Double-stranded RNA molecules for administration to eye

Abstract

The present invention relates to double stranded RNA molecules conjugated to at least one conjugate moiety and pharmaceutical compositions thereof for topical application to the eye. The double-stranded RNA molecule is complementary, such as fully complementary, to a target expressed in the eye, and is capable of inhibiting expression of a target expressed in the eye. The double-stranded RNA molecules can be used to treat conditions and diseases of the eye.

Description

Technical Field

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

[0002] Double-stranded RNA molecules such as siRNA molecules can regulate the expression of target nucleic acids, particularly by inhibiting the expression of target nucleic acids, by binding to complementary mRNA post-transcriptionally, typically resulting in degradation of the target mRNA and loss of translation.

[0003] siRNA molecules are capable of inducing RNA-dependent gene silencing in the cytoplasm of cells via the RNA-induced silencing complex (RISC), in which they interact with the catalytic RISC component argonaute.

[0004] Delivery of double-stranded RNA molecules to tissue sites or specific cells can be impaired or prevented by a variety of factors that reduce the stability of the double-stranded RNA molecules or prevent the double-stranded RNA molecules from being effectively delivered to their target sites. Such factors include, but are not limited to, sensitivity to endogenous RNAases, short half-life and poor stability, recognition by the immune system, large size and charge, and endosomal entrapment.

[0005] One tissue site that may pose particular challenges for delivery is the eye, which can be affected by a variety of conditions, lesions, and diseases. Delivery of double-stranded RNA molecules to the eye may have beneficial therapeutic effects because the double-stranded RNA molecules target and inhibit target genes expressed in the eye that may be involved in causing the lesions.

[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, wherein the double-stranded RNA molecule is capable of binding to a target sequence, wherein 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, wherein the first strand comprises a continuous nucleotide sequence of at least 8 nucleotides that is complementary to the target sequence, and wherein the double-stranded RNA molecule is conjugated with 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 small hairpin RNA (shRNA) molecule.

[0009] In some embodiments, the double-stranded RNA molecule can be capable of inhibiting the 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 or more conjugate moieties can 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 there are more than one conjugate moiety, the conjugate moieties can be a combination of one or more fatty acid molecules and one or more cholesterol molecules, or can be a combination of two or more fatty acid molecules, or can be a combination of two or more cholesterol molecules. In embodiments where there are more than one conjugate moiety, each conjugate moiety is independently selected such that the conjugate moieties attached to the double-stranded RNA molecule can be the same or can be different.

[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. In some embodiments, the fatty acid molecule comprises 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.

[0021] In some embodiments, the fatty acid molecule comprises one or more triple bonds. In some embodiments, the fatty acid molecule comprises 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.

[0022] In some embodiments, the fatty acid molecule is selected from the list consisting of: 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;

[0023] 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;

[0024] C24:5; C24:6; C24:7; C24:8; C24:9; C24:10; C24:11; C25:0; C25:1; C25:2;

[0025] 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; C26:10;

[0026] C26:11; C26:12; C27:0; C27:1; C27:2; C27:3; C27:4; C27:5; C27:6; C27:7;

[0027] C27:8; C27:9; C27:10; C27:11; C27:12; C28:0; C28:1; C28:2; C28:3; C28:4;

[0028] C28:5; C28:6; C28:7; C28:8; C28:9; C28:10; C28:11; C28:12; C28:13; C29:0;

[0029] C29:1; C29:2; C29:3; C29:4; C29:5; C29:6; C29:7; C29:8; C29:9; C29:10;

[0030] C29:11; C29:12; C29:13; C30:0; C30:1; C30:2; C30:3; C30:4; C30:5; C30:6;

[0031] C30:7; C30:8; C30:9; C30:10; C30:11; C30:12; C30:13; C30:14; C31:0; C31:1;

[0032] C31:2; C31:3; C31:4; C31:5; C31:6; C31:7; C31:8; C31:9; C31:10; C31:11;

[0033] C31:12; C31:13; C31:14; C32:0; C32:1; C32:2; C32:3; C32:4; C32:5; 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; C34:16; C35:0; C35:1; C35:2; C35:3; C35:4; C35:5;

[0034] C35:6; C35:7; C35:8; C35:9; C35:10; C35:11; C35:12; C35:13; C35:14; C35:15;

[0035] C35:16; C36:0; C36:1; C36:2; C36:3; C36:4; C36:5; C36:6; C36:7; C36:8;

[0036] 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;

[0037] C38:11; C38:12; C38:13; C38:14; C38:15; C38:16; C38:17; C38:18; C39:0;

[0038] C39:1; C39:2; C39:3; C39:4; C39:5; C39:6; C39:7; C39:8; C39:9; C39:10;

[0039] C39:11; C39:12; C39:13; C39:14; C39:15; C39:16; C39:17; C39:18; C40:0;

[0040] 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.

[0041] In embodiments where there are more than one fatty acid molecule, each fatty acid molecule is independently selected such that the fatty acid molecules attached to the double-stranded RNA molecule can be the same or can be different.

[0042] In some embodiments, the conjugate moiety can be a cholesterol molecule.

[0043] In some embodiments, the cholesterol molecule can be selected from the group consisting of: 3'-cholesteryl-TEGCPG, 5'-cholesterol-TEG-CE phosphoramidite, 5'-cholesterol-CE phosphoramidite, and cholesteryl-TEG-CE phosphoramidite. In embodiments where there is more than one cholesterol moiety, the cholesterol moieties can 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.

[0044] In embodiments where there is more than one cholesterol molecule, each cholesterol molecule is independently selected such that the cholesterol molecules attached to the double-stranded RNA molecule can be the same or can be different.

[0045] In embodiments where there is more than one conjugate moiety, the conjugate moiety can be a combination of one or more selected from C16, C22, or cholesterol molecules.

[0046] In some embodiments, the conjugate moiety can be located at the 5' end or the 3' end of one of the strands of the double-stranded RNA molecule. In some embodiments, the conjugate moiety can be located at the 3' end of the first strand of the double-stranded RNA molecule. The first strand can be in the sense orientation (i.e., the first strand is the sense strand). In some embodiments, the conjugate moiety can be located at the 3' end of the sense strand of the double-stranded RNA molecule.

[0047] In some embodiments, a linker can be located between the double-stranded RNA molecule and the conjugate moiety. In some embodiments, the linker can be C6. In some embodiments, the linker can 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).

[0048] In some embodiments where the double-stranded RNA molecule comprises more than one conjugate moiety, a linker can be located between each of the double-stranded RNA molecule and the conjugate moieties. In some embodiments, a linker can be located between each of the conjugate moieties.

[0049] In some embodiments, the linker can be a cleavable linker.

[0050] In some embodiments, the length of the continuous 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.

[0051] In some embodiments, the length of the continuous nucleotide sequence can be at least 20 nucleotides. In some embodiments, the length of the continuous nucleotide sequence can be 20, 21, 22, 23 or 24 nucleotides.

[0052] In some embodiments, the first strand can consist of a continuous nucleotide sequence.

[0053] In some embodiments, the double-stranded RNA molecule can be used for administration to the anterior part of the eye.

[0054] In some embodiments, the double-stranded RNA molecule can be used for administration to the conjunctiva of the eye or the cornea of the eye. In some embodiments, the double-stranded RNA molecule can be used for administration to the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva and / or fornix conjunctiva.

[0055] In some embodiments, the continuous nucleotide sequence can be at least about 75% complementary to the target sequence. The continuous nucleotide sequence can be at least about 80%, at least about 85%, at least about 90%, at least about 95% or completely (such as about 100%) complementary to the target sequence. In some embodiments, the continuous nucleotide sequence can contain 1, 2, 3, 4, 5, 6, 7, 8 or more mismatches with the target sequence.

[0056] In some embodiments, the target can be AHA-1. In some embodiments, the AHA-1 target can comprise or consist of the following: SEQ ID NO:1 or SEQ ID NO:2.

[0057] In some embodiments, the continuous nucleotide sequence can be complementary to the AHA-1 target sequence. In some embodiments, the continuous nucleotide sequence can contain a nucleotide sequence complementary to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the continuous nucleotide sequence can comprise or consist of the following: SEQ ID NO:3.

[0058] In some embodiments, compared to a control, the expression of a target 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%. It should be understood that the % inhibition of target expression mentioned above will be the % reduction relative to the control, where the term "control" refers to the expression of the target in cells that have not been exposed to the double-stranded RNA molecule of the present invention.

[0059] In some embodiments, the double-stranded RNA molecule can comprise one or more modified nucleosides. The one or more modified nucleosides can 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 can be an affinity-enhanced 2'-sugar modified nucleoside.

[0060] In some embodiments, one or more of the internucleoside bonds between nucleosides located on a continuous nucleotide sequence can be modified. In 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 bonds between nucleosides located on a continuous nucleotide sequence can be modified.

[0061] In some embodiments, one or more or all of the modified internucleoside bonds can comprise phosphorothioate bonds. In some embodiments, all of the internucleoside bonds present in the double-stranded RNA molecule can be phosphorothioate internucleoside bonds.

[0062] 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.

[0063] 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.

[0064] The present invention also provides a pharmaceutical composition comprising the double-stranded RNA molecule of the present invention and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0065] The present invention also provides a method for treating or preventing a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention.

[0066] 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.

[0067] The present invention also provides the use of the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention for the preparation of a medicament for treating or preventing a disease.

[0068] In some embodiments, the disease can be conjunctivitis, dry eye or inflammation.

[0069] 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.

[0070] In some embodiments, the double-stranded RNA molecule can be administered for less than one day or for one, two, three, four, five, six, seven or more than seven days.

[0071] In some embodiments, the double-stranded RNA molecule can be administered for the following periods:

[0072] (i) one week, two weeks, three weeks, four weeks, five weeks, six weeks or more than six weeks; or

[0073] (ii) one month, two months, three months, four months, five months, six months or more than 6 months; or

[0074] (iii) one year, two years, three years, four years, five years or more than five years.

[0075] In some embodiments, the double-stranded RNA molecule can be administered to one eye or both eyes.

[0076] The present invention also provides an in vitro method for regulating the expression of a target in a cell, the method comprising administering to the cell in an effective amount the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention.

[0077] Sequence Listing

[0078] The sequence listing submitted together with the present application is incorporated herein by reference. Brief Description of the Drawings

[0079] Figure 1- AHSA1 expression normalized to HPRT in the palpebral conjunctiva rabbit samples at 96 hours after the last administration. For the siRNA molecules comprising the sequence of SEQ ID NO:3, a knockdown of 20% to 25% was observed throughout the conjunctiva, wherein the C16-conjugated, C22-conjugated, and cholesterol-conjugated AHSA1 siRNAs had superior knockdown compared to naked siRNA (i.e., unconjugated siRNA).

