.

[0016] In some embodiments, the fatty acid molecule can be C16.

[0017] In some embodiments, the fatty acid molecule can be C22.

[0018] In some embodiments, the fatty acid molecule is branched. In some embodiments, the fatty acid molecule is unbranched.

[0019] In some embodiments, the fatty acid molecule is saturated. In some embodiments, the fatty acid molecule is unsaturated.

[0020] In some embodiments, the fatty acid molecule comprises one or more double bonds, hi some embodiments, the fatty acid molecule comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 or more carbon double bonds.

[0021] In some embodiments, the fatty acid molecule comprises one or more triple bonds, hi some embodiments, the fatty acid molecule comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 or more carbon triple bonds.

[0022] In some embodiments, the fatty acid molecule is C3:0; C4:0; C4:1; C5:0; C5:1; C6:0; C6:1; C6:2; C7:0; C7:1; C7:2; C8:0; C8:1; C8:2; C8:3; C9:0; C9:1; C9:2; C9:3; C10:0; C10:1; C10:2; C10:3; C10:4; C11:0; C11:1; C11:2; C11:3; C11:4; C12:0; C12:1; C12:2; C12:3; C12:4; C12:5; C13:0; C13:1; C13:2; C13:3; C13:4; C13:5; C14:0; C14:1; C14:2; C14:3; C14:4; C14:5; C14:6; C15:0; C15:1; C15:2; C15:3; C15:4; C15:5; C15:6; C16:0; C16:1; C16:2; C16:3; C16:4; C16:5; C16:6; C16:7; C17:0; C17:1; C17:2; C17:3; C17:4; C17:5; C17:6; C17:7; C18:0; C18:1; C18:2; C18:3; C18:4; C18:5; C18:6; C18:7; C18:8; C19:0; C19:1; C19:2; C19:3; C19:4; C19:5; C19:6; C19:7; C19:8; C20:0; C20:1; C20:2; C20:3; C20:4; C20:5; C20:6; C20:7; C20:8; C20:9; C21:0; C21:1; C21:2; C21:3; C21:4; C21:5; C21:6; C21:7; C21:8; C21:9; C22:0; C22:1; C22:2; C22:3; C22:4; C22:5; C22:6; C22:7; C22:8; C22:9; C22:10; C23:0; C23:1; C23:2; C23:3; C23:4; C23:5; C23:6; C23:!7; C23:8; C23:9; C23:10; C24:0; C24:1; C24:2; C24:3; C24:4; C24:5; C24:6; C24:7; C24:8; C24:9; C24:10; C24:11; C25:0; C25:1; C25:2; C25:3; C25:4; C25:5; C25:6; C25:7; C25:8; C25:9; C25:10; C25:11; C26:0; C26:1; C26:2; C26:3; C26:4; C26:5; C26:6; C26:7; C26:8; C26:9; It should be noted that there seems to be a possible error in the original text where "C23:!7" is likely a typo. I've translated it as is but it might need to be corrected in the source for a more accurate representation.<h2 style=";text-align:left;direction:ltr">C26:10;C26:11;C26:12;C27:0;C27:1;C27:2;C27:3;C27:4;C27:5;C27:6;C27:7;C27:8;C27:9;C27:10;C27:11;C27:12;C28:0;C28:1;C28:2;C28: 3;C28:4;C28:5;C28:6;C28:7;C28:8;C28:9;C28:10;C28:11;C28:12;C28 :13;C29:0;C29:1;C29:2;C29:3;C29:4;C29:5;C29:6;C29:7;C29:8;C29:9 ;C29:10;C29:11;C29:12;C29:13;C30:0;C30:1;C30:2;C30:3;C30:4;C30 :5;C30:6;C30:7;C30:8;C30:9;C30:10;C30:11;C30:12;C30:13;C30:14;C 31:0;C31:1;C31:2;C31:3;C31:4;C31:5;C31:6;C31:7;C31:8;C31:9;C31 :10;C31:11;C31:12;C31:13;C31:14;C32:0;C32:1;C32:2;C32:3;C32:4;C 32:5;C32:6;C32:7;C32:8;C32:9;C32:10;C32:11;C32:12;C32:13;C32:14;C32:15;C33:0;C33:1;C33:2;C33:3;C33:4;C33:5;C33:6;C33:7;C33:8; C33:9;C33:10;C33:11;C33:12;C33:13;C33:14;C33:15;C34:0;C34:1;C34:2;C34:3;C34:4;C34:5;C34:6;C34:7;C34:8;C34:9;C34:10;C34:11;C34 :12;C34:13;C34:14;C34:15;C34:16;C35:0;C35:1;C35:2;C35:3;C35:4; C35:5;C35:6;C35:7;C35:8;C35:9;C35:10;C35:11;C35:12;C35:13;C35:1 4;C35:15;C35:16;C36:0;C36:1;C36:2;C36:3;C36:4;C36:5;C36:6;C36: 7;C36:8;C36:9;C36:10;C36:11;C36:12;C36:13;C36:14;C36:15;C36:16;C36:17;C37:0;C37:1;C37:2;C37:3;C37:4;C37:5;C37:6;C37:7;C37:8;C37 :9;C37:10;C37:11;C37:12;C37:13;C37:14;C37:15;C37:16;C37:17;C38:0 ;C38:1;C38:2;C38:3;C38:4;C38:5;C38:6;C38:7;C38:8;C38:9;C38:10;C3 8:11;C38:12;C38:13;C38:14;C38:15;C38:16;C38:17;C38:18;C39:0;C39: 1;C39:2;C39:3;C39:4;C39:5;C39:6;C39:7;C39:8;C39:9;C39:10;C39:11; C39:12;C39:13;C39:14;C39:15;C39:16;C39:17;C39:18;C40:0;C40:1;C40 :2;C40:3;C40:4;C40:5;C40:6;C40:7;C40:8;C40:9;C40:10;C40:11;C40:12;C40:13;C40:14;C40:15;C40:16;C40:17;C40:18;C40:19. ;

[0023] In embodiments where more than one fatty acid molecule is present, each fatty acid molecule is independently selected such that the fatty acid molecules attached to the double-stranded RNA molecule may or may not be the same.

[0024] In some embodiments, the conjugate moiety can be a cholesterol molecule.

[0025] In some embodiments, the cholesterol molecule may be selected from the group consisting of 3'-cholesteryl-TEG CPG, 5'-cholesterol-TEG-CE phosphoramidite, 5'-cholesterol-CE phosphoramidite, and cholesteryl-TEG-CE phosphoramidite. In embodiments where more than one cholesterol moiety is present, the cholesterol moieties may be a combination selected from the group consisting of 3'-cholesteryl-TEG CPG, 5'-cholesterol-TEG-CE phosphoramidite, 5'-cholesterol-CE phosphoramidite, and cholesteryl-TEG-CE phosphoramidite.

[0026] In embodiments in which more than one cholesterol molecule is present, each cholesterol molecule is independently selected such that the cholesterol molecules attached to the double-stranded RNA molecule may or may not be the same.

[0027] In embodiments where more than one conjugate moiety is present, the conjugate moieties may be one or a combination of one or more selected from C16, C22, or cholesterol molecules.

[0028] In some embodiments, the conjugate moiety may be located at the 5'-end or 3'-end of one of the strands of the double-stranded RNA molecule. In some embodiments, the conjugate moiety may be located at the 3'-end of the first strand of the double-stranded RNA molecule. The first strand may be in the sense orientation (i.e., the first strand is the sense strand). In some embodiments, the conjugate moiety may be located at the 3'-end of the sense strand of the double-stranded RNA molecule.

[0029] In some embodiments, a linker may be disposed between the double-stranded RNA molecule and the conjugate moiety. In some embodiments, the linker may be C6. In some embodiments, the linker may be TEG. In some embodiments, the linker is a dinucleotide. In some embodiments, the dinucleotide is CA (in other words, in some embodiments, the linker is a CA dinucleotide).

[0030] In some embodiments, where the double-stranded RNA molecule comprises more than one conjugate moiety, a linker may be disposed between the double-stranded RNA molecule and each of the conjugate moieties. In some embodiments, a linker may be disposed between each of the conjugate moieties.

[0031] In some embodiments, the linker may be a cleavable linker.

[0032] In some embodiments, the contiguous nucleotide sequence can be at least 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

[0033] In some embodiments, the contiguous nucleotide sequence may be at least 20 nucleotides in length. In some embodiments, the contiguous nucleotide sequence may be 20, 21, 22, 23, or 24 nucleotides in length.

[0034] In some embodiments, the first strand can consist of a contiguous nucleotide sequence.

[0035] In some embodiments, the double-stranded RNA molecule may be for administration to the front of the eye.

[0036] In some embodiments, the double-stranded RNA molecule can be for administration to the conjunctiva of the eye or the cornea of ​​the eye, hi some embodiments, the double-stranded RNA molecule can be for administration to the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva, and / or conjunctival fornix.

[0037] In some embodiments, the contiguous nucleotide sequence may be at least about 75% complementary to the target sequence. The contiguous nucleotide sequence may be at least about 80%, at least about 85%, at least about 90%, at least about 95%, or completely (e.g., about 100%) complementary to the target sequence. In some embodiments, the contiguous nucleotide sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, or more mismatches to the target sequence.

[0038] In some embodiments, the target may be AHA-1. In some embodiments, the AHA-1 target may comprise or consist of SEQ ID NO:1 or SEQ ID NO:2.

[0039] In some embodiments, the contiguous nucleotide sequence may be complementary to an AHA-1 target sequence. In some embodiments, the contiguous nucleotide sequence may comprise a nucleotide sequence complementary to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the contiguous nucleotide sequence may comprise or consist of SEQ ID NO: 3.

[0040] In some embodiments, target expression can be inhibited by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 95%, or about 100% compared to a control. It will be understood that the percent inhibition of target expression referred to above is a percent reduction compared to a control, and the term "control" refers to target expression in cells that have not been exposed to the double-stranded RNA molecule of the present invention.

[0041] In some embodiments, the double-stranded RNA molecule may comprise one or more modified nucleosides. The one or more modified nucleosides may be one or more 2' sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-O-methoxyethyl-RNA (2'-MOE), 2'-alkoxy-RNA; 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; arabinonucleic acid (ANA); 2'-fluoro-ANA; locked nucleic acid (LNA), and any combination thereof. In some embodiments, the 2' sugar-modified nucleoside may be an affinity-enhancing 2' sugar-modified nucleoside.

[0042] In some embodiments, one or more of the internucleoside linkages located between nucleosides on a contiguous nucleotide sequence may be modified, hi some embodiments, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the internucleoside linkages located between nucleosides on a contiguous nucleotide sequence may be modified.

[0043] In some embodiments, one or more, or all, of the modified internucleoside linkages may comprise phosphorothioate linkages. In some embodiments, all internucleoside linkages present in the double-stranded RNA molecule may be phosphorothioate internucleoside linkages.

[0044] In some embodiments, the double-stranded RNA molecule can be in the form of a pharmaceutically acceptable salt. The salt can be a sodium salt or a potassium salt.

[0045] The double-stranded RNA molecule can be an isolated double-stranded RNA molecule or a purified double-stranded RNA molecule. In some embodiments, the double-stranded RNA molecule of the present invention is a manufactured (artificial) double-stranded RNA molecule.

[0046] The present invention also provides pharmaceutical compositions comprising the double-stranded RNA molecules of the present invention and pharmaceutically acceptable diluents, solvents, carriers, salts and / or adjuvants.

[0047] The present invention also provides a method for treating or preventing a disease in a subject, the method comprising administering a therapeutically or prophylactically effective amount of a double-stranded RNA molecule of the present invention or a pharmaceutical composition of the present invention to a subject in need thereof.

[0048] The present invention also provides the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention for use as a medicament in the treatment of a disease.

[0049] The present invention also provides use of the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention for preparing a medicament for treating or preventing a disease.

[0050] In some embodiments, the disease can be conjunctivitis, dry eye, or inflammation.

[0051] In some embodiments, the double-stranded RNA molecule can be administered to the eye once a day, twice a day, three times a day, or more than three times a day.

[0052] In some embodiments, the double-stranded RNA molecule can be administered for less than 1 day or for 1, 2, 3, 4, 5, 6, 7, or more than 7 days.

[0053] In some embodiments, the double-stranded RNA molecule may be administered for the following periods: (i) 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more than 6 weeks; or (ii) 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more; or (iii) 1 year, 2 years, 3 years, 4 years, 5 years or more.

[0054] In some embodiments, the double-stranded RNA molecule may be administered to one eye or to both eyes.

[0055] The present invention also provides an in vitro method for modulating expression of a target in a cell, the method comprising administering to the cell an effective amount of a double-stranded RNA molecule of the present invention or a pharmaceutical composition of the present invention.