[0080] Figure 2 : AHSA1 siRNA content in the palpebral conjunctiva rabbit samples at 96 hours after the last administration. For the siRNA molecules comprising the sequence of SEQ ID NO:3, an increased content was observed in the conjunctiva, wherein the C16-, C22-, and especially cholesterol-conjugated AHA1 siRNAs had superior content compared to naked siRNA (i.e., unconjugated siRNA).

[0081] Figure 3 . AHSA1 expression normalized to HPRT in the bulbar conjunctiva EYEPRIM rabbit samples at 96 hours after the last administration. A 69% knockdown (C16-conjugated SEQ ID NO:3), 65% knockdown (C22-conjugated SEQ ID NO:3), and 61% knockdown (cholesterol-conjugated SEQ ID NO:3) in the conjunctiva EYEPRIM samples were compared to 4% for naked siRNA (i.e., unconjugated siRNA) (SEQ ID NO:3 (no conjugation)). Compared to naked siRNA, C16- had significantly superior knockdown (p = 0.02, Student's t-test), and the C22- and cholesterol-conjugated AHSA1 siRNAs had superior knockdown.

[0082] Figure 4 . Biophysical analysis of different fatty acid-conjugated AHA-1 specific siRNAs (FA-siRNAs). Column 3 shows the propensity of different fatty acid conjugates to exist in different oligomeric states at a concentration of 25 μM (final oligomeric state measured by AUC); column 4 shows the percentage of monomers of different FA-siRNAs when dissolved in 25 μM PBS; column 5 shows the binding affinity to mouse serum albumin (MSA; determined by ITC); column 6 shows the number of FA-siRNA conjugates bound to MSA.

[0083] Figure 5For naked siRNA molecules containing the sequence of SEQ ID NO:3, C16-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, C22-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, and cholesterol-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, conjunctival tissue ISH staining was observed, indicating that C16, C22, and cholesterol AHSA1 siRNAs have superior tissue staining compared to naked AHSA1 siRNA, and C16 and C22 AHAS1 siRNAs showed the most excellent results compared to naked and cholesterol AHSA1 siRNAs. In addition, the staining of naked AHSA1 siRNA was mainly located in the superficial layer of the conjunctiva, while the staining of C16, C22, and cholesterol AHSA1 siRNAs was also located in the stroma of the conjunctival tissue. Detailed Description of the Invention

[0084] Nucleic acid molecule

[0085] As used herein, the term "nucleic acid molecule" or "therapeutic nucleic acid molecule" is defined as a molecule (i.e., nucleotide sequence) containing two or more covalently linked nucleosides, as commonly understood by those skilled in the art.

[0086] The nucleic acid molecules mentioned in the present invention are generally therapeutic oligonucleotides with a length of less than 50 nucleotides.

[0087] As used herein, the terms "polynucleotide", "nucleotide", "nucleic acid", "nucleic acid molecule", and "nucleic acid sequence" are intended to be synonymous with each other.

[0088] Nucleic acid molecules are generally produced in the laboratory, first synthesized by solid-phase chemistry and then purified and isolated. When referring to the sequence of a nucleic acid molecule, it refers to the sequence or order of the nucleobase part of covalently linked nucleotides or nucleosides or their modifications. The nucleic acid molecules of the present invention are artificial, chemically synthesized, and usually purified or isolated. The nucleic acid molecules of the present invention may contain one or more modified nucleosides or nucleotides.

[0089] 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.

[0090] Oligonucleotide

[0091] As used herein, the term "oligonucleotide" is defined as a molecule containing two or more covalently linked nucleosides, as commonly understood by those skilled in the art. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0092] Double-stranded RNA molecule

[0093] As used herein, the terms "RNA interference (RNAi) molecule", "RNAi molecule", or "RNAi" refer to short, canonical double-stranded RNA molecules capable of inducing RNA-dependent gene silencing via the RNA-induced silencing complex (RISC) in the cytoplasm of a cell, where they interact with the catalytic RISC component argonaute. One type of RNAi molecule is small interfering RNA (siRNA), which is a canonical double-stranded RNA molecule that, by binding to complementary mRNA post-transcriptionally, typically results in degradation of the mRNA and loss of translation. In other words, as used herein, the term "siRNA molecule" is defined as a nucleic acid molecule capable of modulating target expression by binding to a target nucleic acid, particularly to a contiguous sequence on the target nucleic acid. The length of an siRNA molecule is typically 20 to 24 base pairs and usually has a phosphorylated 5' end and a hydroxylated 3' end with two overhanging nucleotides. Small interfering RNA (siRNA) may also be referred to as short interfering RNA or silencing RNA.

[0094] In some embodiments, the double-stranded RNA molecule of the present invention can be a small interfering RNA (siRNA) molecule.

[0095] The double-stranded RNA molecule of the present invention 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.

[0096] A double-stranded RNA molecule can be described as comprising a sense strand and an antisense strand.

[0097] In some embodiments, the first strand can be the sense strand (i.e., in the sense orientation) and the second strand can be the antisense strand (i.e., in the antisense orientation).

[0098] In some embodiments, the first strand can be the antisense strand (i.e., in the antisense orientation) and the second strand can be the sense strand (i.e., in the sense orientation).

[0099] Another type of RNAi molecule is small hairpin RNA (shRNA), which is an artificial RNA molecule with a hairpin structure that, upon expression, is capable of reducing the level of target mRNA via DICER and the RNA-induced silencing complex (RISC). Small hairpin RNA (shRNA) may also be referred to as short hairpin RNA. In some embodiments, the double-stranded RNA molecule of the present invention can be a small hairpin RNA (shRNA) molecule.

[0100] RNAi molecules can be designed based on the RNA sequence of a target gene. The corresponding RNAi molecules can then be chemically synthesized or synthesized by in vitro transcription, or expressed from a vector or PCR product.

[0101] The lengths of siRNA and shRNA molecules are typically between 20 and 50 nucleotides, such as between 25 and 35 nucleotides in length, and can interact with an endonuclease called Dicer, which is thought to process dsRNA into short interfering RNAs (siRNAs) of 19 to 23 base pairs (with characteristic two-base 3' overhangs), and then the siRNA is incorporated into the RNA-induced silencing complex (RISC). An effective extended form of the Dicer substrate has been described in US 8,349,809 and US 8,513,207, which are incorporated herein by reference. After binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing. RNAi reagents can be chemically modified using modified internucleotide linkages and high-affinity nucleosides, such as 2'-4' bicyclic ribose-modified nucleosides, including LNA and cET.

[0102] In some embodiments, the double-stranded RNA molecules of the invention comprise or consist of the following in length: about 8 to 50 nucleotides.

[0103] In some embodiments, the double-stranded RNA molecules of the invention comprise or consist of the following in length: about 12 to 50 nucleotides, such as about 15 to 45 in length, such as about 20 to 40, such as about 25 to 35 consecutive nucleotides.

[0104] In some embodiments, the double-stranded RNA molecules comprise or consist of the following in length: about 18 to 25 nucleotides. In some embodiments, the double-stranded RNA molecules comprise or consist of the following in length: about 18 to 30 nucleotides. In some embodiments, the double-stranded RNA molecules comprise or consist of the following in length: about 18 to 35 nucleotides. In some embodiments, the double-stranded RNA molecules comprise or consist of the following in length: about 20 to 25 nucleotides. In some embodiments, the double-stranded RNA molecules comprise or consist of the following in length: about 20 to 30 nucleotides. In some embodiments, the double-stranded RNA molecules comprise or consist of the following in length: about 20 to 35 nucleotides. It should be understood that any range given herein includes the endpoints of the range. Thus, if it is said that the double-stranded RNA molecule comprises about 10 to 30 nucleotides, then lengths of about 10 and about 30 nucleotides are both included.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 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.

[0108] In some embodiments, the length of the double-stranded RNA molecule can be at least about 20 nucleotides. The length of the double-stranded RNA molecule can be 20, 21, 22, 23, or 24 nucleotides.

[0109] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 18 nucleotides.

[0110] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 19 nucleotides.

[0111] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 20 nucleotides.

[0112] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 21 nucleotides.

[0113] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 22 nucleotides.

[0114] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 23 nucleotides.

[0115] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 24 nucleotides.

[0116] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 25 nucleotides.

[0117] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 26 nucleotides.

[0118] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 27 nucleotides.

[0119] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 28 nucleotides.

[0120] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 29 nucleotides.

[0121] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 30 nucleotides.

[0122] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 31 nucleotides.

[0123] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 32 nucleotides.

[0124] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 33 nucleotides.

[0125] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 34 nucleotides.

[0126] In some embodiments, the double-stranded RNA molecule comprises or consists of, in length: 35 nucleotides.

[0127] The double-stranded RNA molecule binds to a target nucleic acid expressed in an animal eye, particularly a mammalian eye. In some embodiments, the double-stranded RNA molecule is generally used to inhibit and / or regulate the expression of a target nucleic acid sequence expressed in the eye.

[0128] Continuous nucleotide sequence

[0129] The term "continuous nucleotide sequence" refers to the region of the double-stranded RNA molecule that is complementary to the target nucleic acid, and this continuous nucleotide sequence can be or can include an oligonucleotide motif sequence. In this context, the term is interchangeable with "continuous nucleobase sequence".

[0130] One strand of the double-stranded RNA molecule comprises or consists of a contiguous nucleotide sequence. In some embodiments, the first strand comprises or consists of a contiguous nucleotide sequence. In some embodiments, the second strand comprises or consists of a contiguous nucleotide sequence.

[0131] In some embodiments, the strand of the double-stranded RNA molecule that comprises or consists of a contiguous nucleotide sequence may optionally comprise further nucleotides, such as a nucleotide linker region that can be used to attach 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.

[0132] It should be understood that the contiguous nucleotide sequence cannot be longer than the double-stranded RNA molecule (or its strand) itself, and the double-stranded RNA molecule (or its strand) cannot be shorter than the contiguous nucleotide sequence.

[0133] In some embodiments, all of the nucleotides of the first or second strand of the double-stranded RNA molecule may constitute a contiguous nucleotide sequence.

[0134] The contiguous nucleotide sequence is a nucleotide sequence in the first or second strand of the double-stranded RNA molecule of the present invention that is complementary and in some cases fully complementary to the target nucleic acid, target sequence, or target site sequence.

[0135] In some embodiments, the contiguous nucleotide sequence is about 8 to 50 nucleotides in length.

[0136] In some embodiments, the contiguous nucleotide sequence may comprise or consist of the following in length: about 12 to 50 nucleotides, such as about 15 to 45 nucleotides in length, such as about 20 to 40 nucleotides, such as about 25 to 35 contiguous nucleotides.

[0137] In some embodiments, the contiguous nucleotide sequence comprises or consists of the following in length: about 18 to 25 nucleotides. In some embodiments, the contiguous nucleotide sequence comprises or consists of the following in length: about 18 to 30 nucleotides. In some embodiments, the contiguous nucleotide sequence comprises or consists of the following in length: about 18 to 35 nucleotides. In some embodiments, the contiguous nucleotide sequence comprises or consists of the following in length: about 20 to 25 nucleotides. In some embodiments, the contiguous nucleotide sequence comprises or consists of the following in length: about 20 to 30 nucleotides. In some embodiments, the contiguous nucleotide sequence comprises or consists of the following in length: about 20 to 35 nucleotides. It should be understood that any range given herein includes the endpoints of the range. Thus, if it is said that the contiguous nucleotide sequence includes about 10 to 30 nucleotides, then both about 10 and 30 nucleotides are included.

[0138] In some embodiments, the continuous nucleotide sequence can comprise or consist of the following: 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.

[0139] In some embodiments, the continuous nucleotide sequence can comprise or consist of the following: 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.

[0140] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 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.

[0141] In some embodiments, the length of the continuous nucleotide sequence can be at least about 20 nucleotides. The length of the continuous nucleotide sequence can be 20, 21, 22, 23, or 24 nucleotides.

[0142] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 18 nucleotides.

[0143] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 19 nucleotides.

[0144] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 20 nucleotides.

[0145] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 21 nucleotides.

[0146] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 22 nucleotides.

[0147] In some embodiments, the continuous nucleotide sequence comprises or consists of the following in length: 23 nucleotides.

[0148] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 24 nucleotides.

[0149] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 25 nucleotides.

[0150] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 26 nucleotides.

[0151] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 27 nucleotides.

[0152] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 28 nucleotides.

[0153] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 29 nucleotides.

[0154] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 30 nucleotides.

[0155] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 31 nucleotides.

[0156] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 32 nucleotides.

[0157] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 33 nucleotides.

[0158] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 34 nucleotides.

[0159] In some embodiments, the continuous nucleotide sequence comprises or consists of, in length: 35 nucleotides.

[0160] In some embodiments, the continuous nucleotide sequence is the same length as the first strand and / or the second strand of the double-stranded RNA molecule.

[0161] In some embodiments, the first strand or the second strand of the double-stranded RNA molecule consists of a continuous nucleotide sequence.

[0162] In some embodiments, the first strand or the second strand of the double-stranded RNA molecule is a continuous nucleotide sequence.

[0163] Conjugate moiety

[0164] The inventors have identified 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.

[0165] As shown in the examples, administering the double-stranded RNA molecules of the present invention to the eye can inhibit the target, particularly the expression of a target expressed in the eye.

[0166] The double-stranded RNA molecules of the present invention are attached to at least one conjugate moiety. In some embodiments, the double-stranded RNA molecule can be attached to more than one conjugate moiety.

[0167] In some embodiments, one or more conjugate moieties can be referred to as the conjugate of the present invention. In some embodiments, the double-stranded RNA molecule can be covalently attached to at least one conjugate moiety.

[0168] For the double-stranded RNA molecule and the conjugate moiety, the terms "attached", "positioned", "linked", and "conjugated" are interchangeable.

[0169] As used herein, the term "conjugate" refers to a double-stranded RNA molecule linked (such as covalently linked) to a conjugate moiety. The conjugate moiety can be directly linked (such as covalently linked) to the double-stranded RNA molecule, or the conjugate moiety can be linked to the double-stranded RNA molecule via a linker group.

[0170] Oligonucleotide conjugates and their synthesis are also reviewed in Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, edited by S.T. Crooke, Chapter 16, Marcel Dekker, Inc., 2001 and reported in Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

[0171] In some embodiments, the conjugate moiety is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (such as bacterial toxins), vitamins, viral proteins (such as capsids), and combinations thereof.

[0172] In some embodiments, the conjugate moiety can be a fatty acid molecule. In some embodiments, the conjugate moiety can be a fatty acid molecule.

[0173] "Fatty acid" refers to a molecule typically composed of a chain of carbon atoms, usually having hydrogen atoms bonded to the carbon atoms along the length of the chain. In other words, a fatty acid molecule contains a hydrocarbon. Hydrogen atoms are typically also present at one (or the end) of the fatty acid molecule chain, and a carboxyl group (-COOH) is typically present at the other (or the end) of the chain. In fact, it is the carboxyl group that makes the molecule an acid (such as a carboxylic acid). As used herein, the terms "fatty acid" and "fatty acid molecule" are considered interchangeable.

[0174] The term "fatty acid" encompasses a single fatty acid molecule as well as a mixture of two or more fatty acid molecules, such as two or more different fatty acid molecules.

[0175] In embodiments where more than one fatty acid molecule is attached to a double-stranded RNA molecule, each fatty acid molecule is independently selected such that the fatty acid molecules attached to the double-stranded RNA molecule can be the same or can be different.

[0176] In some embodiments, two or more fatty acids can be linked with a linker. It should be understood that the linker linking two or more fatty acids can be attached at any point on each of the two or more fatty acid molecules.

[0177] A fatty acid molecule can be a molecule containing carbon atoms. A fatty acid molecule can have the formula CX, where C represents carbon and X refers to the total number of carbon atoms present in the fatty acid molecule (such as the carbon chain length) (for example, "C12" refers to a fatty acid molecule having 12 carbons).

[0178] In some embodiments, a fatty acid molecule can be a molecule having 3 to 40 carbon atoms (such as C3-C40).

[0179] In some embodiments, the fatty acid molecules are 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.

[0180] In some embodiments, a fatty acid molecule can be a molecule having 12 to 24 carbon atoms (such as C12-C24).

[0181] In some embodiments, the fatty acid molecules are selected from the list consisting of: C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, and C24.

[0182] In some embodiments, the fatty acid molecule is C10.

[0183] In some embodiments, the fatty acid molecule is C16.

[0184] In some embodiments, the fatty acid molecule is C18.

[0185] In some embodiments, the fatty acid molecule is C22.

[0186] In some embodiments, the fatty acid molecule can be a salt of the fatty acid molecule (e.g., fatty acid salt).

[0187] In some embodiments, the fatty acid molecule can be branched or unbranched. In other words, the fatty acid molecule can contain a branch or can contain no branch. It should be understood that "unbranched" refers to a linear chain of atoms, such as a linear chain of carbon atoms bonded to hydrogen atoms (e.g., CH2 groups), where the carbon atoms are also linked by carbon-carbon bonds.

[0188] It should also be understood that "branched" refers to a non-linear chain of atoms, e.g., where one or more carbon groups form a branch by attaching to another carbon atom chain bonded to a hydrogen atom (e.g., CH2 group), where the carbon atoms are also linked by carbon-carbon bonds. For example, a branched fatty acid molecule can contain one or more carbon groups (such as methyl groups) attached to a carbon atom chain. The one or more carbon groups forming the branch can be attached at any point along the carbon chain (i.e., attached to one or more of positions C2 to CN-1, where C represents carbon and N refers to the total number of carbon atoms in the linear chain).

[0189] It should be understood that when two or more fatty acids are linked by a linker, all fatty acids can be branched. It should also be understood that when two or more fatty acids are linked by a linker, all fatty acids can be unbranched. It should also be understood that when two or more fatty acids are linked by a linker, one or more of the fatty acids can be branched and the remaining one or more fatty acids can be unbranched.

[0190] It should be understood that a fatty acid that does not contain a carbon double bond (e.g., C=C bond) can be referred to as a saturated fatty acid. It should be understood that the term "saturated" refers to the maximum possible number of atoms (e.g., hydrogen atoms) bonded to each carbon in the molecule

[0191] In some embodiments, the fatty acid molecule can be a saturated fatty acid. In some embodiments, the fatty acid molecule can be fully saturated (i.e., contain carbon single bonds but no carbon double bonds or carbon triple bonds). In some embodiments, the fatty acid molecule can be partially saturated (i.e., contain a combination of: (i) one or more carbon single bonds; and (ii) one or more carbon non-single bonds (i.e., carbon double bonds and / or carbon triple bonds)).

[0192] In some embodiments, the fatty acid molecule may contain a carbon double bond (e.g., C═C bond). In some embodiments, the fatty acid molecule may contain a carbon triple bond (e.g., C≡C bond). In some embodiments, the fatty acid molecule may contain a combination of a carbon double bond (e.g., C═C bond) and a carbon triple bond (e.g., C≡C bond). It should be understood that when the fatty acid molecule contains one or more carbon double bonds and / or one or more carbon triple bonds, the remaining bonds in the fatty acid molecule may be carbon single bonds (e.g., C-C bond).

[0193] It should be understood that a fatty acid containing one or more carbon double bonds and / or one or more carbon triple bonds may be referred to as an unsaturated fatty acid. An unsaturated fatty acid containing one carbon double bond or one carbon triple bond may be referred to as a monounsaturated fatty acid. An unsaturated fatty acid containing two or more non-single bonds (i.e., carbon double bonds and / or carbon triple bonds) may be referred to as a polyunsaturated fatty acid.

[0194] In some embodiments, the fatty acid molecule may be an unsaturated fatty acid. In some embodiments, the fatty acid molecule may be a monounsaturated fatty acid. In some embodiments, the fatty acid molecule may be a polyunsaturated fatty acid.

[0195] In some embodiments, the fatty acid molecule may be completely unsaturated (i.e., containing carbon double bonds and / or carbon triple bonds but no carbon single bonds). In some embodiments, the fatty acid molecule may be partially unsaturated (i.e., containing a combination of: (i) one or more carbon non-single bonds (i.e., carbon double bonds and / or carbon triple bonds); and (ii) one or more carbon single bonds).

[0196] It should be understood that when two or more fatty acids are linked by a linker, all the fatty acids may be saturated. It should also be understood that when two or more fatty acids are linked by a linker, all the fatty acids may be unsaturated. It should further be understood that when two or more fatty acids are linked by a linker, one or more of the fatty acids may be saturated and the remaining one or more fatty acids may be unsaturated.

[0197] It should be understood that saturated or unsaturated fatty acid molecules may be branched or unbranched.

[0198] In some embodiments, saturated fatty acid molecules may be branched or unbranched.

[0199] In some embodiments, unsaturated fatty acid molecules may be branched or unbranched. In some embodiments, monounsaturated fatty acid molecules may be branched or unbranched. In some embodiments, polyunsaturated fatty acid molecules may be branched or unbranched.

[0200] In some embodiments, the branched-chain fatty acid molecules can be fully saturated, fully unsaturated, or can be a mixture of both saturated and unsaturated. In some embodiments, the non-branched-chain fatty acid molecules can be fully saturated, fully unsaturated, or can be a mixture of both saturated and unsaturated.

[0201] 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 double bonds.

[0202] 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.

[0203] In some embodiments, the fatty acid molecules contain one or more double bonds. In some embodiments, the fatty acid molecules contain one or more triple bonds. In some embodiments, the fatty acid molecules contain a combination of one or more carbon double bonds and one or more carbon triple bonds. It should be understood that when the fatty acid molecules contain one or more carbon double bonds and / or carbon triple bonds, the remaining bonds in the fatty acid molecules can be carbon single bonds (e.g., C-C bonds).

[0204] In some embodiments, the fatty acid molecules contain 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.

[0205] In some embodiments, the fatty acid molecules contain 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.