[0056] Sequence Listing The Sequence Listing submitted with this application is hereby incorporated by reference. [Brief explanation of the drawings]

[0057] [Figure 1] AHSA1 expression normalized to HPRT in palpebral conjunctival rabbit samples 96 hours after the last dose. For siRNA molecules containing SEQ ID NO: 3, 20-25% knockdown in the whole conjunctiva was observed, with greater knockdown for AHSA1 siRNA conjugated to C16, C22, and cholesterol compared to naked (i.e., unconjugated) siRNA. [Figure 2] AHSA1 siRNA content in rabbit palpebral conjunctiva samples 96 hours after the last dose. Increased content in the conjunctiva was observed for siRNA molecules containing the sequence of SEQ ID NO: 3, with greater content for AHA1 siRNA conjugated to C16, C22, and especially cholesterol, compared to naked (i.e., unconjugated) siRNA. [Figure 3]AHSA1 expression normalized to HPRT in bulbar conjunctival EYEPRIM rabbit samples 96 hours after the last dose. 69% knockdown (SEQ ID NO: 3 conjugated to C16), 65% knockdown (SEQ ID NO: 3 conjugated to C22), and 61% knockdown (SEQ ID NO: 3 conjugated to cholesterol) in conjunctival EYEPRIM samples compared to 4% for naked (i.e., unconjugated siRNA) siRNA (SEQ ID NO: 3 (no conjugation)), indicating significantly greater knockdown by C16-conjugated AHSA1 siRNA compared to naked siRNA (p=0.02 Student's t-test), as well as greater knockdown by C22-conjugated and cholesterol-conjugated AHSA1 siRNA. [Figure 4] Biophysical analysis of AHA-1-specific siRNA (FA-siRNA) conjugated to different fatty acids. Column 3 shows the tendency of different fatty acid conjugates to exist in different oligomeric states at a concentration of 25 μM (final oligomeric state as measured by AUC), column 4 shows the percentage of monomer for different FA-siRNA when dissolved in 25 μM PBS, column 5 shows the binding affinity to mouse serum albumin (MSA; determined by ITC), and column 6 shows the number of FA-siRNA conjugates bound to MSA. [Figure 5]Conjunctival tissue ISH staining was observed for naked siRNA molecules containing the sequence of SEQ ID NO: 3, siRNA molecules containing the sequence of SEQ ID NO: 3 conjugated to C16, siRNA molecules containing the sequence of SEQ ID NO: 3 conjugated to C22, and siRNA molecules containing the sequence of SEQ ID NO: 3 conjugated to cholesterol. Compared with naked AHSA1 siRNA, C16, C22, and cholesterol AHSA1 siRNAs showed superior tissue staining, with C16 and C22 AHSA1 siRNAs showing the best results compared with naked and cholesterol AHSA1 siRNAs. Furthermore, while staining for naked AHSA1 siRNA was primarily located in the superficial conjunctiva, staining for C16, C22, and cholesterol AHSA1 siRNAs was also located in the stroma of the conjunctival tissue. DETAILED DESCRIPTION OF THE INVENTION

[0058] nucleic acid molecule As used herein, the term "nucleic acid molecule" or "therapeutic nucleic acid molecule" is defined as it is commonly understood by those of skill in the art as a molecule comprising two or more covalently linked nucleosides (i.e., a nucleotide sequence).

[0059] The nucleic acid molecule(s) referred to in this invention are generally therapeutic oligonucleotides less than 50 nucleotides in length.

[0060] As used herein, the terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule," and "nucleic acid sequence" are intended to be synonymous with each other.

[0061] Nucleic acid molecules are generally produced in laboratories by solid-phase chemical synthesis followed by purification and isolation.When referring to the sequence of nucleic acid molecules, it refers to the sequence or order of the nucleic acid base moiety of covalently linked nucleotides or nucleosides, or their modification.The nucleic acid molecules of the present invention are artificial, are chemically synthesized, and are typically purified or isolated.The nucleic acid molecules of the present invention can comprise one or more modified nucleosides or nucleotides.

[0062] The nucleic acid molecules of the present invention may contain one or more modified nucleosides, such as 2' sugar-modified nucleosides. The nucleic acid molecules of the present invention may contain one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.

[0063] Oligonucleotides As used herein, the term "oligonucleotide" is defined as a molecule containing two or more covalently linked nucleosides, as generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0064] double stranded RNA molecule As used herein, the terms "RNA interference (RNAi) molecule," "RNAi molecule," or "RNAi" refer to short, typically double-stranded RNA molecules that can induce RNA-dependent gene silencing via the RNA-induced silencing complex (RISC) in the cytoplasm of a cell and interact with the catalytic RISC component, Argonaute. One type of RNAi molecule is small interfering RNA (siRNA), which is a typically double-stranded RNA molecule that binds to complementary mRNA after transcription, typically resulting in mRNA degradation and loss of translation. In other words, the term "siRNA molecule," as used herein, is defined as a nucleic acid molecule that can regulate the expression of a target nucleic acid, particularly by binding to a contiguous sequence on the target nucleic acid. siRNA molecules are typically 20-24 base pairs in length and usually have a phosphorylated 5' end and a hydroxylated 3' end with two overhanging nucleotides. Small interfering RNA (siRNA) is also known as small interfering RNA or silencing RNA.

[0065] In some embodiments, the double-stranded RNA molecules of the present invention can be small interfering RNA (siRNA) molecules.

[0066] The double-stranded RNA molecules of the present invention comprise a first strand having a 5' end and a 3' end and a second strand having a 5' end and a 3' end, wherein the first strand is complementary to the second strand.

[0067] A double-stranded RNA molecule can be described in terms of comprising a sense strand and an antisense strand.

[0068] In some embodiments, the first strand can be the sense strand (ie, in the sense orientation) and the second strand can be the antisense strand (ie, in the antisense orientation).

[0069] In some embodiments, the first strand can be the antisense strand (ie, in the antisense orientation) and the second strand can be the sense strand (ie, in the sense orientation).

[0070] Another type of RNAi molecule is short hairpin RNA (shRNA), which is an artificial RNA molecule with hairpin structure that can reduce the level of target mRNA through DICER and RNA-induced (reducing) silencing complex (RISC) when expressed.Small hairpin RNA (shRNA) can also be known as short hairpin RNA.In some embodiments, the double-stranded RNA molecule of the present invention can be short hairpin RNA (shRNA) molecule.

[0071] RNAi molecules can be designed based on the RNA sequence of a gene of interest. The corresponding RNAi molecules can then be synthesized chemically or by in vitro transcription, or expressed from a vector or PCR product.

[0072] siRNA and shRNA molecules are generally 20-50 nucleotides in length, e.g., 25-35 nucleotides in length, and can interact with an endonuclease known as Dicer, which is believed to enzymatically cleave dsRNA into 19-23 base-pair small interfering RNAs (siRNAs) with characteristic two-base 3' overhangs that are then incorporated into the RNA-induced silencing complex (RISC). Effective extended forms of Dicer substrates are described in U.S. Patent Nos. 8,349,809 and 8,513,207, incorporated herein by reference. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing. RNAi agents can be chemically modified using modified internucleotide linkages and high-affinity nucleosides, such as 2'-4' bicyclic ribose-modified nucleosides, including LNA and cET.

[0073] In some embodiments, the double-stranded RNA molecules of the present invention comprise or consist of a length of about 8 to 50 nucleotides.

[0074] In some embodiments, the double-stranded RNA molecule of the present invention comprises or consists of a length of about 12 to 50 nucleotides, such as about 15 to 45, such as about 20 to 40, such as about 25 to 35 consecutive nucleotides.

[0075] In some embodiments, the double-stranded RNA molecule comprises or consists of about 18-25 nucleotides in length. In some embodiments, the double-stranded RNA molecule comprises or consists of about 18-30 nucleotides in length. In some embodiments, the double-stranded RNA molecule comprises or consists of about 18-35 nucleotides in length. In some embodiments, the double-stranded RNA molecule comprises or consists of about 20-25 nucleotides in length. In some embodiments, the double-stranded RNA molecule comprises or consists of about 20-30 nucleotides in length. In some embodiments, the double-stranded RNA molecule comprises or consists of about 20-35 nucleotides in length. Any range provided herein should be understood to include the endpoints of the range. Thus, when a double-stranded RNA molecule is stated to comprise about 10-30 nucleotides, both lengths of about 10 and about 30 nucleotides are included.

[0076] In some embodiments, the double-stranded RNA molecule comprises or consists of about 50 or fewer nucleotides, about 45 or fewer nucleotides, about 40 or fewer nucleotides, about 35 or fewer nucleotides, about 30 or fewer nucleotides, about 25 or fewer nucleotides, about 20 or fewer nucleotides, or about 15 or fewer nucleotides.

[0077] In some embodiments, the double-stranded RNA molecule comprises or consists of about 10 or more nucleotides, about 15 or more nucleotides, about 20 or more nucleotides, about 25 or more nucleotides, about 30 or more nucleotides, about 35 or more nucleotides, about 40 or more nucleotides, or about 45 or more nucleotides.

[0078] In some embodiments, the double-stranded RNA molecule comprises or consists of at least 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0079] In some embodiments, the double-stranded RNA molecule can be at least about 20 nucleotides in length. The double-stranded RNA molecule can be 20, 21, 22, 23, or 24 nucleotides in length.

[0080] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 18 nucleotides.

[0081] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 19 nucleotides.

[0082] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 20 nucleotides.

[0083] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 21 nucleotides.

[0084] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 22 nucleotides.

[0085] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 23 nucleotides.

[0086] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 24 nucleotides.

[0087] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 25 nucleotides.

[0088] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 26 nucleotides.

[0089] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 27 nucleotides.

[0090] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 28 nucleotides.

[0091] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 29 nucleotides.

[0092] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 30 nucleotides.

[0093] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 31 nucleotides.

[0094] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 32 nucleotides.

[0095] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 33 nucleotides.

[0096] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 34 nucleotides.

[0097] In some embodiments, the double-stranded RNA molecule comprises or consists of a length of 35 nucleotides.

[0098] The double-stranded RNA molecule (s) binds to the target nucleic acid expressed in animal eyes, particularly mammalian eyes. In some embodiments, the double-stranded RNA molecule is typically for inhibiting and / or regulating the expression of the target nucleic acid sequence expressed in eyes.

[0099] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of a double-stranded RNA molecule that is complementary to a target nucleic acid, which may be or contain an oligonucleotide motif sequence. This term is used interchangeably with the term "contiguous nucleic acid base sequence" herein.

[0100] One of the strands of the double-stranded RNA molecule comprises or consists of a continuous nucleotide sequence. In some embodiments, the first strand comprises or consists of a continuous nucleotide sequence. In some embodiments, the second strand comprises or consists of a continuous nucleotide sequence.

[0101] In some embodiments, the strand of a double-stranded RNA molecule comprising or consisting of a contiguous nucleotide sequence may optionally comprise a nucleotide linker region that can be used to attach additional nucleotide(s), such as a functional group (e.g., a conjugate group), to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0102] It is understood that the contiguous nucleotide sequence cannot be longer than the double-stranded RNA molecule (or strand thereof) itself, and that the double-stranded RNA molecule (or strand thereof) cannot be shorter than the contiguous nucleotide sequence.

[0103] In some embodiments, all of the nucleosides of the first or second strand of the double-stranded RNA molecule may constitute a contiguous nucleotide sequence.

[0104] A contiguous nucleotide sequence is a sequence of nucleotides in the first or second strand of a double-stranded RNA molecule of the present invention that is complementary, and in some cases completely complementary, to a target nucleic acid, target sequence or target site sequence.

[0105] In some embodiments, the contiguous nucleotide sequence is about 8 to 50 nucleotides in length.

[0106] In some embodiments, the contiguous nucleotide sequence may comprise or consist of a length of about 12 to 50 nucleotides, such as about 15 to 45, such as about 20 to 40, such as about 25 to 35 contiguous nucleotides.

[0107] In some embodiments, a contiguous nucleotide sequence comprises or consists of about 18-25 nucleotides in length. In some embodiments, a contiguous nucleotide sequence comprises or consists of about 18-30 nucleotides in length. In some embodiments, a contiguous nucleotide sequence comprises or consists of about 18-35 nucleotides in length. In some embodiments, a contiguous nucleotide sequence comprises or consists of about 20-25 nucleotides in length. In some embodiments, a contiguous nucleotide sequence comprises or consists of about 20-30 nucleotides in length. In some embodiments, a contiguous nucleotide sequence comprises or consists of about 20-35 nucleotides in length. Any range provided herein should be understood to include the endpoints of the range. Thus, when a contiguous nucleotide sequence is stated to comprise about 10-30 nucleotides, both about 10 and about 30 nucleotides are included.

[0108] In some embodiments, the contiguous nucleotide sequence may comprise or consist of about 50 or fewer nucleotides, about 45 or fewer nucleotides, about 40 or fewer nucleotides, about 35 or fewer nucleotides, about 30 or fewer nucleotides, about 25 or fewer nucleotides, about 20 or fewer nucleotides, or about 15 or fewer nucleotides.

[0109] In some embodiments, the contiguous nucleotide sequence may comprise or consist of about 10 or more nucleotides, about 15 or more nucleotides, about 20 or more nucleotides, about 25 or more nucleotides, about 30 or more nucleotides, about 35 or more nucleotides, about 40 or more nucleotides, or about 45 or more nucleotides.

[0110] In some embodiments, the contiguous nucleotide sequence comprises or consists of 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

[0111] In some embodiments, the contiguous nucleotide sequence can be at least about 20 nucleotides in length. The contiguous nucleotide sequence can be 20, 21, 22, 23, or 24 nucleotides in length.

[0112] In some embodiments, the contiguous nucleotide sequence comprises or consists of 18 nucleotides in length.

[0113] In some embodiments, the contiguous nucleotide sequence comprises or consists of 19 nucleotides in length.

[0114] In some embodiments, the contiguous nucleotide sequence comprises or consists of 20 nucleotides in length.

[0115] In some embodiments, the contiguous nucleotide sequence comprises or consists of 21 nucleotides in length.

[0116] In some embodiments, the contiguous nucleotide sequence comprises or consists of 22 nucleotides in length.

[0117] In some embodiments, the contiguous nucleotide sequence comprises or consists of 23 nucleotides in length.

[0118] In some embodiments, the contiguous nucleotide sequence comprises or consists of 24 nucleotides in length.

[0119] In some embodiments, the contiguous nucleotide sequence comprises or consists of 25 nucleotides in length.

[0120] In some embodiments, the contiguous nucleotide sequence comprises or consists of 26 nucleotides in length.

[0121] In some embodiments, the contiguous nucleotide sequence comprises or consists of 27 nucleotides in length.

[0122] In some embodiments, the contiguous nucleotide sequence comprises or consists of 28 nucleotides in length.

[0123] In some embodiments, the contiguous nucleotide sequence comprises or consists of 29 nucleotides in length.

[0124] In some embodiments, the contiguous nucleotide sequence comprises or consists of 30 nucleotides in length.