[0206] In some embodiments, the fatty acid molecule comprises a combination of the following: 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 and carbon triple bonds.

[0207] It should be understood that more than nineteen carbon double bonds can be used in the present invention. It should be understood that more than nineteen carbon triple bonds can be used in the present invention.

[0208] The carbon double bond can be in a cis configuration or a trans configuration. The carbon triple bond can be in a cis configuration or a trans configuration. It should be understood that the cis configuration means that two functional groups (such as hydrogen atoms) adjacent to the carbon double bond or carbon triple bond protrude on the same side of the chain, while the trans configuration means that two functional groups (such as hydrogen atoms) adjacent to the carbon double bond or carbon triple bond are located on opposite sides of the chain.

[0209] In some embodiments, the fatty acid molecule can be a cis-unsaturated fatty acid molecule.

[0210] In some embodiments, the fatty acid molecule can be a trans-unsaturated fatty acid molecule.

[0211] In some embodiments, the fatty acid molecule can comprise a combination of a cis-unsaturated fatty acid component and a trans-unsaturated fatty acid component. In some embodiments, the fatty acid molecule can comprise a combination of cis and trans double bonds. In some embodiments, the fatty acid molecule can comprise a combination of cis and trans triple bonds. In some embodiments, the fatty acid molecule can comprise a combination of cis and trans double bonds and cis and trans triple bonds.

[0212] The fatty acid molecule can have the formula CX:Y, where C represents carbon, where X refers to the total number of carbon atoms present in the fatty acid molecule (i.e., the carbon chain length), and where Y refers to the total number of unsaturated / non-single carbon bonds (i.e., carbon double bonds and / or carbon triple bonds) present in the fatty acid molecule (e.g., "C16:5" refers to a fatty acid molecule having a length of 16 carbons, which has a total of 5 unsaturated / non-single carbon bonds in total (i.e., there can be 5 carbon double bonds, or there can be 5 carbon triple bonds, or there can be a combination of a total of 5 non-single carbon bonds of carbon double bonds and carbon triple 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 unsaturated / non-single bonds (i.e., double bonds 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.).

[0213] However, for a given number X (in CX:Y), the maximum number Y of double bonds and / or triple bonds is equal to X / 2 - 1 (i.e., Y = (X / 2) - 1). In other words, for a given number X of carbons in a fatty acid molecule, the number Y of double bonds and / or triple bonds can be any non-negative number (i.e., 0, 1, 2, 3, etc.), up to a maximum of Y = (X / 2) - 1. Thus, in some embodiments where the fatty acid molecule has the formula CX:Y, Y can be equal to the maximum value X / 2 - 1 (i.e., Y = (X / 2) - 1). If X is odd, the result (as a half number) must be rounded down to the nearest natural number. For example, when X = 7, the result of (X / 2) - 1 is 2.5, which must be rounded down to 2. In other words, for X = 7, the maximum number of double bonds and / or triple bonds is Y = 2; for X = 9, the maximum number of double bonds and / or triple bonds is Y = 3, etc. When X is even, 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 for X = 8 or 9 (i.e., for fatty acid molecules having 8 or 9 carbons), the possible maximum number of double bonds and / or triple bonds is 3; and the Y value in the notation C8:Y or C9:Y can be any natural number from 0 to 3 (i.e., 0, 1, 2, or 3). In other words, for X = 8, the possible fatty acids are C8:0, C8:1, C8:2, and C8:3; and for X = 9, the possible fatty acids are C9:0, C9:1, C9:2, and C9:3. This also applies to any other X value (where X is a positive natural number, i.e., non-zero and non-negative).

[0214] In some embodiments, the fatty acid molecule is selected from the list consisting of: 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;

[0215] 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;

[0216] C17:3; C17:4; C17:5; C17:6; C17:7; C18:0; C18:1; C18:2; C18:3; C18:4;

[0217] C18:5; C18:6; C18:7; C18:8; C19:0; C19:1; C19:2; C19:3; C19:4; C19:5;

[0218] C19:6; C19:7; C19:8; C20:0; C20:1; C20:2; C20:3; C20:4; C20:5; C20:6;

[0219] C20:7; C20:8; C20:9; C21:0; C21:1; C21:2; C21:3; C21:4; C21:5; C21:6;

[0220] C21:7; C21:8; C21:9; C22:0; C22:1; C22:2; C22:3; C22:4; C22:5; C22:6;

[0221] C22:7; C22:8; C22:9; C22:10; C23:0; C23:1; C23:2; C23:3; C23:4; C23:5;

[0222] C23:6; C23:7; C23:8; C23:9; C23:10; C24:0; C24:1; C24:2; C24:3; C24:4;

[0223] C24:5; C24:6; C24:7; C24:8; C24:9; C24:10; C24:11; C25:0; C25:1; C25:2;

[0224] C25:3; C25:4; C25:5; C25:6; C25:7; C25:8; C25:9; C25:10; C25:11; C26:0;

[0225] C26:1; C26:2; C26:3; C26:4; C26:5; C26:6; C26:7; C26:8; C26:9; C26:10;

[0226] C26:11; C26:12; C27:0; C27:1; C27:2; C27:3; C27:4; C27:5; C27:6; C27:7;

[0227] C27:8; C27:9; C27:10; C27:11; C27:12; C28:0; C28:1; C28:2; C28:3; C28:4;

[0228] C28:5; C28:6; C28:7; C28:8; C28:9; C28:10; C28:11; C28:12; C28:13; C29:0;

[0229] C29:1; C29:2; C29:3; C29:4; C29:5; C29:6; C29:7; C29:8; C29:9; C29:10;

[0230] C29:11; C29:12; C29:13; C30:0; C30:1; C30:2; C30:3; C30:4; C30:5; C30:6;

[0231] C30:7; C30:8; C30:9; C30:10; C30:11; C30:12; C30:13; C30:14; C31:0; C31:1;

[0232] C31:2; C31:3; C31:4; C31:5; C31:6; C31:7; C31:8; C31:9; C31:10; C31:11;

[0233] C31:12; C31:13; C31:14; C32:0; C32:1; C32:2; C32:3; C32:4; C32:5; C32:6;

[0234] C32:7; C32:8; C32:9; C32:10; C32:11; C32:12; C32:13; C32:14; C32:15;

[0235] C33:0; C33:1; C33:2; C33:3; C33:4; C33:5; C33:6; C33:7; C33:8; C33:9;

[0236] C33:10; C33:11; C33:12; C33:13; C33:14; C33:15; C34:0; C34:1; C34:2;

[0237] C34:3; C34:4; C34:5; C34:6; C34:7; C34:8; C34:9; C34:10; C34:11; C34:12;

[0238] C34:13; C34:14; C34:15; C34:16; C35:0; C35:1; C35:2; C35:3; C35:4; C35:5;

[0239] C35:6; C35:7; C35:8; C35:9; C35:10; C35:11; C35:12; C35:13; C35:14; C35:15;

[0240] C35:16; C36:0; C36:1; C36:2; C36:3; C36:4; C36:5; C36:6; C36:7; C36:8;

[0241] C36:9; C36:10; C36:11; C36:12; C36:13; C36:14; C36:15; C36:16; C36:17;

[0242] C37:0; C37:1; C37:2; C37:3; C37:4; C37:5; C37:6; C37:7; C37:8; C37:9;

[0243] 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;

[0244] C38:11; C38:12; C38:13; C38:14; C38:15; C38:16; C38:17; C38:18; C39:0;

[0245] C39:1; C39:2; C39:3; C39:4; C39:5; C39:6; C39:7; C39:8; C39:9; C39:10;

[0246] C39:11; C39:12; C39:13; C39:14; C39:15; C39:16; C39:17; C39:18; C40:0;

[0247] 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.

[0248] The carbon-carbon double bond can be located at any point in the fatty acid molecule (i.e., between any adjacent carbon atom pairs). The carbon-carbon double bond can be located at any point in the fatty acid molecule (i.e., between multiple adjacent carbon atom pairs). The carbon-carbon triple bond can be located at any point in the fatty acid molecule (i.e., between any adjacent carbon atom pairs). The carbon-carbon triple bond can be located at any point in the fatty acid molecule (i.e., between multiple adjacent carbon atom pairs).

[0249] In some embodiments, the fatty acid molecule can include one or more modifications and / or substitutions. In some embodiments, the fatty acid molecule can include one or more amino acids. In some embodiments, the fatty acid molecule can include one or more sugars or carbohydrate molecules. It should be understood that saturated, unsaturated, monounsaturated, and polyunsaturated fatty acid molecules can be modified and / or substituted. It should also be understood that branched-chain and non-branched-chain fatty acid molecules can be modified and / or substituted.

[0250] It should be understood that as Figure 4 shown, the binding strength of the double-stranded RNA molecule of the present invention can be purposefully controlled (e.g., adjusted) by the length of the fatty acid molecule / portion used. Without wishing to be bound by theory, it is believed that the binding strength of the double-stranded RNA molecule of the present invention (i.e., conjugated with the fatty acid molecule) may be proportional to the length of the fatty acid, as Figure 4 exemplified by the C10, C16, and C22 fatty acid conjugates, especially in the case where albumin is used as a transport vehicle.

[0251] In some embodiments, the fatty acid molecule / portion used as the conjugate portion can be selected based on the desired binding strength according to the needs of a specific intended use of the double-stranded RNA molecule of the present invention.

[0252] In some embodiments, the double-stranded RNA molecule of the present invention can be administered in combination with albumin. The albumin can be serum albumin, such as mouse serum albumin or human serum albumin.

[0253] Without wishing to be bound by theory, the 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 (e.g., into cells).

[0254] Without wishing to be bound by theory, the binding strength of the double-stranded RNA molecule of the present invention can be utilized to affect (i) the circulation 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 intracellular uptake of the double-stranded RNA of the present invention into disease-related tissues.

[0255] In some embodiments, the fatty acid molecule of the double-stranded RNA molecule of the present invention can be a fatty acid molecule having a suitable binding strength with albumin to affect the circulation 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.

[0256] In some embodiments, the fatty acid molecule conjugated to the double-stranded RNA of the present invention can be a fatty acid molecule having a suitable binding strength with albumin to affect the stability of the double-stranded RNA of the present invention in a biological fluid (such as tears). In some embodiments, the suitable binding strength can be a binding strength suitable for binding to albumin.

[0257] In some embodiments, the fatty acid molecule conjugated to the double-stranded RNA of the present invention can be a fatty acid molecule having a suitable binding strength with albumin to affect the intracellular uptake of the double-stranded RNA of the present invention into disease-related tissues. In some embodiments, the suitable binding strength can be a binding strength suitable for binding to albumin.

[0258] In some embodiments, the conjugate moiety is a cholesterol molecule. In some embodiments, the conjugate moiety can be a cholesterol molecule. It should be understood that the terms "cholesterol molecule" and "cholesterol moiety" are interchangeable.

[0259] In embodiments where 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 can be the same or can be different.