[0125] In some embodiments, the contiguous nucleotide sequence comprises or consists of 31 nucleotides in length.

[0126] In some embodiments, the contiguous nucleotide sequence comprises or consists of 32 nucleotides in length.

[0127] In some embodiments, the contiguous nucleotide sequence comprises or consists of 33 nucleotides in length.

[0128] In some embodiments, the contiguous nucleotide sequence comprises or consists of 34 nucleotides in length.

[0129] In some embodiments, the contiguous nucleotide sequence comprises or consists of 35 nucleotides in length.

[0130] In some embodiments, the contiguous nucleotide sequence is the same length as the first and / or second strand of the double-stranded RNA molecule.

[0131] In some embodiments, the first or second strand of the double-stranded RNA molecule consists of a contiguous nucleotide sequence.

[0132] In some embodiments, the first or second strand of the double-stranded RNA molecule is a contiguous nucleotide sequence.

[0133] Conjugate moiety The present inventors have demonstrated that double-stranded RNA molecules capable of binding to a target sequence can be effectively administered to the eye by conjugating the double-stranded RNA molecule to at least one conjugate moiety.

[0134] As illustrated in the Examples, administration of the double-stranded RNA molecules of the present invention to the eye can inhibit the expression of targets, particularly targets expressed in the eye.

[0135] The double-stranded RNA molecules of the present invention are linked to at least one conjugate moiety. In some embodiments, the double-stranded RNA molecules may be linked to more than one conjugate moiety.

[0136] In some embodiments, one conjugate moiety or multiple conjugate moieties may be referred to as a conjugate of the present invention. In some embodiments, the double-stranded RNA molecule is covalently linked to at least one conjugate moiety.

[0137] The terms "bound," "positioned," "linked," and "conjugated" are interchangeable with respect to the double-stranded RNA molecule and the conjugated moiety.

[0138] The term "conjugate" as used herein refers to a double-stranded RNA molecule linked, for example, covalently linked, to a conjugate moiety. The conjugate moiety may be directly linked, for example, covalently linked, to the double-stranded RNA molecule, or the conjugate moiety may be linked to the double-stranded RNA molecule via a linker group.

[0139] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

[0140] In some embodiments, the conjugate moiety is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), and combinations thereof.

[0141] In some embodiments, the conjugate moiety can be one fatty acid molecule. In some embodiments, the conjugate moiety can be multiple fatty acid molecules.

[0142] A "fatty acid" is typically a molecule consisting of a chain of carbon atoms, with hydrogen atoms typically attached to the carbon atoms along the length of the chain. In other words, a fatty acid molecule comprises a hydrocarbon. A hydrogen atom is typically also found at one end (or terminus) of the chain of a fatty acid molecule, and a carboxyl group (-COOH) is typically found at the other end (or terminus) of the chain. In fact, it is the carboxyl group that makes the molecule an acid (e.g., a carboxylic acid). As used herein, the terms "fatty acid" and "fatty acid molecule" are considered interchangeable.

[0143] The term "fatty acid" encompasses one single fatty acid molecule as well as a mixture of two or more fatty acid molecules, for example two or more different fatty acid molecules.

[0144] In embodiments in which more than one fatty acid molecule is attached to the double-stranded RNA molecule, each fatty acid molecule is independently selected such that the fatty acid molecules attached to the double-stranded RNA molecule may or may not be the same.

[0145] In some embodiments, two or more fatty acids may be connected with a linker, and it will be understood that the linker connecting two or more fatty acids may be attached to any point on each of the two or more fatty acid molecules.

[0146] A fatty acid molecule can be a molecule made up of carbon atoms. A fatty acid molecule can have the formula CX, where C means carbon and X refers to the total number of carbon atoms (e.g., the carbon chain length) present in the fatty acid molecule (e.g., "C12" refers to a 12-carbon fatty acid molecule).

[0147] In some embodiments, the fatty acid molecule can be a molecule having between 3 and 40 carbon atoms (eg, C3-C40).

[0148] In some embodiments, the fatty acid molecule is selected from the list consisting of C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39 and C40.

[0149] In some embodiments, the fatty acid molecule can be a molecule having 12 to 24 carbon atoms (eg, C12 to C24).

[0150] In some embodiments, the fatty acid molecule is selected from the list consisting of C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24.

[0151] In some embodiments, the fatty acid molecule is C10.

[0152] In some embodiments, the fatty acid molecule is C16.

[0153] In some embodiments, the fatty acid molecule is C18.

[0154] In some embodiments, the fatty acid molecule is C22.

[0155] In some embodiments, the fatty acid molecule can be a salt of the fatty acid molecule (eg, a fatty acid salt).

[0156] In some embodiments, the fatty acid molecule may be branched or unbranched. In other words, the fatty acid molecule may include a branched chain or an unbranched chain. "Unbranched" refers to a straight chain of atoms, e.g., a straight chain of carbon atoms bonded to hydrogen atoms (e.g., CH groups), with the understanding that the carbon atoms may also be linked by carbon-carbon bonds.

[0157] "Branched" will also be understood to refer to a non-linear chain of atoms, for example, where one or more carbon groups form a branch by being attached to another chain of carbon atoms bonded to a hydrogen atom (e.g., a CH2 group), and the carbon atoms are also linked by carbon-carbon bonds. For example, a branched chain fatty acid molecule can include one or more carbon groups (such as methyl group(s)) attached to a chain of carbon atoms. The one or more carbon groups forming the branch(es) can be attached at any point along the carbon chain (i.e., can be attached to one or more of positions C2 through C-N-1, where C means carbon and N refers to the total number of carbon atoms in the linear chain).

[0158] It is understood that when two or more fatty acids are connected by a linker, all of the fatty acids may be branched.It is also understood that when two or more fatty acids are connected by a linker, all of the fatty acids may not be branched.It is also understood that when two or more fatty acids are connected by a linker, one or more of the fatty acids may be branched, and the remaining one or more fatty acids may not be branched.

[0159] It will be understood that fatty acids that do not contain carbon double bonds (e.g., C=C bonds) may be referred to as saturated fatty acids. It will be understood that the term "saturated" means that the maximum possible number of atoms (e.g., hydrogen atoms) are attached to each carbon in the molecule.

[0160] In some embodiments, the fatty acid molecule may be a saturated fatty acid. In some embodiments, the fatty acid molecule may be fully saturated (i.e., containing a single carbon bond but no double or triple carbon bonds). In some embodiments, the fatty acid molecule may be partially saturated (i.e., containing a combination of (i) one or more single carbon bonds; and (ii) one or more non-single carbon bonds (i.e., double and / or triple carbon bonds)).

[0161] In some embodiments, a fatty acid molecule may contain a double carbon bond (e.g., a C=C bond). In some embodiments, a fatty acid molecule may contain a triple carbon bond (e.g., a C≡C bond). In some embodiments, a fatty acid molecule may contain a combination of double carbon bonds (e.g., a C=C bond) and triple carbon bonds (e.g., a C≡C bond). It will be understood that when a fatty acid molecule contains double carbon bond(s) and / or triple carbon bond(s), the remaining bonds within the fatty acid molecule may be single carbon bonds (e.g., C≡C bonds).

[0162] It is understood that fatty acids containing one or more carbon-carbon double bonds and / or one or more carbon-carbon triple bonds can be referred to as unsaturated fatty acids. Unsaturated fatty acids containing one carbon-carbon double bond or one carbon-carbon triple bond can be referred to as monounsaturated fatty acids. Unsaturated fatty acids containing two or more non-single bonds (i.e., carbon-carbon double bonds and / or carbon-carbon triple bonds) can be referred to as polyunsaturated fatty acids.

[0163] In some embodiments, the fatty acid molecule can be an unsaturated fatty acid. In some embodiments, the fatty acid molecule can be a monounsaturated fatty acid. In some embodiments, the fatty acid molecule can be a polyunsaturated fatty acid.

[0164] In some embodiments, the fatty acid molecule can be fully unsaturated (i.e., contains double and / or triple carbon bonds, but no single carbon bonds). In some embodiments, the fatty acid molecule can be partially unsaturated (i.e., contains a combination of (i) one or more non-single carbon bonds (i.e., double and / or triple carbon bonds) and (ii) one or more single carbon bonds).

[0165] It is understood that when two or more fatty acids are connected by a linker, all of the fatty acids may be saturated.It is also understood that when two or more fatty acids are connected by a linker, all of the fatty acids may be unsaturated.It is also understood that when two or more fatty acids are connected by a linker, one or more of the fatty acids may be saturated, and the remaining one or more fatty acids may be unsaturated.

[0166] It will be understood that the saturated or unsaturated fatty acid molecules may be branched or unbranched.

[0167] In some embodiments, the saturated fatty acid molecule can be branched or unbranched.

[0168] In some embodiments, unsaturated fatty acid molecules can be branched or unbranched. In some embodiments, monounsaturated fatty acid molecules can be branched or unbranched. In some embodiments, polyunsaturated fatty acid molecules can be branched or unbranched.

[0169] In some embodiments, branched fatty acid molecules may be fully saturated, fully unsaturated, or a mixture of both saturated and unsaturated, while in some embodiments, unbranched fatty acid molecules may be fully saturated, fully unsaturated, or a mixture of both saturated and unsaturated.

[0170] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the fatty acid molecules contain a double bond.

[0171] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the fatty acid molecules contain triple bonds.

[0172] In some embodiments, the fatty acid molecule contains one or more double bonds. In some embodiments, the fatty acid molecule contains one or more triple bonds. In some embodiments, the fatty acid molecule contains a combination of one or more double carbon bonds and one or more triple carbon bonds. It will be understood that when a fatty acid molecule contains one or more double carbon bonds and / or triple carbon bonds, the remaining bonds in the fatty acid molecule may be single carbon bonds (e.g., C-C bonds).

[0173] In some embodiments, the fatty acid molecule comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 or more carbon double bonds.

[0174] In some embodiments, the fatty acid molecule comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 or more carbon triple bonds.

[0175] In some embodiments, the fatty acid molecule comprises a combination of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 or more carbon double and triple bonds.

[0176] It will be appreciated that more than 19 carbon double bonds may be used in the present invention. It will be appreciated that more than 19 carbon triple bonds may be used in the present invention.

[0177] The carbon-carbon double bond may be in a cis or trans configuration. The carbon-carbon triple bond may be in a cis or trans configuration. It will be understood that a cis configuration means that the two functional groups (e.g., hydrogen atoms) adjacent to the carbon-carbon double or triple bond protrude from the same side of the chain, and that a trans configuration means that the two functional groups (e.g., hydrogen atoms) adjacent to the carbon-carbon double or triple bond are on opposite sides of the chain.

[0178] In some embodiments, the fatty acid molecule can be a cis-unsaturated fatty acid molecule.

[0179] In some embodiments, the fatty acid molecule can be a trans-unsaturated fatty acid molecule.

[0180] In some embodiments, the fatty acid molecules may contain a combination of cis-unsaturated and trans-unsaturated fatty acid components. In some embodiments, the fatty acid molecules may contain a combination of cis- and trans-double bonds. In some embodiments, the fatty acid molecules may contain a combination of cis- and trans-triple bonds. In some embodiments, the fatty acid molecules may contain a combination of cis- and trans-double bonds and cis- and trans-triple bonds.

[0181] A fatty acid molecule may have the formula CX:Y, where C means carbon, X refers to the total number of carbon atoms present in the fatty acid molecule (i.e., the carbon chain length), and Y refers to the total number of unsaturated / non-single carbon bonds (i.e., double carbon bonds and / or triple carbon bonds) present in the fatty acid molecule (e.g., "C16:5" refers to a fatty acid molecule 16 carbons in length with a total of 5 unsaturated / non-single carbon bonds (i.e., there can be 5 double carbon bonds or there can be 5 triple carbon bonds). (There may be a combination of carbon-carbon double and carbon-carbon triple bonds, or there may be a combined mix of carbon-carbon double and carbon-carbon triple bonds, with a total of five non-single bonds). In other words, the number X before the colon specifies the number of carbon atoms, and the number Y after the colon specifies the total number of unsaturations / non-single bonds (i.e., double and / or triple bonds) in the fatty acid molecule. X can be any positive natural number (i.e., 1, 2, 3, etc.). X cannot be 0 or a negative value. Y can be any non-negative number (i.e., 0, 1, 2, 3, etc.).

[0182] However, for a given number of X (in CX:Y), the maximum number of double and / or triple bonds, Y, is equal to X / 2-1 (i.e., Y=(X / 2)-1). In other words, for a given number of carbons, X, in a fatty acid molecule, the number of double and / or triple bonds, Y, can be any non-negative number (i.e., 0, 1, 2, 3, etc.) up to Y=(X / 2)-1. Thus, in some embodiments in which a fatty acid molecule has the formula CX:Y, Y can be equal to the maximum value of X / 2-1 (i.e., Y=(X / 2)-1). If X is an odd number, the result (a number ending in 0.5) must be rounded down to the nearest whole number. For example, if X=7, the result of (X / 2)-1 is 2.5, which must be rounded down to 2. In other words, if X=7, the maximum number of double and / or triple bonds is Y=2; if X=9, the maximum number of double and / or triple bonds is Y=3, etc. If X is an even number, the result for Y does not need to be rounded down: if X=8, then Y=3; if X=10, then Y=4, etc. This means that when X=8 or 9 (i.e., for fatty acid molecules with 8 or 9 carbons), the maximum number of possible double and / or triple bonds is 3, and note that the value Y in C8:Y or C9:Y can be any whole number between 0 and 3 (i.e., 0, 1, 2, or 3). In other words, when X=8, the possible fatty acids are C8:0, C8:1, C8:2, and C8:3; when X=9, the possible fatty acids are C9:0, C9:1, C9:2, and C9:3. The same is true for any other value of X (where X is a positive whole number, i.e., non-zero and non-negative).

[0183] In some embodiments, the fatty acid molecule is C3:0; C4:0; C4:1; C5:0; C5:1; C6:0; C6:1; C6:2; C7:0; C7:1; C7:2; C8:0; C8:1; C8:2; C8:3; C9:0; C9:1; C9:2; C9:3; C10:0; C10:1; C10:2; C10:3; C10:4; C11:0; C11:1; C11:2; C11:3; C11:4; C12:0; C12:1; C12:2; C12:3; C12:4; C12:5; C13:0; C13:1; C13:2; C13:3; C13:4; C13:5; C14:0; C14:1; C14:2; C14:3; C14:4; C14:5; C14:6; C15:0; C15:1; C15:2; C15:3; C15:4; C15:5; C15:6; C16:0; C16:1; C16:2; C16:3; C16:4; C16:5; C16:6; C16:7; C17:0; C17:1; C17:2; C17:3; C17:4; C17:5; C17:6; C17:7; C18:0; C18:1; C18:2; C18:3; C18:4; C18:5; C18:6; C18:7; C18:8; C19:0; C19:1; C19:2; C19:3; C19:4; C19:5; C19:6; C19:7; C19:8; C20:0; C20:1; C20:2; C20:3; C20:4; C20:5; C20:6; C20:7; C20:8; C20:9; C21:0; C21:1; C21:2; C21:3; C21:4; C21:5; C21:6; C21:7; C21:8; C21:9; C22:0; C22:1; C22:2; C22:3; C22:4; C22:5; C22:6; C22:7; C22:8; C22:9; C22:10; C23:0; C23:1; C23:2; C23:3; C23:4; C23:5; C23:6; C23:7; C23:8; C23:9; C23:10; C24:0; C24:1; C24:2; C24:3; C24:4; C24:5; C24:6; C24:7; C24:8; C24:9; C24:10; C24:11; C25:0; C25:1; C25:2; C25:3; C25:4; C25:5; C25:6; C25:7; C25:8; C25:9; C25:10; C25:11; C26:0; C26:1; C26:2; C26:3; C26:4; C26:5; C26:6; C26:7; C26:8; C26:9;<h2 style=";text-align:left;direction:ltr">C26:10;C26:11;C26:12;C27:0;C27:1;C27:2;C27:3;C27:4;C27:5;C27:6;C27:7;C27:8;C27:9;C27:10;C27:11;C27:12;C28:0;C28:1;C28:2;C28: 3;C28:4;C28:5;C28:6;C28:7;C28:8;C28:9;C28:10;C28:11;C28:12;C28 :13;C29:0;C29:1;C29:2;C29:3;C29:4;C29:5;C29:6;C29:7;C29:8;C29:9 ;C29:10;C29:11;C29:12;C29:13;C30:0;C30:1;C30:2;C30:3;C30:4;C30 :5;C30:6;C30:7;C30:8;C30:9;C30:10;C30:11;C30:12;C30:13;C30:14;C 31:0;C31:1;C31:2;C31:3;C31:4;C31:5;C31:6;C31:7;C31:8;C31:9;C31 :10;C31:11;C31:12;C31:13;C31:14;C32:0;C32:1;C32:2;C32:3;C32:4;C 32:5;C32:6;C32:7;C32:8;C32:9;C32:10;C32:11;C32:12;C32:13;C32:14;C32:15;C33:0;C33:1;C33:2;C33:3;C33:4;C33:5;C33:6;C33:7;C33:8; C33:9;C33:10;C33:11;C33:12;C33:13;C33:14;C33:15;C34:0;C34:1;C34:2;C34:3;C34:4;C34:5;C34:6;C34:7;C34:8;C34:9;C34:10;C34:11;C34 :12;C34:13;C34:14;C34:15;C34:16;C35:0;C35:1;C35:2;C35:3;C35:4; C35:5;C35:6;C35:7;C35:8;C35:9;C35:10;C35:11;C35:12;C35:13;C35:1 4;C35:15;C35:16;C36:0;C36:1;C36:2;C36:3;C36:4;C36:5;C36:6;C36: 7;C36:8;C36:9;C36:10;C36:11;C36:12;C36:13;C36:14;C36:15;C36:16;C36:17;C37:0;C37:1;C37:2;C37:3;C37:4;C37:5;C37:6;C37:7;C37:8;C37 :9;C37:10;C37:11;C37:12;C37:13;C37:14;C37:15;C37:16;C37:17;C38:0 ;C38:1;C38:2;C38:3;C38:4;C38:5;C38:6;C38:7;C38:8;C38:9;C38:10;C3 8:11;C38:12;C38:13;C38:14;C38:15;C38:16;C38:17;C38:18;C39:0;C39: 1;C39:2;C39:3;C39:4;C39:5;C39:6;C39:7;C39:8;C39:9;C39:10;C39:11; C39:12;C39:13;C39:14;C39:15;C39:16;C39:17;C39:18;C40:0;C40:1;C40 :2;C40:3;C40:4;C40:5;C40:6;C40:7;C40:8;C40:9;C40:10;C40:11;C40:12;C40:13;C40:14;C40:15;C40:16;C40:17;C40:18;C40:19. ;

[0184] One carbon-carbon double bond can be located at any one point within a fatty acid molecule (i.e., between any pair of adjacent carbon atoms). Multiple carbon-carbon double bonds can be located at any multiple points within a fatty acid molecule (i.e., between multiple pairs of adjacent carbon atoms). One carbon-carbon triple bond can be located at any one point within a fatty acid molecule (i.e., between any pair of adjacent carbon atoms). Multiple carbon-carbon triple bonds can be located at any multiple points within a fatty acid molecule (i.e., between multiple pairs of adjacent carbon atoms).

[0185] In some embodiments, the fatty acid molecule may contain one or more modifications and / or substitutions. In some embodiments, the fatty acid molecule may contain one or more amino acids. In some embodiments, the fatty acid molecule may contain one or more sugar or carbohydrate molecules. It will be understood that modifications and / or substitutions can be made to saturated, unsaturated, monounsaturated, and polyunsaturated fatty acid molecules. It will also be understood that modifications and / or substitutions can be made to branched and unbranched fatty acid molecules.

[0186] It will be understood that the binding strength of the double-stranded RNA molecules of the present invention can be intentionally controlled (e.g., adjusted) by the length of the fatty acid molecule / moiety used, as shown in Figure 4. Without wishing to be bound by theory, it can be thought that the binding strength of the double-stranded RNA molecules of the present invention (i.e., conjugated to a fatty acid molecule) can be proportional to the length of the fatty acid, as exemplified in Figure 4 using C10, C16, and C22 fatty acid conjugates, particularly when albumin is used as a transport vehicle.

[0187] In some embodiments, the fatty acid molecules / moieties used as conjugate moieties can be selected based on the required binding strength, depending on the needs of the specific intended use of the double-stranded RNA molecules of the present invention.

[0188] In some embodiments, the double-stranded RNA molecules of the present invention can be administered in combination with albumin, which can be serum albumin such as mouse serum albumin or human serum albumin.

[0189] Without wishing to be bound by theory, binding of the double-stranded RNA molecule of the present invention to albumin may be one of the mechanisms by which the double-stranded RNA molecule of the present invention can be transported, for example, into cells.

[0190] Without wishing to be bound by theory, the binding strength of the double-stranded RNA molecules of the present invention can be utilized to affect (i) the circulating profile of the double-stranded RNA of the present invention in plasma; (ii) the stability of the double-stranded RNA of the present invention in biological fluids (e.g., tears); and (iii) the cellular uptake of the double-stranded RNA of the present invention into disease-related tissues.

[0191] In some embodiments, the fatty acid molecule of the double-stranded RNA molecule of the present invention can be a fatty acid molecule with binding strength to albumin suitable for affecting the circulating profile of the double-stranded RNA of the present invention in plasma. In some embodiments, the suitable binding strength can be a binding strength suitable for binding to albumin.

[0192] In some embodiments, the fatty acid molecule conjugated to the double-stranded RNA of the present invention may have a binding strength to albumin suitable for affecting the stability of the double-stranded RNA of the present invention in biological fluids (e.g., tears). In some embodiments, the appropriate binding strength may be a binding strength suitable for binding to albumin.

[0193] In some embodiments, the fatty acid molecule conjugated to the double-stranded RNA of the present invention may have a binding strength to albumin suitable for influencing the cellular uptake of the double-stranded RNA of the present invention into disease-related tissues. In some embodiments, the suitable binding strength may be a binding strength suitable for binding to albumin.

[0194] In some embodiments, one conjugate moiety is one cholesterol molecule. In some embodiments, multiple conjugate moieties can be multiple cholesterol molecules. It will be understood that the terms "cholesterol molecule" and "cholesterol moiety" are interchangeable.

[0195] In embodiments in which more than one cholesterol molecule is attached to the double-stranded RNA molecule, each cholesterol molecule is independently selected such that the cholesterol molecules attached to the double-stranded RNA molecule may or may not be the same.

[0196] In some embodiments, two or more cholesterol molecules may be connected with a linker, and it will be understood that the linker connecting two or more cholesterol molecules may be attached to any point on each of the two or more cholesterol molecules.

[0197] In some embodiments, the cholesterol moiety is selected from the group comprising 3'-cholesteryl-TEG CPG, 5'-cholesterol-TEG-CE phosphoramidite, 5'-cholesterol-CE phosphoramidite or cholesteryl-TEG-CE phosphoramidite (TEG = triethylene glycol, CPG = Controlled Pore Glass Synthesis Supports, also known as CPG supports).

[0198] In some embodiments, the cholesterol moiety is 3'-cholesteryl-TEG CPG. In some embodiments, the cholesterol moiety is derived from 3'-cholesteryl-TEG CPG. For example, 3'-cholesteryl-TEG CPG can be used as an agent for introducing a cholesterol moiety into the 3' end of one strand of a double-stranded RNA molecule.

[0199] In some embodiments, the cholesterol moiety is 5'-cholesterol-TEG-CE phosphoramidite. In some embodiments, the cholesterol moiety is derived from 5'-cholesterol-TEG-CE phosphoramidite. For example, 5'-cholesterol-TEG-CE phosphoramidite can be used as an agent for introducing a cholesterol moiety into the 5' end of one strand of a double-stranded RNA molecule.

[0200] In some embodiments, the cholesterol moiety is 5'-cholesterol-CE phosphoramidite. In some embodiments, the cholesterol moiety is derived from 5'-cholesterol-CE phosphoramidite. For example, 5'-cholesterol-CE phosphoramidite can be used as an agent for introducing a cholesterol moiety into the 5' end of one strand of a double-stranded RNA molecule.

[0201] In some embodiments, the cholesterol moiety is cholesteryl-TEG-CE phosphoramidite. In some embodiments, the cholesterol moiety is derived from cholesteryl-TEG-CE phosphoramidite. For example, cholesteryl-TEG-CE phosphoramidite can be used as an agent for introducing a cholesterol moiety into one end of a chain of a double-stranded RNA molecule.

[0202] In some embodiments, the strands of a double-stranded RNA molecule can be synthesized by using normal amidites or inverted amidites.

[0203] In some embodiments, a cholesterol molecule or cholesterol moiety is attached to the 3' end of one of the strands of a double-stranded RNA molecule by using an inverted amidite (e.g., a nucleoside phosphoramidite). In other words, a strand can be constructed using an inverted amidite, and then cholesterol can be attached to the 3' end of one of the strands by using any 5' cholesterol amidite at the end of the synthesis, thereby placing the cholesterol at the 3' end of the strand.

[0204] In some embodiments, a conjugate moiety (eg, a fatty acid molecule or a cholesterol molecule) is positioned at the 5' or 3' end of one of the strands of the double-stranded RNA molecule.

[0205] In some embodiments, the conjugate moiety is positioned at the 5'-end of the first strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is positioned at the 3'-end of the first strand of the double-stranded RNA molecule.

[0206] In some embodiments, the conjugate moiety is positioned at the 5'-end of the second strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is positioned at the 3'-end of the second strand of the double-stranded RNA molecule.

[0207] A double-stranded RNA molecule can be described in terms of comprising a sense strand and an antisense strand.

[0208] In some embodiments, the conjugate moiety is positioned at the 5'-end of the sense strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is positioned at the 3'-end of the sense strand of the double-stranded RNA molecule.

[0209] In some embodiments, the conjugate moiety is positioned at the 5'-end of the antisense strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is positioned at the 3'-end of the antisense strand of the double-stranded RNA molecule.

[0210] In some embodiments, the conjugate moiety is not located at the terminal position of either strand (i.e., the conjugate moiety is not located at the 5' or 3' end of either strand). For example, the conjugate moiety may be attached to a position within the middle or central region of the contiguous nucleotide sequence. As used herein, the terms "middle" and "middle" are intended to indicate that the conjugate moiety is not located at either end of the strand, and do not indicate that the conjugate moiety is located equidistant from each end.

[0211] In some embodiments, the conjugate moiety is located at any position in the contiguous nucleotide sequence. In some embodiments, the conjugate moiety is located at any position on the double-stranded RNA molecule.

[0212] Linker Linkage, linker or spacer is the connection between two atoms, which connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.Conjugate moiety can be directly or through linking moiety (such as linker or spacer) to double-stranded RNA molecule.Linker plays the role of covalently connecting conjugate moiety to double-stranded RNA molecule or its continuous nucleotide sequence.

[0213] As used herein, the terms "linker" and "spacer" are interchangeable.

[0214] In some embodiments of the present invention, the double-stranded RNA molecule of the present invention may include a linker (also referred to as a "linker region") disposed between the double-stranded RNA molecule and the conjugate moiety. The linker may be attached to a continuous nucleotide sequence of the strand of the double-stranded RNA molecule that is complementary to the target nucleic acid and the conjugate moiety.

[0215] In some embodiments, the linker is C6.

[0216] In some embodiments, the linker is TEG.

[0217] In some embodiments, the linker is a dinucleotide. In some embodiments, the linker is a CA dinucleotide.