[0260] In some embodiments, two or more cholesterol molecules can be linked with a linker. It should be understood that the linker connecting two or more cholesterol molecules can be attached at any point on each of the two or more cholesterol molecules.

[0261] In some embodiments, the cholesterol moiety is selected from the group consisting of: 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 support, also known as CPG support).

[0262] 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 a reagent for introducing a cholesterol moiety at the 3' end of one of the strands in the double-stranded RNA molecule.

[0263] 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 a reagent to introduce a cholesterol moiety at the 5'-end of one of the strands in a double-stranded RNA molecule.

[0264] 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 a reagent to introduce a cholesterol moiety at the 5'-end of one of the strands in a double-stranded RNA molecule.

[0265] 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 a reagent to introduce a cholesterol moiety at the end of one of the strands in a double-stranded RNA molecule.

[0266] In some embodiments, the strands of the double-stranded RNA molecule can be synthesized using regular amidites or reverse amidites.

[0267] In some embodiments, the cholesterol molecule or cholesterol moiety is attached at the 3'-end of one of the strands in a double-stranded RNA molecule using a reverse amidite (such as a nucleoside phosphoramidite). In other words, by constructing the strand using a reverse amidite and then using any 5'-cholesterol amidite at the synthetic end, cholesterol can be attached to the 3'-end of one of the strands, which will place the cholesterol at the 3'-end of the strand.

[0268] In some embodiments, the conjugate moiety (e.g., a fatty acid molecule or a cholesterol molecule) is located at the 5'-end or 3'-end of one of the strands in a double-stranded RNA molecule.

[0269] In some embodiments, the conjugate moiety is located at the 5'-end of the first strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is located at the 3'-end of the first strand of the double-stranded RNA molecule.

[0270] In some embodiments, the conjugate moiety is located at the 5'-end of the second strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is located at the 3'-end of the second strand of the double-stranded RNA molecule.

[0271] The double-stranded RNA molecule can be described as comprising a sense strand and an antisense strand.

[0272] In some embodiments, the conjugate moiety is located at the 5'-end of the sense strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is located at the 3'-end of the sense strand of the double-stranded RNA molecule.

[0273] In some embodiments, the conjugate moiety is located at the 5'-end of the antisense strand of the double-stranded RNA molecule. In some embodiments, the conjugate moiety is located at the 3'-end of the antisense strand of the double-stranded RNA molecule.

[0274] In some embodiments, the conjugate moiety is not located at the terminal end position of either strand (i.e., the conjugate moiety is not located at the 5'-end or 3'-end of either strand). For example, the conjugate moiety can be attached to a position in the middle or central region of a continuous nucleotide sequence. As used herein, the terms "middle" and "central" are intended to mean that the conjugate moiety is not located at either end of the strand and do not mean that the position of the conjugate moiety is equidistant from each end.

[0275] In some embodiments, the conjugate moiety is located at any position of a continuous nucleotide sequence. In some embodiments, the conjugate moiety is located at any position on the double-stranded RNA molecule.

[0276] Linker

[0277] A bond, linker or spacer is a connection between two atoms that couples one target chemical group or segment to another target chemical group or segment via one or more covalent bonds. The conjugate moiety can be attached to the double-stranded RNA molecule directly or through a linking moiety (such as a linker or spacer). A linker is used to covalently connect the conjugate moiety to the double-stranded RNA molecule or its continuous nucleotide sequence.

[0278] As used herein, the terms "linker" and "spacer" are interchangeable.

[0279] In some embodiments of the present invention, the double-stranded RNA molecule of the present invention can comprise a linker (also referred to as a "linker region") located between the double-stranded RNA molecule and the conjugate moiety. The linker can be attached to the continuous nucleotide sequence of the strand of the double-stranded RNA molecule that is complementary to the target nucleic acid and the conjugate moiety.

[0280] In some embodiments, the linker is C6.

[0281] In some embodiments, the linker is TEG.

[0282] In some embodiments, the linker is a dinucleotide. In some embodiments, the linker is the CA dinucleotide.

[0283] In some embodiments, the linker is a biodegradable linker. A biodegradable linker comprises or consists of a physiologically labile bond that is cleavable under conditions normally encountered in or similar to those in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidation or reduction conditions or reagents, and salt concentrations encountered in mammalian cells or similar salt concentrations. Conditions within mammalian cells also include enzyme activities normally present in mammalian cells, such as those from proteolytic enzymes or hydrolases or nucleases. In some embodiments, the biodegradable linker is sensitive to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker comprises from 1 to 5 nucleosides, such as one or more DNA nucleosides comprising at least two consecutive phosphodiester linkages. For a detailed description of the phosphodiester containing a biodegradable linker, see WO 2014 / 076195.

[0284] In some embodiments, the linker is not a biodegradable linker. Linkers that are not necessarily biodegradable but are primarily used to covalently attach a conjugate moiety to an oligonucleotide are known. These linkers can comprise a chain structure or oligomer of repeating structures such as ethylene glycol, amino acid units or aminoalkyl groups or combinations thereof. In some embodiments, the linker is aminoalkyl, such as a C2-C36 aminoalkyl group, including for example a C6 to C12 aminoalkyl group.

[0285] Additional 5' and / or 3' nucleosides

[0286] In some embodiments, one or both strands of the double-stranded RNA molecule of the invention can further comprise additional 5' and / or 3' nucleosides. In other words, in some embodiments, the double-stranded RNA molecule of the invention can comprise 5' and / or 3' nucleosides in addition to the continuous nucleotide sequence.

[0287] The additional 5' and / or 3' nucleosides can be complementary or non-complementary (such as fully complementary) to the target nucleic acid.

[0288] The addition of further 5' and / or 3' nucleosides can be for the purpose of conjugating the continuous nucleotide sequence to a conjugate moiety or another functional group. When used to conjugate the continuous nucleotide sequence to a conjugate moiety, it can serve as a biodegradable linker. Alternatively, it can be used to provide exonuclease protection or facilitate synthesis or manufacture.

[0289] The further 5' and / or 3' nucleosides can independently comprise or consist of: 1, 2, 3, 4, 5 or more than 5 additional nucleotides, which can be complementary or non-complementary to the target nucleic acid.

[0290] Further 5' and / or 3' nucleotides can serve as nuclease-sensitive biodegradable linkers. In some embodiments, additional 5' and / or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biodegradable linkers suitable for such uses are disclosed in WO2014 / 076195, which, for example, includes phosphodiester-linked DNA dinucleotides. The use of biodegradable linkers in polyoligonucleotide constructs is disclosed in WO2015 / 113922, where they are used to link multiple antisense constructs within a single oligonucleotide.

[0291] In some embodiments, the internucleoside bond between the additional 5' and / or 3' nucleotides and the contiguous nucleotide sequence is a phosphodiester bond.

[0292] Administered to the eye

[0293] The double-stranded RNA molecule of the present invention is topically administered to the eye (e.g., by ocular administration).

[0294] It should be understood that the double-stranded RNA molecule of the present invention can be administered to any part of the eye.

[0295] In some embodiments, the double-stranded RNA molecule is used for administration to the anterior part of the eye.

[0296] The conjunctiva and the cornea are both components of the eye. In some embodiments, the double-stranded RNA molecule is used for administration to the conjunctiva. In some embodiments, the double-stranded RNA molecule is used 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.

[0297] The conjunctiva includes the bulbar conjunctiva, the palpebral conjunctiva, the ocular conjunctiva, and the fornix conjunctiva. In some embodiments, the double-stranded RNA molecule is used for administration to the bulbar conjunctiva. In some embodiments, the double-stranded RNA molecule is used for administration to the palpebral conjunctiva. In some embodiments, the double-stranded RNA molecule is used for administration to the ocular conjunctiva. In some embodiments, the double-stranded RNA molecule is used for administration to the fornix conjunctiva. In some embodiments, the double-stranded RNA molecule is used for administration to one or more of the bulbar conjunctiva, the palpebral conjunctiva, the ocular conjunctiva, and the fornix conjunctiva. 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 fornix conjunctiva. In some embodiments, the double-stranded RNA molecule targets one or more of the bulbar conjunctiva, the palpebral conjunctiva, the ocular conjunctiva, and the fornix conjunctiva.

[0298] In some embodiments, the double-stranded RNA molecule can be used for administration to the cornea and the conjunctiva. In some embodiments, the double-stranded RNA molecule can target the cornea and the conjunctiva.

[0299] In some embodiments, the double-stranded RNA molecules of the present invention can be administered in combination with albumin. The albumin can be serum albumin, such as mouse serum albumin or human serum albumin.

[0300] Complementarity to the target sequence

[0301] The continuous nucleotide sequence of the double-stranded RNA molecule of the present invention can be complementary to a target nucleic acid sequence (e.g., the nucleic acid sequence of a target mRNA).

[0302] In some embodiments, the continuous nucleotide sequence can be completely (such as about 100%) complementary to the target nucleic acid sequence.

[0303] In some embodiments, the continuous nucleotide sequence can be partially complementary to the target nucleic acid sequence, for example, 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.

[0304] In some embodiments, the continuous nucleotide sequence can include 1, 2, 3, 4, 5, 6, 7, 8 or more mismatches, where a mismatch is a nucleotide within the continuous nucleotide sequence that does not base pair with its target base.

[0305] The target nucleic acid sequence can be a target nucleic acid sequence expressed or present in the eye, such as an mRNA sequence.

[0306] In some embodiments, the target can be any target present in the eye. In some embodiments, the target nucleic acid sequence can be any nucleic acid sequence expressed or present in the eye. In some embodiments, the target nucleic acid sequence can be any mRNA sequence expressed or present in the eye. It should be understood that the reference to the eye herein includes any part of the eye. It should also be understood that the reference to the eye herein includes any combination of parts of the eye, including but not limited to the front part of the eye or all parts of the eye.

[0307] In some embodiments, the target can be any target associated with an eye lesion, eye disease or eye disorder. In some embodiments, the target can be any target associated with one or more eye lesions, eye diseases or eye disorders.

[0308] In some embodiments, the target nucleic acid sequence can be any nucleic acid sequence associated with an eye lesion, eye disease, or eye disorder. In some embodiments, the target nucleic acid sequence can be any nucleic acid sequence associated with one or more eye lesions, eye diseases, or eye disorders.

[0309] In some embodiments, the target nucleic acid sequence can be any mRNA sequence associated with an eye lesion, eye disease, or eye disorder. In some embodiments, the target nucleic acid sequence can be any mRNA sequence associated with one or more eye lesions, eye diseases, or eye disorders.

[0310] It should be understood that the target (or target nucleic acid sequence or target mRNA sequence) associated with an eye lesion, eye disease, or eye disorder 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 an eye lesion, eye disease, or eye disorder.

[0311] An example of a specific target that can be used in the present invention is AHA-1. It should be understood that AHA-1 is a non-limiting example of a target. It should also be understood that the present invention encompasses multiple other targets.

[0312] AHA1 (or AHA-1), particularly the AHA1 gene, is also known as p38; AHSA1; hAha1; C14orf3. In the context of the present invention, it should be understood that "AHA1", "p38", "AHSA1", "hAha1", and "C14orf3" are interchangeable terms.

[0313] In some embodiments, the target is the AHA-1 nucleic acid sequence.

[0314] The AHA-1 target nucleic acid sequence can comprise or consist of the following: SEQ ID NO:1 or SEQ ID NO:2.

[0315] SEQ ID NO:1 (human AHA1 target sequence: positions 488 - 507, NCBI reference sequence: NM_012111.3):

[0316] 5'-AATCTCGTGGCCTTAATGAAA-3'

[0317] SEQ ID NO:2 (rabbit AHA1 target sequence: positions 548 - 567, NCBI reference sequence: XM_002719625.3):

[0318] 5'-AATCTCGTGGCCTTAATGAAG-3

[0319] It should be understood that a "wobble" base pair mismatch exists in SEQ ID NO:2 as compared to SEQ ID NO:1 (i.e., A to G in the last nucleotide position differs between SEQ ID NO:1 and SEQ ID NO:2). Without wishing to be bound by theory, this mismatch should not affect the activity of the double-stranded RNA, since either A or G can still hybridize with the complementary U in the contiguous nucleotide sequence of the strand of the double-stranded RNA molecule (e.g., by wobble base pairing). If a wobble base pair mismatch is present, it should be understood that this mismatch should not affect the activity of the double-stranded RNA molecule.

[0320] In some embodiments, the contiguous nucleotide sequence comprises or consists of: a nucleotide sequence complementary to the AHA-1 target sequence.

[0321] In some embodiments, the contiguous nucleotide sequence comprises or consists of: a nucleotide sequence complementary to SEQ ID NO:1.

[0322] In some embodiments, the contiguous nucleotide sequence comprises or consists of: SEQ ID NO:2.

[0323] In some embodiments, the contiguous nucleotide sequence comprises or consists of: SEQ ID NO:3.

[0324] SEQ ID NO:3 (AHA-1 siRNA antisense strand):

[0325] 5'-UUUCAUUAAGGCCACGAGAUU-3'

[0326] In some embodiments, AHA-1 may be expressed in the bulbar conjunctiva and / or palpebral conjunctiva. In some embodiments, a decrease in AHA-1 expression may be observed in the bulbar conjunctiva and / or palpebral conjunctiva.

[0327] Inhibiting the target

[0328] In some embodiments, the double-stranded RNA molecule of the invention may be capable of inhibiting the expression of a target. In other words, the double-stranded RNA molecule may be capable of reducing the expression level of the target.

[0329] The target may be a target nucleic acid sequence (such as a target mRNA sequence) or a target protein.

[0330] In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting the 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% relative to a control.

[0331] In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting the expression of 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% relative to a control.

[0332] In some embodiments, the double-stranded RNA molecules of the present invention may be capable of inhibiting the 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% relative to a control.

[0333] It should be understood that the % inhibition of target expression mentioned above will be the % reduction relative to a control, where the term "control" refers to the expression of the target in cells that have not been exposed to the double-stranded RNA molecules of the present invention.

[0334] In some embodiments, the control may be a mock transfection, such as treating cells with PBS. Herein, the reduction in target expression includes a reduction in target mRNA level and / or a reduction in target protein level.

[0335] Nucleotides and nucleosides

[0336] Nucleotides and nucleosides are components of oligonucleotides and polynucleotides and, for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides and nucleosides. In nature, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate ester groups (which are not present in nucleosides). Nucleosides and nucleotides may also be interchangeably referred to as "units" or "monomers".

[0337] Modified double-stranded RNA molecules

[0338] The double-stranded RNA molecules of the present invention may be modified double-stranded RNA molecules.

[0339] The term "modified double-stranded RNA molecule" encompasses double-stranded RNA molecules that contain one or more sugar-modified nucleosides and / or modified internucleoside linkages.

[0340] In some embodiments, the double-stranded RNA molecule or its contiguous nucleotide sequence can include modified nucleobases that act as the indicated nucleobases in base pairing. For example, 5-methylcytosine can be used in place of methylcytosine. Inosine can be used as a universal base.

[0341] It should be understood that a contiguous nucleobase sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity for a target nucleic acid.

[0342] The pattern of incorporating modified nucleosides (such as high-affinity modified nucleosides) into an oligonucleotide sequence is generally referred to as oligonucleotide design.

[0343] In some embodiments, high-affinity modified nucleosides can be used.

[0344] In one embodiment, the double-stranded RNA molecule contains at least 1 modified nucleoside, such as 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 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.

[0345] Suitable modifications are described herein.

[0346] Modified internucleoside linkages

[0347] In some embodiments, the double-stranded RNA molecules of the invention can contain one or more modified internucleoside linkages.

[0348] The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently couples two nucleosides, as is commonly understood by those skilled in the art. The double-stranded RNA molecules of the invention can thus contain one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.

[0349] 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.

[0350] In some embodiments, one or more or all of the modified internucleoside linkages comprise phosphorothioate linkages.

[0351] In some embodiments, at least 50% of the internucleoside linkages in the double-stranded RNA molecule or its contiguous nucleotide sequence are phosphorothioates, 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 phosphorothioates. In some embodiments, all of the internucleoside linkages in the double-stranded RNA molecule or its contiguous nucleotide sequence are phosphorothioates.

[0352] In further embodiments, the double-stranded RNA molecule can comprise at least one modified internucleoside linkage. In some embodiments, at least 75% (such as all) of the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioates or boranophosphonate internucleoside linkages.

[0353] In some embodiments, all of the internucleoside linkages in the contiguous nucleotide sequence of the double-stranded RNA molecule can be phosphorothioates, or all of the internucleoside linkages of the double-stranded RNA molecule can be phosphorothioate linkages.

[0354] Modified nucleoside

[0355] In some embodiments, the double-stranded RNA molecule of the present invention can comprise one or more modified nucleosides.

[0356] As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified by the introduction of one or more modifications to the sugar moiety or the (nucleo)base moiety as compared to an equivalent DNA or RNA nucleoside.

[0357] In some embodiments, one or more of the modified nucleosides of the double-stranded RNA molecules of the present invention may comprise a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the terms "nucleoside analogue" or modified "unit" or modified "monomer". Nucleosides having an unmodified DNA or RNA sugar moiety are referred to herein as DNA or RNA nucleosides. Nucleosides having a modified nucleoside in the base region of a DNA or RNA nucleoside are generally still referred to as DNA or RNA if they permit Watson Crick base pairing. Exemplary modified nucleosides that may be used in the double-stranded RNA molecules of the present invention include LNA, 2'-O-MOE, 2'oMe, and morpholino nucleoside analogues.

[0358] Nucleobase

[0359] The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization.

[0360] In the context of the present invention, the term "nucleobase" also includes modified nucleobases, which may be different from the naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to the naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as 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.

[0361] In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazole-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazole-uracil, 2-thiouracil, 2'-thiothymine, inosine, 2,6-diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0362] The nucleobase moiety can be represented by the letter code of each corresponding nucleobase, such as A, T, G, C, or U, where each letter may optionally include a modified nucleobase having the same function. For example, in an exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA spacer oligomers, 5-methylcytosine LNA nucleosides can be used. 5-Methylcytosine can be represented as "E".

[0363] High-affinity modified nucleosides

[0364] High-affinity modified nucleosides are modified nucleosides that, when incorporated into an oligonucleotide, enhance the affinity of the oligonucleotide for its complementary target, for example, as measured by the melting temperature (T m m). The high-affinity modified nucleosides of the present invention preferably increase the melting temperature of each modified nucleoside by between +0.5°C and +12°C, more preferably between +1.5°C and +10°C, and most preferably between +3°C and +8°C. Many high-affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNA) (see, for example, Freier & Altmann, Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).

[0365] Sugar modifications

[0366] When compared to the ribose moiety found in DNA and RNA, the double-stranded RNA molecules of the present invention can contain one or more nucleosides having a modified sugar moiety (i.e., modification of the sugar moiety).

[0367] Many modified nucleosides having a ribose sugar moiety have been prepared, mainly for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0368] Such modifications include those in which the ribose ring structure is modified, for example, by replacing the ribose ring structure with a hexose ring (HNA) or a bicyclic ring, which typically has a double-base bridge between the C2 and C4 carbon atoms of the ribose ring (LNA), or an unlinked ribose ring that typically lacks a bond between C2 and C3 (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acid (WO 2011 / 017521) or tricyclic nucleic acid (WO 2013 / 154798). Modified nucleosides also include those in which the sugar moiety is replaced by a non-sugar moiety, such as in the case of peptide nucleic acid (PNA) or morpholino nucleic acid.

[0369] Sugar modifications also include modifications made by changing a substituent on the ribose ring to a group other than hydrogen or the 2'-OH group naturally present in DNA and RNA nucleosides. For example, substituents can be introduced at the 2', 3', 4' or 5' positions.

[0370] 2'-sugar modified nucleoside

[0371] A 2'-sugar modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2'-substituted nucleoside) or contains a 2'-linked bivalent group capable of forming a bridge between the 2' carbon and the second carbon in the ribose ring, such as an LNA (2'-4' bivalent bridged) nucleoside.

[0372] In fact, a great deal of effort has been spent developing 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 provide enhanced binding affinity for oligonucleotides and / or increased nuclease resistance. Examples of 2'-substituted modified nucleosides are 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 more 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 Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Shown below are schematic diagrams of some 2'-substituted modified nucleosides.

[0373]

[0374] For the purposes of the present invention, 2'-substituted sugar modified nucleosides do not include 2'-bridged nucleosides such as LNA.

[0375] In some embodiments, the double-stranded RNA molecule comprises one or more sugar modified nucleosides, such as 2'-sugar modified nucleosides.

[0376] 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.

[0377] In some embodiments, the double-stranded RNA molecule of the present invention comprises 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.

[0378] In some embodiments, the 2'-sugar-modified nucleoside is an affinity-enhanced 2'-sugar-modified nucleoside.

[0379] Locked nucleic acid nucleoside (LNA nucleoside)

[0380] An "LNA nucleoside" is a 2'-modified nucleoside that comprises a bi-linkage (also referred to as a "2'-4' bridge") connecting the C2' and C4' of the ribose ring of the nucleoside, which restricts or locks the conformation of the ribose ring.

[0381] These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). When LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule, the locking of the ribose conformation is associated with an enhancement of the hybridization affinity (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0382] Non-limiting exemplary LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett., 12, 73-76, Seth et al., J. Org. Chem., 2010, Vol75(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.

[0383] Other non-limiting exemplary LNA nucleosides are disclosed in Scheme 1.

[0384] Scheme 1:

[0385]

[0386] Particular LNA nucleosides are β-D-oxy-LNA, 6'-methyl-β-D-oxy LNA such as (S)-6'-methyl-β-D-oxy-LNA (ScET), and ENA.