[0218] In some embodiments, the linker is a biocleavable linker. A biocleavable linker comprises or consists of a physiologically labile bond that is cleavable under conditions normally encountered or similar to those encountered in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In some embodiments, the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker comprises one to five nucleosides, such as DNA nucleosides containing at least two consecutive phosphodiester linkages. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195.

[0219] In some embodiments, the linker is not a biocleavable linker. Linkers that are not necessarily biocleavable but function primarily to covalently connect the conjugate moiety to the oligonucleotide are known. These linkers may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups, or combinations thereof. In some embodiments, the linker is an aminoalkyl, such as a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group.

[0220] Additional 5' and / or 3' nucleosides In some embodiments, one or both strands of the double-stranded RNA molecule of the present invention may further comprise additional 5' and / or 3' nucleosides. In other words, in some embodiments, the double-stranded RNA molecule of the present invention may comprise 5' and / or 3' nucleosides in addition to the contiguous nucleotide sequence.

[0221] The additional 5' and / or 3' nucleosides may or may not be complementary, eg, perfectly complementary, to the target nucleic acid.

[0222] The addition of additional 5' and / or 3' nucleosides can be used to join consecutive nucleotide sequences to a conjugate moiety or another functional group. When used to join consecutive nucleotide sequences to a conjugate moiety, it can function as a biocleavable linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0223] The additional 5' and / or 3' nucleosides may independently comprise or consist of 1, 2, 3, 4, 5 or more than 5 additional nucleotides that may be complementary or non-complementary to the target nucleic acid.

[0224] Additional 5' and / or 3' nucleosides can function as nuclease-sensitive biocleavable linkers. In some embodiments, the additional 5' and / or 3' terminal nucleotides are linked by phosphodiester linkages and are DNA or RNA. Suitable nucleotide-based biocleavable linkers for such use are disclosed in WO 2014 / 076195, including, for example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015 / 113922, and they are used to link multiple antisense constructs within a single oligonucleotide.

[0225] In some embodiments, the internucleoside linkages located between the additional 5' and / or 3' nucleosides and the contiguous nucleotide sequence are phosphodiester linkages.

[0226] Ocular administration The double-stranded RNA molecules of the present invention are administered locally to the eye (eg, ocular administration).

[0227] It should be understood that the double-stranded RNA molecules of the present invention can be administered to any part of the eye.

[0228] In some embodiments, the double-stranded RNA molecule is for administration to the anterior segment of the eye.

[0229] Both conjunctiva and cornea are components of the eye.In some embodiments, the double-stranded RNA molecule is intended for administration to the conjunctiva.In some embodiments, the double-stranded RNA molecule is intended for administration to the cornea.In some embodiments, the double-stranded RNA molecule targets the conjunctiva.In some embodiments, the double-stranded RNA molecule targets the cornea.

[0230] The conjunctiva includes the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva, and conjunctival fornix. In some embodiments, the double-stranded RNA molecule is for administration to the bulbar conjunctiva. In some embodiments, the double-stranded RNA molecule is for administration to the palpebral conjunctiva. In some embodiments, the double-stranded RNA molecule is for administration to the ocular conjunctiva. In some embodiments, the double-stranded RNA molecule is for administration to the conjunctival fornix. In some embodiments, the double-stranded RNA molecule is for administration to one or more of the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva, and conjunctival fornix. In some embodiments, the double-stranded RNA molecule targets the bulbar conjunctiva. In some embodiments, the double-stranded RNA molecule targets the palpebral conjunctiva. In some embodiments, the double-stranded RNA molecule targets the ocular conjunctiva. In some embodiments, the double-stranded RNA molecule targets the conjunctival fornix. In some embodiments, the double-stranded RNA molecule targets one or more of the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva, and conjunctival fornix.

[0231] In some embodiments, the double-stranded RNA molecule may be for administration to the cornea and conjunctiva. In some embodiments, the double-stranded RNA molecule may target the cornea and conjunctiva.

[0232] In some embodiments, the double-stranded RNA molecules of the present invention can be administered in combination with albumin, which can be serum albumin such as mouse serum albumin or human serum albumin.

[0233] Complementarity to the target sequence The contiguous nucleotide sequence of the double-stranded RNA molecule of the present invention can be complementary to a target nucleic acid sequence, for example, a nucleic acid sequence of a target mRNA.

[0234] In some embodiments, the contiguous nucleotide sequence can be perfectly complementary, eg, about 100%, to the target nucleic acid sequence.

[0235] In some embodiments, the contiguous nucleotide sequence may be partially complementary to the target nucleic acid sequence, e.g., at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary to the target nucleic acid sequence.

[0236] In some embodiments, the contiguous nucleotide sequence can contain 1, 2, 3, 4, 5, 6, 7, 8 or more mismatches, where a mismatch is a nucleotide within the contiguous nucleotide sequence that does not base pair with its target.

[0237] The target nucleic acid sequence can be a target nucleic acid sequence, such as an mRNA sequence, expressed or present in the eye.

[0238] In some embodiments, the target may be any target present in the eye. In some embodiments, the target nucleic acid sequence may be any nucleic acid sequence expressed or present in the eye. In some embodiments, the target nucleic acid sequence may be any mRNA sequence expressed or present in the eye. Reference to the eye herein will be understood to include any part of the eye. Reference to the eye herein will also be understood to include any combination of parts of the eye, including, but not limited to, the anterior part of the eye or all parts of the eye.

[0239] In some embodiments, the target can be any target associated with an ocular condition, ocular disease, or ocular condition. In some embodiments, the target can be any target associated with one or more ocular conditions, ocular diseases, or ocular conditions.

[0240] In some embodiments, the target nucleic acid sequence can be any nucleic acid sequence associated with an ocular condition, ocular disease, or ocular symptom. In some embodiments, the target nucleic acid sequence can be any nucleic acid sequence associated with one or more ocular conditions, ocular diseases, or ocular symptoms.

[0241] In some embodiments, the target nucleic acid sequence can be any mRNA sequence associated with an ocular condition, ocular disease, or ocular symptom. In some embodiments, the target nucleic acid sequence can be any mRNA sequence associated with one or more ocular conditions, ocular diseases, or ocular symptoms.

[0242] It will be understood that a target (or target nucleic acid sequence or target mRNA sequence) associated with an ocular condition, ocular disease or ocular symptom includes, but is not limited to, any target (or target nucleic acid sequence or target mRNA sequence) that causes, contributes to, or is a marker of the ocular condition, ocular disease or ocular symptom.

[0243] An example of a specific target that can be used in the present invention is AHA-1. It will be understood that AHA-1 is a non-limiting example of a target. It will also be understood that multiple other targets are encompassed by the present invention.

[0244] AHA1 (or AHA-1), in particular the AHA1 gene, is also known as p38; AHSA1; hAha1; C14orf3. In the context of the present invention, it will be understood that "AHA1," "p38," "AHSA1," "hAha1," and C14orf3 are interchangeable terms.

[0245] In some embodiments, the target is an AHA-1 nucleic acid sequence.

[0246] The AHA-1 target nucleic acid sequence can include or consist of SEQ ID NO:1 or SEQ ID NO:2.

[0247] SEQ ID NO: 1 (human AHA1 target sequence: positions 488-507, NCBI reference sequence: NM_012111.3): 5'-AATCTCGTGGCCTTAATGAAA-3' SEQ ID NO: 2 (Rabbit AHA1 target sequence: positions 548-567, NCBI reference sequence: XM_002719625.3): 5'-AATCTCGTGGCCTTAATGAAG-3

[0248] It will be understood that there is a "wobble" base pair mismatch in SEQ ID NO:2 compared to SEQ ID NO:1 (i.e., SEQ ID NO:1 differs from SEQ ID NO:2 in that an A becomes a G at the last nucleotide position). Without wishing to be bound by theory, this mismatch should not affect the activity of the double-stranded RNA because either A or G can still hybridize (e.g., by wobble base pairing) to a complementary U in the consecutive nucleotide sequence of the strands of the double-stranded RNA molecule. It will be understood that if a wobble base pair mismatch is present, this mismatch should not affect the activity of the double-stranded RNA molecule.

[0249] In some embodiments, the contiguous nucleotide sequence comprises or consists of a nucleotide sequence complementary to an AHA-1 target sequence.

[0250] In some embodiments, the contiguous nucleotide sequence comprises or consists of a nucleotide sequence complementary to SEQ ID NO:1.

[0251] In some embodiments, the contiguous nucleotide sequence comprises or consists of a nucleotide sequence complementary to SEQ ID NO:2.

[0252] In some embodiments, the contiguous nucleotide sequence comprises or consists of SEQ ID NO:3.

[0253] SEQ ID NO: 3 (AHA-1 siRNA antisense strand): 5'-UUUCAUUAAGGCCACGAGAUU-3'

[0254] In some embodiments, AHA-1 can be expressed in the bulbar and / or palpebral conjunctiva. In some embodiments, a decrease in AHA-1 expression can be observed in the bulbar and / or palpebral conjunctiva.

[0255] Target Inhibition In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting the expression of a target, in other words, the double-stranded RNA molecules may be capable of reducing the level of expression of a target.

[0256] The target can be a target nucleic acid sequence (such as a target mRNA sequence) or a target protein.

[0257] In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting expression of a target by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a control.

[0258] In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting expression of a target mRNA by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a control.

[0259] In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting expression of a target protein by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a control.

[0260] It will be understood that the % inhibition of target expression referred to above is the % reduction compared to the control, the term "control" referring to the expression of the target in cells not exposed to the double-stranded RNA molecule of the present invention.

[0261] In some embodiments, a control can be a mock transfection, e.g., treatment of cells with PBS, where reduced expression of the target includes reduced levels of target mRNA and / or reduced levels of target protein.

[0262] Nucleotides and Nucleosides Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and in the present invention, include both naturally occurring nucleotides and nucleosides and non-naturally occurring nucleotides and nucleosides.Naturally, nucleotides, such as DNA nucleotides and RNA nucleotides, comprise a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (not present in nucleosides).Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

[0263] Modified double-stranded RNA molecules The double-stranded RNA molecule of the present invention may be a modified double-stranded RNA molecule.

[0264] The term "modified double-stranded RNA molecule" encompasses double-stranded RNA molecules comprising one or more sugar-modified nucleosides and / or modified internucleoside linkages.

[0265] In some embodiments, the double-stranded RNA molecule or its contiguous nucleotide sequence may contain modified nucleobases that function as the designated nucleobase in base pairing, for example, 5-methylcytosine may be used instead of methylcytosine. Inosine may be used as a universal base.

[0266] It will be understood that the consecutive nucleobase sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid.

[0267] The pattern in which modified nucleosides (such as high affinity modified nucleosides) are incorporated into an oligonucleotide sequence is commonly referred to as the oligonucleotide design.

[0268] In some embodiments, high affinity modified nucleosides may be used.

[0269] In one embodiment, the double-stranded RNA molecule contains at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least At least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49 or at least 50 modified nucleosides.

[0270] Suitable modifications are described herein.

[0271] Modified internucleoside linkages In some embodiments, the double-stranded RNA molecules of the present invention may contain one or more modified internucleoside linkages.

[0272] The term "modified internucleoside linkage" is defined as commonly understood by those skilled in the art as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides to one another. Thus, the double-stranded RNA molecules of the present invention can contain one or more modified internucleoside linkages, for example, one or more phosphorothioate internucleoside linkages.

[0273] In some embodiments, at least 50% of the internucleoside linkages in the double-stranded RNA molecule or its contiguous nucleotide sequence are modified, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or more of the internucleoside linkages in the double-stranded RNA molecule or its contiguous nucleotide sequence are modified. In some embodiments, all of the internucleoside linkages in the double-stranded RNA molecule or its contiguous nucleotide sequence are modified.

[0274] In some embodiments, one or more, or all, of the modified internucleoside linkages comprise a phosphorothioate linkage.

[0275] In some embodiments, at least 50% of the internucleoside linkages in the double-stranded RNA molecule or a contiguous nucleotide sequence thereof are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or more of the internucleoside linkages in the double-stranded RNA molecule or a contiguous nucleotide sequence thereof are phosphorothioate. In some embodiments, all of the internucleoside linkages in the double-stranded RNA molecule or a contiguous nucleotide sequence thereof are phosphorothioate.

[0276] In further embodiments, the double-stranded RNA molecule may contain at least one modified internucleoside linkage. In some embodiments, at least 75%, for example all, of the internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages.

[0277] In some embodiments, all internucleoside linkages of a contiguous nucleotide sequence of a double-stranded RNA molecule can be phosphorothioate, or all internucleoside linkages of a double-stranded RNA molecule can be phosphorothioate linkages.

[0278] Modified Nucleosides In some embodiments, the double-stranded RNA molecules of the present invention may contain one or more modified nucleosides.

[0279] The term "modified nucleoside" or "nucleoside modification," as used herein, refers to a nucleoside that is modified relative to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications in the sugar moiety or (nucleic acid) base moiety.

[0280] In some embodiments, one or more of the modified nucleosides of the double-stranded RNA molecules of the present invention may contain a modified sugar moiety. The term "modified nucleoside" may also be used interchangeably herein with the terms "nucleoside analog" or modified "unit" or modified "monomer." Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if they are capable of Watson-Crick base pairing. Exemplary modified nucleosides that can be used in the double-stranded RNA molecules of the present invention include LNA, 2'-O-MOE, 2'oMe, and morpholino nucleoside analogs.

[0281] Nucleic acid bases The term nucleobase includes the purine (eg, adenine and guanine) and pyrimidine (eg, uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization.

[0282] In the context of the present invention, the term "nucleobase" also encompasses modified nucleobases that may be different from naturally occurring nucleobases, but are functional during nucleic acid hybridization.In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-naturally occurring variants.Such variants are described, for example, in Hirao et al., 2012, Accounts of Chemical Research, 45, 2055-2065 and Bergstrom, 2009, Curr.Protoc.Nucleic Acid Chem., 37, 1.4.1-1.4.32.

[0283] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0284] Nucleobase moiety can be represented by the letter code of each corresponding nucleobase, for example, A, T, G, C or U, and each letter can optionally include a modified nucleobase of equivalent function.For example, in the exemplified oligonucleotide, nucleobase moiety is selected from A, T, G, C and 5-methylcytosine.Optionally, for LNA gapmer, 5-methylcytosine LNA nucleoside can be used.5-methylcytosine can be represented as "E".

[0285] High-affinity modified nucleosides High affinity modified nucleosides, when incorporated into oligonucleotides, can, for example, increase the melting temperature (Tm The high affinity modified nucleosides of the present invention are modified nucleosides that increase the affinity of an oligonucleotide for its complementary target, as measured by the affinity of the oligonucleotide for its complementary target. The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).

[0286] sugar modification The double-stranded RNA molecules of the present invention may include one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

[0287] Numerous nucleosides with modifications in the ribose sugar moiety have been created primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0288] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA), or a bicyclic ring, typically having a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring, typically lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0289] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0290] 2' sugar-modified nucleosides A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (2' substituted nucleoside), or a nucleoside containing a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon of the ribose ring, such as an LNA (2'-4' biradical bridge) nucleoside.

[0291] Indeed, much attention has been focused on the development of 2'-sugar-substituted nucleosides, and many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can confer improved binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleosides include 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'oMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213, and Deleavy and Damha, Chemistry and Biology 2012, 19, 937. Below are examples of some 2'-substituted modified nucleosides. [ka]

[0292] In the context of the present invention, 2'-substituted sugar modified nucleosides do not include 2'-bridged nucleosides such as LNA.

[0293] In some embodiments, the double-stranded RNA molecule comprises one or more sugar-modified nucleosides, for example, 2' sugar-modified nucleosides.

[0294] In some embodiments, the double-stranded RNA molecule comprises 1, 2, 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 sugar-modified nucleosides.

[0295] In some embodiments, the double-stranded RNA molecules of the present invention comprise one or more 2' sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'oMe), 2'-O-methoxyethyl-RNA (2'MOE), 2'-alkoxy-RNA, 2'-amino-DNA, 2'-fluoro-RNA 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. In some embodiments, one or more of the modified nucleosides may be LNA.

[0296] In some embodiments, the 2' sugar modified nucleoside is an affinity-enhancing 2' sugar modified nucleoside.

[0297] Locked nucleic acid nucleosides (LNA nucleosides) An "LNA nucleoside" is a 2'-modified nucleoside that contains a biradical linking C2' and C4' of the ribose sugar ring of the nucleoside (also referred to as a "2'-4' bridge"), which restricts or fixes the conformation of the ribose ring.

[0298] These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). The steric fixation of ribose is associated with improved hybridization affinity (duplex stabilization) when LNA is incorporated into oligonucleotides to complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0299] Non-limiting, exemplary LNA nucleosides include those described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al. al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al., J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry, 2016, 59, 9645-9667.

[0300] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1: [ka]

[0301] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxyLNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

[0302] A particularly advantageous LNA is beta-D-oxy-LNA.

[0303] Morpholino oligonucleotides In some embodiments, the double-stranded RNA molecules of the present invention comprise or consist of morpholino nucleosides (i.e., morpholino oligomers, as phosphorodiamidate morpholino oligomers (PMOs)). Splice-modulating morpholino oligonucleotides have been approved for clinical use; see, for example, eteplirsen, a 30-nt morpholino oligonucleotide that targets frameshift mutations in Duchenne muscular dystrophy (DMD). Morpholino oligonucleotides have nucleases attached to a six-membered morpholino ring rather than a ribose, such as methylene morpholine rings linked via phosphorodiamidate groups, as shown in the following diagram of four consecutive morpholino nucleotides: [ka]

[0304] In some embodiments, the double-stranded RNA molecules of the present invention can be, for example, 8 to 50 morpholino nucleotides in length.

[0305] Complementarity The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U).

[0306] Oligonucleotides may contain nucleosides having modified nucleobases, e.g., 5-methylcytosine is often substituted for cytosine, and it will be understood that the term complementarity therefore encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).

[0307] The term "% complementary," as used herein, refers to the proportion (in percent) of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) across the contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned nucleobases that are complementary (form Watson-Crick base pairs) between two sequences (when aligning the target sequence 5'-3' with the oligonucleotide sequence from 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in calculating the % complementarity of a contiguous nucleotide sequence.

[0308] It will be understood that in determining complementarity, chemical modifications of the nucleobases will be disregarded so long as the nucleobases retain their functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0309] identity The term "identity," as used herein, refers to the proportion (expressed as a percentage) of nucleotides of a contiguous nucleotide sequence within a nucleic acid molecule (e.g., an siRNA molecule) that are identical to a reference sequence (e.g., a sequence motif) over the contiguous nucleotide sequence.

[0310] Thus, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleobases between two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. It will be understood that chemical modifications of nucleobases are disregarded in determining identity, so long as the nucleobase retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating percent identity).

[0311] It is therefore understood that a relationship exists between identity and complementarity, such that a contiguous nucleotide sequence within a double-stranded RNA molecule of the present invention that is complementary to a target sequence also shares a percentage of identity with the complementary sequence.

[0312] Hybridization As used herein, the term "hybridize" or "hybridizing" should be understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is often measured by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) in physiological conditions, T mis not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° more accurately represents binding affinity, ΔG° = -RTln(K d ) to calculate the dissociation constant (K d ), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction in which the aqueous concentration is 1M, pH is 7, and the temperature is 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and the ΔG° in the case of a spontaneous reaction is less than zero. ΔG° can be experimentally measured by using isothermal titration calorimetry (ITC) method, for example, as described in Hansen et al., 1965, Chem.Comm.36-38 and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will know that commercially available devices are available for measuring ΔG°. ΔG° can be estimated numerically by using the nearest neighbor model described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, using appropriately derived thermodynamic parameters as described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.

[0313] In some embodiments, the degree or strength of hybridization is measured by the Gibbs free energy ΔG° under standard conditions. The oligonucleotide may hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal, e.g., less than -15 kcal, e.g., less than -20 kcal, and e.g., less than -25 kcal. In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of -10 to -60 kcal, e.g., -12 to -40, e.g., -15 to -30 kcal, or -16 to -27 kcal, e.g., -18 to -25 kcal.

[0314] salt The term "salt" as used herein conforms to its commonly known meaning, i.e., an ionic collection of anions and cations.

[0315] The present invention provides a pharmaceutically acceptable salt of the double-stranded RNA molecule of the present invention. In other words, the present invention provides a double-stranded RNA molecule of the present invention in the form of a pharmaceutically acceptable salt.

[0316] In some embodiments, the pharmaceutically acceptable salt may be a sodium salt or a potassium salt.

[0317] The present invention provides pharmaceutically acceptable sodium salts of the double-stranded RNA molecules of the present invention.

[0318] The present invention provides pharmaceutically acceptable potassium salts of the double-stranded RNA molecules of the present invention.

[0319] Delivery of double-stranded RNA molecules The double-stranded RNA molecules of the present invention can be encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

[0320] This may be for the purpose of delivering the double-stranded RNA molecule of the present invention to target cells and / or to improve the pharmacokinetics of the double-stranded RNA molecule.

[0321] Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0322] It will be appreciated that because the double-stranded RNA molecules of the present invention are conjugated to at least one conjugate moiety, encapsulation of the double-stranded RNA molecule is performed in addition to conjugation of the double-stranded RNA molecule to at least one conjugate moiety, which may provide additional advantages compared to conjugation to a conjugate moiety alone.

[0323] In some embodiments, the double-stranded RNA molecule of the present invention can be administered in combination with albumin.Albumin can be serum albumin, such as mouse serum albumin or human serum albumin.Without wishing to be bound by theory, albumin can function as a transport vehicle and / or can promote or enhance the uptake of the double-stranded RNA molecule of the present invention into relevant tissues or cells.

[0324] Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising the double-stranded RNA molecules of the present invention and pharmaceutically acceptable diluents, solvents, carriers, salts and / or adjuvants.

[0325] The present invention provides a pharmaceutical composition comprising the double-stranded RNA molecule of the present invention and a pharmaceutically acceptable salt. For example, the salt may contain a metal cation, such as a sodium salt or a potassium salt.

[0326] The present invention provides a pharmaceutical composition of the present invention, which comprises a double-stranded RNA molecule of the present invention and an aqueous diluent or solvent.

[0327] The present invention provides solutions, such as phosphate buffered saline solutions, of the double-stranded RNA molecules of the present invention. In some embodiments, the solutions, such as phosphate buffered saline solutions, of the present invention are sterile solutions.

[0328] The pharmaceutical compositions of the present invention can be administered locally to the eye (e.g., ocularly). It is understood that the double-stranded RNA molecules of the present invention can be administered to any part of the eye.

[0329] In some embodiments, the pharmaceutical composition is for administration to the anterior segment of the eye. In some embodiments, the pharmaceutical composition is for administration to the conjunctiva. In some embodiments, the pharmaceutical composition is for administration to the cornea. In some embodiments, the pharmaceutical composition is for administration to the bulbar conjunctiva. In some embodiments, the pharmaceutical composition is for administration to the palpebral conjunctiva. In some embodiments, the pharmaceutical composition is for administration to the ocular conjunctiva. In some embodiments, the pharmaceutical composition is for administration to the conjunctival fornix. In some embodiments, the pharmaceutical composition is for administration to one or more of the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva, and conjunctival fornix.

[0330] In some embodiments, the pharmaceutical compositions of the present invention may comprise albumin. The albumin may be a serum albumin, such as mouse serum albumin or human serum albumin.

[0331] Manufacturing method In a further aspect, the present invention provides a method for producing a double-stranded RNA molecule of the present invention, comprising reacting nucleotide units to form consecutive covalently linked nucleotide units contained within an oligonucleotide. The method may use phosphoramidite chemistry (see, e.g., Caruthers et al. (1987) Methods in Enzymology, vol. 154, pp. 287-313). In a further embodiment, the method further comprises reacting the consecutive nucleotide sequence with a conjugate moiety to attach (e.g., covalently link) the conjugate moiety to the double-stranded RNA molecule. In a further aspect, a method for producing a composition of the present invention is provided, comprising mixing a double-stranded RNA molecule of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0332] treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Thus, it will be recognized that the treatment referred to herein may, in some embodiments, be prophylactic.

[0333] The present invention provides a method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease.

[0334] The present invention provides the double-stranded RNA molecule of the present invention for use as a medicament for the treatment of a disease.

[0335] The present invention provides the double-stranded RNA molecules of the present invention for use in therapy.

[0336] The present invention provides a double-stranded RNA molecule of the present invention for preparing a medicament for treating or preventing a disease.

[0337] The present invention provides a pharmaceutical composition of the present invention for use as a medicament.

[0338] The present invention provides a pharmaceutical composition of the present invention for use in therapy.

[0339] The present invention provides a pharmaceutical composition of the present invention for the preparation of a medicament for the treatment or prevention of a disease.

[0340] The disease may be an eye-related disease. The disease may be a disease affecting the eye.

[0341] The disease may be an eye (or eye-related) infection. The disease may be a type of eye (or eye-related) inflammation.

[0342] In some embodiments, the disease can be conjunctivitis, dry eye, or ocular inflammation.

[0343] In some embodiments, the subject to be treated is an animal, preferably a mammal, such as a mouse, rat, hamster, or monkey, or preferably a human. In some embodiments, the subject is a human.

[0344] In some embodiments, the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention is for use in a combination therapy with another therapeutic agent.

[0345] Dosing Strategy It will be understood that the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention can be administered to a subject once, or can be administered over a period of hours, days, weeks, months, or years.

[0346] In some embodiments, administration of the double-stranded RNA molecule or pharmaceutical composition can be chronic administration.

[0347] In some embodiments, the double-stranded RNA molecule or pharmaceutical composition of the present invention is administered to only one eye.

[0348] In some embodiments, the double-stranded RNA molecule or pharmaceutical composition of the present invention is administered to both eyes. Administration to both eyes can be simultaneous or sequential.

[0349] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye once a day, twice a day, three times a day, or more than three times a day. For example, the double-stranded RNA molecule can be administered to one (or each) eye 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a day.

[0350] In some embodiments, the double-stranded RNA molecule can be administered to one (or each) eye every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In some embodiments, the double-stranded RNA molecule can be administered to one (or each) eye every 4 hours.

[0351] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for less than one day, or one day, or for two, three, four, five, six, seven, eight, nine, ten, or more than ten consecutive days. For example, the double-stranded RNA molecule can be administered to one (or each) eye for 1, 2, 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, or more than 31 days.

[0352] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more than 6 weeks.For example, the double-stranded RNA molecule can be administered to one (or each) eye for 1, 2, 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 weeks or more than 52 weeks.

[0353] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more than 6 months. For example, the double-stranded RNA molecule can be administered to one (or each) eye for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more than 12 months.

[0354] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for a period of 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 months. For example, the double-stranded RNA molecule can be administered to one (or each) eye for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 years or more than 25 years.

[0355] Methods for regulating expression The present invention provides a method for modulating expression of a target in a cell, the method comprising administering to the cell an effective amount of a double-stranded RNA molecule of the present invention or a pharmaceutical composition of the present invention.

[0356] In some embodiments, the method is an in vitro method.

[0357] In some embodiments, the method is an in vivo method.

[0358] In some embodiments, the cell is an animal cell, preferably a mammalian cell, such as a mouse cell, rat cell, hamster cell, or monkey cell, or preferably a human cell.

[0359] In some embodiments, the cell is a mammalian cell.

[0360] In some embodiments, the cells are human cells.

[0361] In some embodiments, the cells are ocular cells. In some embodiments, the cells are human ocular cells.

[0362] In some embodiments, the cells may be collected by a sampling medical device. In some embodiments, the cells may be derived from a population of conjunctival cells from the ocular surface of the eye.