[0387] A particularly advantageous LNA is β-D-oxy-LNA.

[0388] Morpholino oligonucleotides

[0389] In some embodiments, the double-stranded RNA molecules of the invention comprise morpholino nucleosides or consist of morpholino nucleosides (i.e., are morpholino oligomers and phosphorodiamidate morpholino oligomers (PMO)). Splice-modulating morpholino oligonucleotides have been approved for clinical use - see, e.g., eteplirsen, a 30 nt morpholino oligonucleotide targeting a frameshift mutation in DMD, for the treatment of Duchenne muscular dystrophy. Morpholino oligonucleotides have nucleic acid bases attached to a six-membered morpholine ring rather than a ribose, such as a methylene morpholine ring attached via a phosphorodiamidate group, as illustrated by the example of the following 4 consecutive morpholino nucleotides:

[0390]

[0391] In some embodiments, the length of the double-stranded RNA molecule of the present invention can be, for example, 8 to 50 morpholino nucleotides.

[0392] Complementarity

[0393] The term "complementarity" describes the ability of nucleosides / nucleotides to Watson-Crick base pair. The Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U).

[0394] It should be understood that oligonucleotides can contain nucleosides with modified nucleobases, for example, 5-methylcytosine is often used instead of cytosine, and thus, the term complementarity includes Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).

[0395] As used herein, the term "percentage of complementarity" refers to the proportion (expressed as a percentage) of nucleotides in a nucleic acid molecule (such as an oligonucleotide) that are complementary to a reference sequence (such as a target sequence or a sequence motif) in a continuous nucleotide sequence of the nucleic acid molecule across the continuous 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 the two sequences (when aligning the oligonucleotide sequence with the target sequence 5'-3' and 3'-5'), dividing it by the total number of nucleotides in the oligonucleotide, and then multiplying by 100. In this comparison, unaligned (forming base pairs) nucleobases / nucleotides are referred to as mismatches. Insertions and deletions are not allowed when calculating the percentage of complementarity of a continuous nucleotide sequence.

[0396] It should be understood that when determining complementarity, chemical modifications of the nucleobases are not considered as long as the functional ability of the nucleobases to form Watson-Crick base pairs is retained (for example, 5'-methylcytosine is considered the same as cytosine when calculating the percentage of complementarity).

[0397] Identity

[0398] The term "identity" as used herein refers to the proportion (expressed as a percentage) of nucleotides in a nucleic acid molecule (such as an siRNA molecule) that are the same as a reference sequence (such as a sequence motif) in a continuous nucleotide sequence of the nucleic acid molecule across the continuous nucleotide sequence.

[0399] Thus, the percent identity is calculated by counting the number of aligned nucleobases that are the same (matched) in 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, percent identity = (number of matches x 100) / length of the alignment region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percent identity of the contiguous nucleotide sequence. It should be understood that in determining identity, chemical modifications of the nucleobases (e.g., 5-methylcytosine is considered the same as cytosine in calculating percent identity) are not taken into account as long as the functional ability of the nucleobase to form Watson Crick base pairs is retained.

[0400] Thus, it should be understood that there is a relationship between identity and complementarity such that a contiguous nucleotide sequence that is complementary to a target sequence within a double-stranded RNA molecule of the invention also shares a certain percentage of identity with the complementary sequence.

[0401] Hybridization

[0402] As used herein, the term “hybridizing / hybridizes” is to be understood as the formation of hydrogen bonds between base pairs on opposite strands of two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid), thereby forming a duplex. The affinity of binding between the two nucleic acid strands is the strength of the hybridization. It is typically described in terms of the melting temperature (T m ), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. Under physiological conditions, T m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515–537). The standard state Gibbs free energy ΔG° is a more accurate representation of the binding affinity and is related to the dissociation constant (K d ) of the reaction by ΔG° = -RTln(K d) is related, where R is the gas constant and T is the absolute temperature. Thus, a very low ΔG° for the reaction between the oligonucleotide and the target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy related to a reaction where the water concentration is 1 M, the pH is 7, and the temperature is 37 °C. Hybridization of the oligonucleotide with the target nucleic acid is a spontaneous reaction, and for a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, by using the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36–38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercial devices are available for measuring ΔG°. The value of ΔG° can also be estimated by using the nearest neighbor model as described in Santa Lucia 1998, Proc Natl Acad Sci USA. 95:1460–1465, appropriately using the derived thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211–11216 and McTigue et al., 2004, Biochemistry 43:5388–5405.

[0403] In some embodiments, the degree or strength of hybridization is measured according to the standard state Gibbs free energy ΔG°. The oligonucleotide can hybridize with the target nucleic acid at estimated ΔG° values below the -10 kcal range, such as below -15 kcal, such as below -20 kcal, and such as below -25 kcal. In some embodiments, the oligonucleotide hybridizes with the target nucleic acid at estimated ΔG° values of -10 to -60 kcal, such as -12 to -40 kcal, such as -15 to -30 kcal, or -16 to -27 kcal, such as -18 to -25 kcal.

[0404] Salt

[0405] As used herein, the term "salt" conforms to its generally known meaning, i.e., an ionic combination of an anion and a cation.

[0406] 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, wherein the double-stranded RNA molecule is in the form of a pharmaceutically acceptable salt.

[0407] In some embodiments, the pharmaceutically acceptable salt can be a sodium salt or a potassium salt.

[0408] The present invention provides a pharmaceutically acceptable sodium salt of the double-stranded RNA molecule of the present invention.

[0409] The present invention provides a pharmaceutically acceptable potassium salt of the double-stranded RNA molecule of the present invention.

[0410] Delivery of double-stranded RNA molecules

[0411] The double-stranded RNA molecules of the present invention can be encapsulated in lipid-based delivery vehicles, covalently linked to or encapsulated in dendrimers, or conjugated with aptamers.

[0412] This may be for delivering the double-stranded RNA molecules of the present invention to target cells and / or improving the pharmacokinetics of the double-stranded RNA molecules.

[0413] Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0414] It should be understood that when the double-stranded RNA molecules of the present invention are conjugated to at least one conjugate moiety, in addition to conjugating the double-stranded RNA molecules to at least one conjugate moiety, encapsulating the double-stranded RNA molecules can provide additional advantages compared to conjugating only to the conjugate moiety.

[0415] In some embodiments, the double-stranded RNA molecules of the present invention can be administered in combination with albumin. The albumin can be serum albumin, such as mouse serum albumin or human serum albumin. Without wishing to be bound by theory, albumin can act as a transport vehicle and / or can facilitate or enhance the uptake of the double-stranded RNA molecules of the present invention into relevant tissues or cells.

[0416] Pharmaceutical compositions

[0417] The present invention provides a pharmaceutical composition comprising the double-stranded RNA molecules of the present invention and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0418] The present invention provides a pharmaceutical composition comprising the double-stranded RNA molecules of the present invention and a pharmaceutically acceptable salt. For example, the salt can comprise a metal cation, such as a sodium salt or a potassium salt.

[0419] The present invention provides the pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises the double-stranded RNA molecules of the present invention and an aqueous diluent or solvent.

[0420] The present invention provides a solution, such as a phosphate buffered saline solution of the double-stranded RNA molecules of the present invention. In some embodiments, the solution of the present invention, such as a phosphate buffered saline solution, is a sterile solution.

[0421] The pharmaceutical compositions of the present invention can be administered topically to the eye (e.g., by ophthalmic administration). It should be understood that the double-stranded RNA molecules of the present invention can be administered to any part of the eye.

[0422] In some embodiments, the pharmaceutical composition is for administration to the anterior part 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 fornix conjunctiva. In some embodiments, the pharmaceutical composition is for administration to one or more of the bulbar conjunctiva, palpebral conjunctiva, ocular conjunctiva, and fornix conjunctiva.

[0423] In some embodiments, the pharmaceutical composition of the present invention may comprise albumin. The albumin may be serum albumin, such as mouse serum albumin or human serum albumin.

[0424] Manufacturing method

[0425] In a further aspect, the present invention provides a method for manufacturing the double-stranded RNA molecule of the present invention, the method comprising reacting nucleotide units and thereby forming covalently linked consecutive nucleotide units contained in an oligonucleotide. The method may use phosphoramidite chemistry (see, for example, Caruthers et al., 1987, Methods in Enzymology vol. 154, pages 287-313). In another embodiment, the method further comprises reacting the consecutive nucleotide sequence with a conjugate moiety to attach (e.g., covalently attach) the conjugate moiety to the double-stranded RNA molecule. In another aspect, there is provided a method for preparing the composition of the present invention, the method comprising mixing the double-stranded RNA molecule of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0426] Treatment

[0427] As used herein, the term "treatment" refers to the treatment of an existing disease (such as the diseases or disorders referred to herein) or the prevention of a disease, i.e., prophylaxis. Thus, it will be recognized that in some embodiments, the treatment referred to herein may be prophylactic.

[0428] The present invention provides a method for treating or preventing a disease, the method comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of the double-stranded RNA molecule of the present invention or the pharmaceutical composition of the present invention.

[0429] The present invention provides the double-stranded RNA molecule of the present invention for use as a medicament in the treatment of a disease.

[0430] The present invention provides the double-stranded RNA molecule of the present invention for use in therapy.

[0431] The present invention provides a double-stranded RNA molecule of the present invention for use in the preparation of a medicament for treating or preventing a disease.

[0432] The present invention provides a pharmaceutical composition of the present invention for use as a medicament.

[0433] The present invention provides a pharmaceutical composition of the present invention for use in therapy.

[0434] The present invention provides a pharmaceutical composition of the present invention for use in the preparation of a medicament for treating or preventing a disease.

[0435] The disease can be a disease associated with the eye. The disease can be a disease affecting the eye.

[0436] The disease can be an infection of the eye (or associated with the eye). The disease can be a type of inflammation of the eye (or associated with the eye).

[0437] In some embodiments, the disease can be conjunctivitis, dry eye, or inflammation of the eye.

[0438] 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.

[0439] In some embodiments, the double-stranded RNA molecule or pharmaceutical composition of the present invention is used for combination therapy with another therapeutic agent.

[0440] Administration strategy

[0441] It should 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.

[0442] In some embodiments, the administration of the double-stranded RNA molecule or pharmaceutical composition can be long-term administration.

[0443] In some embodiments, the double-stranded RNA molecule or pharmaceutical composition of the present invention is administered to only one eye.

[0444] In some embodiments, the double-stranded RNA molecule or pharmaceutical composition of the present invention is administered to both eyes. It can be administered to both eyes simultaneously or sequentially.

[0445] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye once, twice, three times, 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.

[0446] In some embodiments, the double-stranded RNA molecule can be administered to one (or each) eye every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, or every 12 hours. In some embodiments, the double-stranded RNA molecule can be administered to one (or each) eye every 4 hours.

[0447] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for less than one day, or for one day, or for two consecutive days, three consecutive days, four consecutive days, five consecutive days, six consecutive days, seven consecutive days, eight consecutive days, nine consecutive days, ten consecutive days, 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.