[0363] Purpose The double-stranded RNA molecules of the present invention can be utilized, for example, as research reagents for diagnosis, therapy, and prevention.

[0364] In studies, such double-stranded RNA molecules may be used to inhibit the expression of targets expressed in the eyes of experimental animals, thereby facilitating functional analysis of the target or evaluation of its usefulness as a target for therapeutic intervention.

[0365] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in practicing or testing embodiments of the present disclosure. Numeric ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction. Amino acid sequences are written from left to right in the amino to carboxy direction, respectively.

[0366] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications are prior art to the appended claims.

[0367] The present invention will now be further illustrated by examples, which are intended to serve as aids to those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. [Example]

[0368] Example 1: New Zealand White rabbits were dosed with 20 μl (500 μg / dose) of a 25 μg / μL solution of either naked siRNA molecules containing the sequence of SEQ ID NO: 3 or siRNA molecules conjugated to C16, C22, or cholesterol, formulated in PBS, by topical administration to both the left and right eyes, three times daily with at least four hours between doses for five days. Four days after the final dose, the rabbits were sacrificed, and palpebral conjunctival samples were collected from each eye (saline n=4, siRNA treatment group n=6) and analyzed for AHSA1 mRNA knockdown by digital droplet PCR.

[0369] After adding metal beads, conjunctival tissue was homogenized in 500 μL of MagnaPure Tissue Lysis Buffer (Roche LifeScience) using a TissueLyser II (Qiagen). mRNA was extracted from 350 μL of lysis buffer using the MagNAPure 96 System according to the manufacturer's instructions (Roche LifeScience) and extracted with 50 μL of RNAse-free water. cDNA synthesis was performed with 4 μL of input RNA using the IScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad). 2 μL was used as input for digital droplet PCR using ddPCR supermix for probes (without dUTP) (Bio-Rad) according to the manufacturer's protocol.

[0370] The following TaqMan gene expression assays were used: AHSA1 (FAM): Oc06762465_g1 (Cat. No.: 4351372, TaqMan Thermofisher Scientific) and HPRT1 (VIC): Oc03399461_m1 (Cat. No.: 4331182, TaqMan Thermofisher Scientific)

[0371] AHSA1 mRNA concentrations were quantified relative to the housekeeping gene HPRT using QuantaSoft Software (Bio-Rad) and normalized to PBS-treated rabbits (set to a value of 1).

[0372] The results are shown in Figure 1. For naked siRNA molecules containing the sequence of SEQ ID NO: 3, siRNA molecules containing the sequence of SEQ ID NO: 3 conjugated to C16, siRNA molecules containing the sequence of SEQ ID NO: 3 conjugated to C22, and siRNA molecules containing the sequence of SEQ ID NO: 3 conjugated to cholesterol, 20-25% knockdown in the whole conjunctiva was observed, indicating superior knockdown by C16, C22, and cholesterol AHSA1 siRNA compared to naked AHSA1 siRNA.

[0373] Example 2: New Zealand White rabbits were administered 20 μl (500 μg / dose) of a 25 μg / μL solution of either naked siRNA molecules containing the sequence of SEQ ID NO: 3 or siRNA molecules conjugated to C16, C22, or cholesterol in PBS by topical administration to the eye, three times daily at least four hours apart, for five days. Four days after the final administration, the rabbits were sacrificed, and palpebral conjunctival samples were collected (saline n=4, siRNA treatment group n=6) and analyzed for AHSA1 siRNA content by hELISA.

[0374] After adding metal beads, conjunctival tissue (approximately 5 mg) was homogenized in 500 μL of MagnaPure Tissue Lysis buffer (Roche LifeScience) using a TissueLyser II (Qiagen). siRNA content was determined using hELISA with a biotinylated capture probe and a detection probe that binds to a digoxigenin-conjugated antisense siRNA strand. The resulting lysate was diluted and incubated with 35 nM biotinylated capture probe and 30 nM digoxigenin-conjugated detection probe in 5x saline sodium citrate buffer [SSC Buffer 20x Concentrate, Sigma-Aldrich, no. 6639] containing 0.05% Tween 20 [Sigma-Aldrich, no. P9416] in a 96-well plate at room temperature for 30 minutes. The assembled complexes were then captured on streptavidin-coated ELISA plates (Nunc 436014) for 1 hour. After three washes with 2x SSCT buffer, each well was incubated with anti-digoxigenin-alkaline phosphatase (AP)-Fab fragment (Roche, no. 11093274910) for 1 hour at room temperature. After three further washes, BluePhos substrate (Kirkegaard & Perry Labs [KPL], no. 50-88-00) was added to the plate, and color development was measured spectrophotometrically at 615 nm after 20 minutes. For oligo content analysis, several dilutions of each sample (50x, 100x, 200x, 400x, 800x, and 1600x) were measured. Drug concentrations in nmol / g conjunctival tissue weight were obtained by back-calculating the respective tissue weights and averaging valid values ​​(i.e., readings within the linear range of the standard curve) from the different dilutions.

[0375] The results are shown in Figure 2. Increased content in the conjunctiva was observed for naked siRNA molecules comprising the sequence of SEQ ID NO: 3, siRNA molecules comprising the sequence of SEQ ID NO: 3 conjugated to C16, siRNA molecules comprising the sequence of SEQ ID NO: 3 conjugated to C22, and siRNA molecules comprising the sequence of SEQ ID NO: 3 conjugated to cholesterol, indicating superior content for AHA1 siRNA conjugated to C16, C22, and especially AHA1 siRNA conjugated to cholesterol, compared to naked siRNA.

[0376] Example 3: New Zealand White rabbits were dosed with 20 μl (500 μg / dose) of a 25 μg / μL solution of either naked siRNA molecules containing the sequence of SEQ ID NO: 3 or siRNA molecules conjugated to C16, C22, or cholesterol in PBS by topical administration to the eye three times daily with at least four hours between doses for five days. Four days after the final dose, EYEPRIM (OPIA Technologies) samples were collected from the bulbar conjunctiva (saline n = 4, siRNA treatment n = 6). After euthanasia, the bulbar conjunctiva was exposed, and the EYEPRIM membrane was pressed against the inferior bulbar conjunctiva for 3 seconds. The membrane was then removed from the EYEPRIM. The membrane was grasped with forceps during removal to avoid dropping or scattering. The membrane was then flash-frozen in a 2 mL Eppendorf tube.

[0377] After adding metal beads, EYEPRIM samples were homogenized in 500 μL of MagnaPure Tissue Lysis buffer (Roche LifeScience) using a TissueLyser II (Qiagen). mRNA was extracted from 350 μL of lysis buffer using the MagNAPure 96 system according to the manufacturer's instructions (Roche LifeScience) and extracted with 50 μL of RNAse-free water. cDNA synthesis was performed with 4 μL of input RNA using the IScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad). 2 μL was used as input for digital droplet PCR using ddPCR supermix for probes (without dUTP) (Bio-Rad) according to the manufacturer's protocol.

[0378] The following TaqMan gene expression assays were used: AHSA1 (FAM): Oc06762465_g1 (Cat. No.: 4351372, TaqMan Thermofisher Scientific) and HPRT1 (VIC): Oc03399461_m1 (Cat. No.: 4331182, TaqMan Thermofisher Scientific)

[0379] AHSA1 mRNA concentrations were quantified relative to the housekeeping gene HPRT using QuantaSoft Software (Bio-Rad) and normalized to PBS-treated rabbits (PBS set to 1).

[0380] The results are shown in Figure 3. 69% knockdown (SEQ ID NO: 3 conjugated to C16), 65% knockdown (SEQ ID NO: 3 conjugated to C22), and 61% knockdown (SEQ ID NO: 3 conjugated to cholesterol) in conjunctival EYEPRIM samples was compared to 54% for naked siRNA (SEQ ID NO: 3), indicating significantly greater knockdown by AHSA1 siRNA conjugated to C16 compared to naked siRNA (p=0.02 Student's t-test), as well as greater knockdown by AHSA1 siRNA conjugated to C22 and cholesterol.

[0381] Example 4 Isothermal titration calorimetry (ITC) Experiments were performed on an auto-PEAQ ITC (Malvern Panalytical, Malvern, UK) in an assay setup where the target protein was present in a syringe and injected into a FA-siRNA sample placed in a cell. The concentrations of mouse serum albumin (MSA, catalog number A3139-100 mg, essentially fatty acid-free) in the syringe (25–450 μM) and FA-siRNA in the cell (3–60 μM) were adjusted using filtered and degassed buffer from dialysis based on their measured stoichiometry and binding affinity. ITC analysis was performed at 25 °C with the following experimental parameters: stirrer speed: 750 rpm, interval: 150 s, injection duration: 3 s, and reference output: 10 μcal / s. The initial delay was set to 60 s and the filter duration to 5 s. Thirteen or 19 injections were performed with a titration volume of 2.0 or 3.0, respectively, to collect enthalpy data for the analyzed interactions. Control titrations were performed to correct for the heat of dilution (FA-siRNA in buffer, MSA in buffer, and buffer in buffer). MSA was dissolved in DPBS (catalog no. 14190-094, Gibco / Thermo Fisher Scientific, Basel, Switzerland) and further dialyzed using DPBS with a Slide-A-Lyzer Dialysi Cassette 20K MWCO (catalog no. 66003, Thermo Scientific™, Waltham, MA) prior to ITC analysis. FA-siRNA powder was solubilized in DPBS. The concentrations of the dialyzed protein and FA-siRNA were determined using UV spectroscopy at 280 and 260 nm, respectively (according to the Beer-Lambert law). The offset and MSA heat of dilution were subtracted from the heat of reaction generated during the FA-siRNA titration. The baseline was adjusted manually (if necessary), and the corrected heat of reaction was fitted using a single-site binding model in MicroCal PEAQ ITC analysis software, version 1.3 (Malvern Instrument, Malvern, UK). Prior to the FA-siRNA ITC study, a control ITC experiment of MSA titration into warfarin (DPBS + 2% DMSO (v / v)) was performed.

[0382] Analytical Ultracentrifugation (AUC) Samples were analyzed in sedimentation velocity (SV) and / or sedimentation equilibrium (SE) experiments in an analytical ultracentrifuge XLI and Optima-AUC (Beckman Coulter, CA) using absorbance detection (260–295 nm range) depending on the tested concentration (1–75 μM). Individual samples were dissolved in DPBS (catalog no. 14190-094, Gibco / Thermo Fisher Scientific, Basel, Switzerland) and diluted with DPB. AUC-SV experiments were performed at 60,000 rpm and 20°C using 3 and 12 mm Ti centerpieces (Nanolytics Instruments, Potsdam, Germany) and an An-60 Ti rotor (Beckman Coulter, CA). SE experiments were performed in multispeed mode (rotor speeds of 10, 15, and 20 krpm or 7, 10, and 14 krpm for FA Bis C16 and FA C24 conjugates, respectively), with FA-siRNA conjugate concentrations set at 6 and 25 μM. A 3 mm Ti centerpiece (Nanolytics Instruments, Potsdam, Germany) and an An-60 Ti rotor (Beckman Coulter, California) were used for SE analysis. The density and viscosity of the buffer were experimentally determined using a densitometer and viscometer (DMA 5000 M and AMVn, Anton Paar, Aarau, Switzerland, respectively). The interaction of FA-siRNA conjugates with MSA was analyzed in SV mode by absorbance (290–305 nm) and / or fluorescence detection using a fluorescence detection system (Aviv Biomedical, New Jersey).

[0383] result The results are shown in Figure 4, which provides a biophysical analysis of AHA-1-specific siRNA (FA-siRNA) conjugated to different fatty acids. Column 3: Tendency of different fatty acid conjugates to exist in different oligomeric states at a concentration of 25 μM (final oligomeric state as measured by AUC); Column 4: Percentage of monomers of different FA-siRNAs when dissolved in 25 μM PBS; Column 5: Binding affinity to mouse serum albumin (MSA; determined by ITC); Column 6: Number of FA-siRNA conjugates bound to MSA.

[0384] The results demonstrate that (i) the binding strength of siRNA molecules conjugated to fatty acids can be adjusted / modified by the length of the fatty acid; (ii) the introduction of a double bond (e.g., stearic acid vs. oleic acid) does not affect the binding of fatty acids to albumin; and (iii) albumin binding can be utilized as a delivery vehicle for siRNA molecules conjugated to fatty acids, with the binding strength being adjusted as needed.

[0385] Example 5: New Zealand White rabbits were dosed with 20 μl (500 μg / dose) of a 25 μg / μL solution of either naked siRNA molecules containing SEQ ID NO:3 or siRNA molecules conjugated to C16, C22, or cholesterol formulated in PBS by topical administration to both eyes, three times daily with at least four hours between doses, for five days. Three days after the final dose, the rabbits were sacrificed, and samples of the upper and lower palpebral conjunctiva were collected from each eye (n = 2 eyes). Histological sections (n ​​= 3-8) were analyzed for siRNA molecule distribution in the conjunctival tissue using in situ hybridization (ISH).

[0386] Conjunctival tissue was dissected and fixed overnight in 10% NBF, followed by a standard dehydration process and embedding in paraffin the following day. Transverse conjunctival tissue sections (5 μm) were stained using an automated Ventana Discovery ULTRA autostainer (Roche Diagnostics). An automated protocol was designed for baking (60°C for 20 minutes), deparaffinization (69°C for 24 minutes), and ISH protease 3 treatment (1 drop, 32 minutes). Slides were treated with DISC inhibitor (1 drop, 12 minutes) and washed with reaction buffer (1x). A DIG-labeled probe targeting SEQ ID NO: 3 was diluted in miRCURY LNA miRNA ISH Buffer (1x in RNase-free water) to a final concentration of 0.25 nM and manually applied to the slides. Slides were denatured at 90°C for 8 minutes, followed by hybridization at 65°C for 16 minutes. The slides were then washed with 0.1x SSC (5 times for 4 minutes at 54°C). DIG molecules were detected using anti-DIG-HRP (1 drop, 4°C, no heat), followed by DISC AMP TSA BF and DISC AMP H2O2 BF (1 drop each, 4 minutes without heat), and DISC anti-BF HRP (1 drop each, 4 minutes without heat). HRP was detected using a DAB (Brown) detection kit. Slides were counterstained with hematoxylin II (4 minutes without heat), followed by treatment with a blue dye (4 minutes without heat), and then mounted in EcoMount mounting medium. To visualize the results, slides were scanned at 20x magnification using an Olympus VS120 slide scanner. Quantification of positive staining was performed using Halo, Indica Labs (V 3.6.4134.166).

[0387] The following reagents were used: ISH-Protease 3 (05273331001), Disc. Inhibitor (07017944001), anti-DIG HRP (07256299001), Disc. Anti-BF HRP (07529422001), Disc. AMP TSA BF (07529422001), hematoxylin II (05277965001), bluing reagent (05266769001), SSC solution (10X) (05353947001), Reaction Buffer Concentrate (10X) (05353955001), and Disc. ChromoMap DAB RUO (05266645001) were all purchased from Roche Diagnostics. miRCURY LNA miRNA ISH buffer (#1108512) and a DIG-labeled probe targeting SEQ ID NO: 3 (Custom miRCURY LNA / 5DiGN / CGTGGCCTTAATGAA / 3DiG_N / ) were purchased from Qiagen.

[0388] The results are shown in Figure 5. Conjunctival tissue ISH staining of 0.3-0.4% was observed for naked siRNA molecules containing SEQ ID NO:3, siRNA molecules containing SEQ ID NO:3 conjugated to C16, siRNA molecules containing SEQ ID NO:3 conjugated to C22, and siRNA molecules containing SEQ ID NO:3 conjugated to cholesterol. Compared to naked AHSA1 siRNA, C16, C22, and cholesterol AHSA1 siRNAs showed superior tissue staining, with C16 and C22 AHSA1 siRNAs showing the best results compared to naked and cholesterol AHSA1 siRNAs. Furthermore, while staining for naked AHSA1 siRNA was primarily located in the superficial conjunctiva, staining for C16, C22, and cholesterol AHSA1 siRNAs was also located in the stroma of the conjunctival tissue.

[0389] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A double-stranded ribonucleic acid (RNA) molecule for topical administration to the eye, wherein the double-stranded RNA molecule can bind to a target sequence, and the double-stranded RNA molecule comprises a first strand having a 5' end and a 3' end, and a second strand having a 5' end and a 3' end, wherein the first strand is complementary to the second strand, and the first strand comprises a continuous nucleotide sequence of at least eight nucleotides that is complementary to the target sequence, and the double-stranded RNA molecule is conjugated to at least one conjugate portion.

2. The double-stranded RNA molecule according to claim 1, wherein the double-stranded RNA molecule is an siRNA molecule.

3. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the double-stranded RNA molecule can inhibit the expression of a target.

4. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the conjugate portion is a fatty acid molecule or a cholesterol molecule.

5. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is selected from the list consisting of C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, and C40.

6. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is C16.

7. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is C22.

8. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is branched.

9. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is not branched.

10. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is saturated.

11. The double-stranded RNA molecule according to claim 4, wherein the fatty acid molecule is unsaturated.

12. The double-stranded RNA molecule according to claim 11, wherein the fatty acid molecule contains one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or nineteen or more carbon double bonds.

13. The double-stranded RNA molecule according to claim 11, wherein the fatty acid molecule contains one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or nineteen or more carbon triple bonds.

14. The fatty acid molecules are C3:0; C4:0; C4:1; C5:0; C5:1; C6:0; C6:1; C6:2; C7:0; C7:1; C7:2; C8:0; C8:1; C8:2; C8:3; C9:0; C9:1; C9:2; C9:3; C10:0; C10:1; C10:2; C10:3; C10:4; C11:0; C11:1; C11:2; C11:3; C11:4; C12:0; C12:1; C12:2; C12:3; C12:4; C12:5; C13:0; C13:1; C13:2; C13:3; C13:4; C13:5; C14:0; C14:1; C14:2; C14:3; C14:4; C14:5; C14:6; C15:0; C15:1; C15:2; C15:3; C15:4; C15:5; C15:6; C16:0; C16:1; C16:2; C16:3; C16:4; C16:5; C16:6; C16:7; C17:0; C17:1; C17:2; C17:3; C17:4; C17:5; C17:6; C17:7; C18:0; C18:1; C18:2; C18:3; C18:4; C18:5; C18:6; C18:7; C18:8; C19:0; C19:1; C19:2; C19:3; C19:4; C19:5; C19:6; C19:7; C19:8; C20:0; C20:1; C20:2; C20:3; C20:4; C20:5; C20:6; C20:7; C20:8; C20:9; C21:0; C21:1; C21:2; C21:3; C21:4; C21:5; C21:6; C21:7; C21:8; C21:9; C22:0; C22:1; C22:2; C22:3; C22:4; C22:5; C22:6; C22:7; C22:8; C22:9; C22:10; C23:0; C23:1; C23:2; C23:3; C23:4; CC26:12;C27:0;C27:1;C27:2;C27:3;C27:4;C27:5;C27:6;C277;C27:8;C 27:9;C27:10;C27:11;C27:12;C28:0 ;C28:1;C28:2;C28:3;C28:4;C28:5; C28:6;C28:7;C28:8;C28:9;C28:10; C28:11;C28:12;C28:13;C29:0;C29: 1;C29:2;C29:3;C29:4;C29:5;C29:6 ;C29:7;C29:8;C29:9;C29:10;C29:11 ;C29:12;C29:13;C30:0;C30:1;C30: 2;C30:3;C30:4;C30:5;C30:6;C30:7 ;C30:8;C30:9;C30:10;C30:11;C30: 12;C30:13;C30:14;C31:0;C31:1;C31 :2;C31:3;C31:4;C31:5;C31:6;C31: 7;C31:8;C31:9;C31:10;C31:11;C31 :12;C31:13;C31:14;C32:0;C32:1;C 32:2;C32:3;C32:4;C32:5;C32:6;C32 :7;C32:8;C32:9;C32:10;C32:11;C3 2:12;C32:13;C32:14;C32:15;C33:0 ;C33:1;C33:2;C33:3;C33:4;C33:5;C33:6;C33:7;C33:8;C33:9;C33:10; C33:11;C33:12;C33:13;C33:14;C33:15;C34:0;C34:1;C34:2;C34:3;C34 :4;C34:5;C34:6;C34:7;C34:8;C34: 9;C34:10;C34:11;C34:12;C34:13;C3 4:14;C34:15;C34:16;C35:0;C35:1; C35:2;C35:3;C35:4;C35:5;C35:6;C 35:7;C35:8;C35:9;C35:10;C35:11; C35:12;C35:13;C35:14;C35:15;C35: 16;C36:0;C36:1;C36:2;C36:3;C36: 4;C36:5;C36:6;C36:7;C36:8;C36:9 ;C36:10;C36:11;C36:12;C36:13;C3 6:14;C36:15;C36:16;C36:17;C37:0;C37:1; C37:2; C37:3; C37:4; C37:5; C37:6; C37:7; C37:8; C37:9; C37:10; C37 :11;C37:12;C37:13;C37:14;C37:15;C37:16;C37:17;C38:0;C38:1;C38:2;C 38:3; C38:4; C38:5; C38:6; C38:7; C38:8; C38:9; C38:10; C38:11; C38:12; C38 :13;C38:14;C38:15;C38:16;C38:17;C38:18;C39:0;C39:1;C39:2;C39:3;C3 A double-stranded RNA molecule according to claim 11, selected from the list consisting of 9:4;C39:5;C39:6;C39:7;C39:8;C39:9;C39:10;C39:11;C39:12;C39:13;C39:14;C39:15;C39:16;C39:17;C39:18;C40:0;C40:1;C40:2;C40:3;C40:4;C40:5;C40:6;C40:7;C40:8;C40:9;C40:10;C40:11;C40:12;C40:13;C40:14;C40:15;C40:16;C40:17;C40:18;C40:

19.

15. The double-stranded RNA molecule according to claim 4, wherein the cholesterol molecule is selected from the group consisting of 3'-cholesteryl-TEG CPG, 5'-cholesterol-TEG-CE phosphoramidite, 5'-cholesterol-CE phosphoramidite, and cholesteryl-TEG-CE phosphoramidite.

16. The double-stranded RNA molecule according to claim 1 or 2, wherein the conjugate portion is (i) located at the 5' end or the 3' end of the first strand, or (ii) located at the 5' end or the 3' end of the second strand.

17. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the conjugate portion is located at the 3' end of the first strand.

18. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the linker is positioned between the double-stranded RNA molecule and the conjugate portion.

19. The double-stranded RNA molecule according to claim 1 or 2, wherein the linker is (i) C6; (ii) TEG; or (iii) dinucleotide, and optionally the dinucleotide is CA.

20. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the continuous nucleotide sequence is at least 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.

21. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the continuous nucleotide sequence is at least 20 nucleotides long.

22. The double-stranded RNA molecule according to claim 21, wherein the continuous nucleotide sequence has a length of 20, 21, 22, 23, or 24 nucleotides.

23. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the first strand consists of the continuous nucleotide sequence.

24. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the double-stranded RNA molecule is for administration to the anterior part of the eye.

25. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the double-stranded RNA molecule is for administration to the conjunctiva or cornea of ​​the eye.

26. The double-stranded RNA molecule according to claim 25, wherein the double-stranded RNA molecule is for administration to the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva and / or conjunctival fornix.

27. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the consecutive nucleotide sequence is at least 75% complementary to the target sequence.

28. The double-stranded RNA molecule according to claim 27, wherein the consecutive nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the target sequence.

29. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the consecutive nucleotide sequence contains one, two, three, four, five, six, seven, eight or more mismatches with respect to the target sequence.

30. The double-stranded RNA molecule according to claim 2, wherein the target is AHA-1.

31. The double-stranded RNA molecule according to claim 30, wherein the AHA-1 target includes SEQ ID NO: 1 or SEQ ID NO: 2, or consists of SEQ ID NO: 1 or SEQ ID NO:

2.

32. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the continuous nucleotide sequence is complementary to the AHA-1 target sequence.

33. The double-stranded RNA molecule according to claim 32, wherein the consecutive nucleotide sequence includes a nucleotide sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO:

2.

34. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the continuous nucleotide sequence includes or consists of SEQ ID NO:

3.

35. The double-stranded RNA molecule according to claim 2, wherein the expression of the target is inhibited by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, or 100% compared to a control.

36. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the double-stranded RNA molecule comprises one or more modified nucleosides.

37. The double-stranded RNA molecule according to claim 36, wherein the one or more modified nucleosides are one or more 2'-saccharide-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA; 2'-O-methylRNA (2'-OMe); 2'-O-methoxyethyl-RNA (2'-MOE); 2'-alkoxy-RNA; 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA; locked nucleic acid (LNA); and any combination thereof.

38. The double-stranded RNA molecule according to claim 37, wherein the 2'-glycosylated nucleoside is an affinity-enhancing 2'-glycosylated nucleoside.

39. A double-stranded RNA molecule according to claim 1 or claim 2, wherein one or more internucleoside bonds located between nucleosides on the continuous nucleotide sequence are modified.

40. The double-stranded RNA molecule according to claim 39, wherein at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the internucleoside bonds located between nucleosides on the consecutive nucleotide sequence are modified.

41. The double-stranded RNA molecule according to claim 39, wherein one, more, or all of the modified nucleoside bonds include a phosphorothioate bond.

42. The double-stranded RNA molecule according to claim 41, wherein all nucleoside bonds present within the double-stranded RNA molecule are phosphorothioate nucleoside bonds.

43. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the double-stranded RNA molecule is in the form of a pharmaceutically acceptable salt.

44. The double-stranded RNA molecule according to claim 43, wherein the salt is a sodium salt or a potassium salt.

45. A pharmaceutical composition comprising a double-stranded RNA molecule as described in Claim 1 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

46. A method for treating or preventing a disease in a subject, comprising administering a therapeutically effective amount or a preventively effective amount of a double-stranded RNA molecule according to claim 1 or claim 2 or a pharmaceutical composition according to claim 45 to a subject in need thereof.

47. A double-stranded RNA molecule according to claim 1 or claim 2, or a pharmaceutical composition according to claim 45, for use as a pharmaceutical in the treatment of a disease.

48. Use of a double-stranded RNA molecule according to claim 1 or claim 2 or a pharmaceutical composition according to claim 45 for the preparation of a medicament for the treatment or prevention of a disease.

49. A double-stranded RNA molecule or pharmaceutical composition for use according to claim 47, wherein the disease is conjunctivitis, dry eye, or inflammation.

50. The double-stranded RNA molecule or pharmaceutical composition for use according to claim 47, wherein the double-stranded RNA molecule is administered to the eye once, twice, three times, or more than three times a day, and optionally, the double-stranded RNA molecule is administered to both eyes.

51. The double-stranded RNA molecule or pharmaceutical composition for use according to claim 47, wherein the double-stranded RNA molecule is administered for less than one day, or for one, two, three, four, five, six, seven, or more than seven days, and optionally, the double-stranded RNA molecule is administered to both eyes.

52. The double-stranded RNA molecule or pharmaceutical composition for use according to claim 47, wherein the double-stranded RNA molecule is administered for a period of (i) one week, two weeks, three weeks, four weeks, five weeks, six weeks or more than six weeks, or (ii) one month, two months, three months, four months, five months, six months or more than six months, or (iii) one year, two years, three years, four years, five years or more than five years, and optionally the double-stranded RNA molecule is administered to both eyes.

53. An in vitro method for regulating the expression of a target in cells, comprising administering an effective amount of a double-stranded RNA molecule according to claim 1 or claim 2 or a pharmaceutical composition according to claim 45 to the cells.

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