[0448] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for a period of one week, two weeks, three weeks, four weeks, five weeks, six weeks, or more than six 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, or more than 52 weeks.

[0449] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for a period of one month, two months, three months, four months, five months, six months, or more than six 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, or more than 12 months.

[0450] In some embodiments, the double-stranded RNA molecule is administered to one (or each) eye for a period of one year, two years, three years, four years, five years, or more than five 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, or more than 25 years.

[0451] Method for regulating expression

[0452] The present invention provides a method for modulating the 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.

[0453] In some embodiments, the method is an in vitro method.

[0454] In some embodiments, the method is an in vivo method.

[0455] In some embodiments, the cell is an animal cell, preferably a mammalian cell such as a mouse cell, a rat cell, a hamster cell or a monkey cell or preferably a human cell.

[0456] In some embodiments, the cell is a mammalian cell.

[0457] In some embodiments, the cell is a human cell.

[0458] In some embodiments, the cell is a cell of the eye. In some embodiments, the cell is a cell of the human eye.

[0459] In some embodiments, the cells can be collected by sampling a medical device. In some embodiments, the cells can be from a collection of conjunctival cells on the ocular surface of the eye.

[0460] Applications

[0461] The double-stranded RNA molecules of the present invention can be used as research reagents, for example, for diagnosis, treatment and prevention.

[0462] In research, such double-stranded RNA molecules can be used to inhibit the expression of a target expressed in the eye of an experimental animal, thereby facilitating functional analysis of the target or evaluating its utility as a therapeutic intervention target.

[0463] The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in a 5' to 3' orientation; amino acid sequences are written left to right in an amino to carboxyl orientation.

[0464] Prior to the filing date of the present application, the publications discussed herein are provided only for their disclosure. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0465] The present invention will now be further described by way of examples, which are intended to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

[0466] Example

[0467] Example 1:

[0468] New Zealand white rabbits were administered by topical application in both the left and right eyes, 3 times a day, with an administration interval of at least 4 h, for 5 days, using 20 μl of a 25 μg / μL solution (500 μg per dose) of naked or C16-, C22-, or cholesterol-conjugated siRNA molecules (containing the sequence of SEQ ID NO:3 formulated in PBS). Four days after the last administration, the rabbits were sacrificed, and palpebral conjunctiva samples were collected from each eye (saline n = 4, siRNA-treated group n = 6), and AHSA1 mRNA knockdown was analyzed by digital droplet PCR.

[0469] After adding metal beads, the conjunctival tissue was homogenized in 500 μL of MagnaPure tissue lysis buffer (Roche LifeScience) using a TissueLyser II (Qiagen), and mRNA was extracted from 350 μL of the lysis buffer and eluted in 50 μL of RNase-free water according to the manufacturer's instructions (Roche LifeScience). cDNA synthesis was performed using 4 μL of input RNA with the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad), and according to the manufacturer's protocol, 2 μL was used as the input for digital droplet PCR, using ddPCR supermix as the probe (without dUTP) (Bio-Rad).

[0470] The following TaqMan gene expression assays were used:

[0471] AHSA1 (FAM): Oc06762465_g1 (catalog number: 4351372, TaqMan Thermofisher Scientific) and HPRT1 (VIC): Oc03399461_m1 (catalog number: 4331182, TaqMan Thermofisher Scientific)

[0472] The concentration of AHSA1 mRNA was quantified relative to the housekeeping gene HPRT using QuantaSoft software (Bio-Rad) and normalized to PBS-treated rabbits (set to value 1).

[0473] The results are shown in Figure 1Shown in. For naked siRNA molecules containing the sequence of SEQ ID NO:3, C16-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, C22-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, and cholesterol-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, 20% to 25% knockdown was observed throughout the conjunctiva, indicating that C16, C22, and cholesterol AHSA1 siRNAs have superior knockdown compared to naked AHSA1 siRNA.

[0474] Example 2:

[0475] New Zealand white rabbits were administered in the eyes by topical application three times a day at least 4 h apart for 5 days with 20 μl of a 25 μg / μL solution (500 μg per dose) of naked or C16-, C22-, or cholesterol-conjugated siRNA molecules (containing the sequence of SEQ ID NO:3 in PBS). Four days after the last administration, the rabbits were sacrificed and palpebral conjunctiva samples were collected (saline n = 4, siRNA-treated group n = 6), and AHSA1 siRNA content analysis was performed by hELISA.

[0476] After adding metal beads, conjunctival tissue (∼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 biotinylated capture probes and digoxigenin-conjugated detection probes that bind to the antisense siRNA strand. The resulting lysate was diluted and incubated with 35 nM biotinylated capture probe and 30 nM digoxigenin-conjugated detection probe in 96-well plates in SSCT buffer (5x sodium citrate buffered saline [SSC buffer 20x concentrate, Sigma-Aldrich, cat. no. 6639], containing 0.05% Tween 20 [Sigma-Aldrich, cat. no. P9416]) for 30 minutes at room temperature. The assembled complexes were then captured on streptavidin-coated ELISA plates (Nunc 436014) for 1 hour, and after three washing steps with 2xSSCT buffer, each well was incubated with anti-digoxigenin alkaline phosphatase (AP)-Fab fragments (Roche, cat. no. 11093274910) for 1 hour at room temperature. After three additional washing steps, BluePhos substrate (Kirkegaard & Perry Labs [KPL], cat. no. 50-88-00) was added to the plates, and the color development was measured spectrophotometrically at 615 nm after 20 minutes. For oligonucleotide content analysis, several dilutions (50x, 100x, 200x, 400x, 800x, and 1,600x) of each sample were measured. The respective tissue weights were calculated and the valid values (i.e., readings within the linear range of the standard curve) for the different dilutions were averaged to generate the drug concentration in nmol / g of conjunctival tissue weight.

[0477] Results are shown in Figure 2 For naked siRNA molecules containing the sequence of SEQ ID NO:3, C16-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, C22-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, and cholesterol-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, increased content was observed in the conjunctiva, indicating that C16-, C22-, and especially cholesterol-conjugated AHA1 siRNAs have superior content compared to naked siRNA.

[0478] Example 3:

[0479] New Zealand white rabbits were administered the drug in the eyes by topical application, three times a day, at least 4 h apart, for 5 days, using 20 μl of a 25 μg / μL solution (500 μg per dose) of naked or C16-, C22- or cholesterol-conjugated siRNA molecules (sequences of SEQ ID NO:3 contained in PBS). Four days after the last administration, EYEPRIM (OPIA technologies) samples were collected from the bulbar conjunctiva (saline n = 4, siRNA-treated group n = 6). After euthanizing the animals, the bulbar conjunctiva was exposed and the EYEPRIM membrane was pressed onto the inferior bulbar conjunctiva for 3 seconds. Then the membrane was removed from the EYEPRIM. When doing so, the membrane was clamped with forceps during ejection to avoid the membrane falling / flying away. The membrane was snap-frozen into a 2 mL Eppendorf tube.

[0480] After adding metal beads, the EYEPRIM samples were homogenized in 500 μL of MagnaPure tissue lysis buffer (Roche LifeScience) using a TissueLyser II (Qiagen), and the pre-mRNA was advanced from 350 μL of the lysis buffer and extracted in 50 μL of RNase-free water according to the manufacturer's instructions (Roche LifeScience). cDNA synthesis was performed using 4 μL of input RNA with the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad), and according to the manufacturer's protocol, 2 μL was used as the input for digital droplet PCR, using ddPCR supermix as the probe (without dUTP) (Bio-Rad).

[0481] The following TaqMan gene expression assays were used:

[0482] AHSA1 (FAM): Oc06762465_g1 (catalog number: 4351372, TaqMan Thermofisher Scientific) and HPRT1 (VIC): Oc03399461_m1 (catalog number: 4331182, TaqMan Thermofisher Scientific).

[0483] The AHSA1 mRNA concentration was quantified relative to the housekeeping gene HPRT using QuantaSoft software (Bio-Rad) and normalized to PBS-treated rabbits (PBS was set to 1)

[0484] The results are in Figure 3Shown in. Comparing 69% knockdown (C16-conjugated SEQ ID NO:3), 65% knockdown (C22-conjugated SEQ ID NO:3), and 61% knockdown (cholesterol-conjugated SEQ ID NO:3) in the conjunctival EYEPRIM sample with 54% of the naked siRNA (SEQ ID NO:3), C16 had significantly superior knockdown compared to the naked siRNA (p = 0.02, Student's t-test), and C22- and cholesterol-conjugated AHSA1 siRNAs had superior knockdown.

[0485] Example 4

[0486] Isothermal titration calorimetry (ITC)

[0487] The experiments were carried out in an automated PEAQ ITC (Malvern Panalytical, Malvern, United Kingdom) in the assay setup, where the target protein was present in the syringe and injected into the FA-siRNA sample placed in the cell. Mouse serum albumin (MSA, catalog number A3139-100 mg, substantially free of fatty acids) (25 - 450 μM) in the syringe and FA-siRNA (3 - 60 μM) in the cell were adjusted using buffer from dialysis filtered and degassed according to their measured stoichiometry and binding affinity. ITC analysis was performed at 25 °C with the following experimental parameters: stirring speed: 750 rpm, interval: 150 s, injection duration: 3 s, reference power 10 μcal / s. The initial delay was set to 60 s and the filter period was set to 5 s, with 13 or 19 injections performed respectively, and titration volumes of 2.0 or 3.0 to collect enthalpy data for the analysis of the interaction. Control titrations were performed to correct for the heat of dilution (buffer titrated into FA-siRNA, MSA titrated into buffer, and buffer titrated into buffer). MSA was dissolved in DPBS (catalog number 14190-094, Gibco / Thermo Fisher Scientific, Basel, Switzerland) and further dialyzed using DPBS (catalog number 66003, Thermo ScientificTM, Waltham, MA) with a Slide-A-Lyzer dialysis cassette 20K MWCO prior to ITC analysis. FA-siRNA powder was dissolved in DPBS. The concentrations of the dialyzed protein and FA-siRNA were determined by ultraviolet spectroscopy at 280 nm and 260 nm respectively (according to the Beer-Lambert law equation). The offset and the heat of dilution of MSA 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 the MicroCal PEAQ ITC analysis software, version 1.3 (Malvern instrument, Malvern, United Kingdom). A control ITC experiment of titrating MSA into warfarin (DPBS + 2% DMSO (v / v)) was performed prior to the FA-siRNA ITC study.

[0488] Analytical ultracentrifugation (AUC)

[0489] Sedimentation velocity (SV) and sedimentation equilibrium (SE) experiments were performed on a analytical ultracentrifuge XLI and Optima-AUC (Beckman Coulter, CA), where absorbance detection (range 260 - 295 nm) was carried out according to the test concentration (1 - 75 μM). Individual samples were dissolved in DPBS (Catalog No. 14190-094, Gibco / Thermo Fisher Scientific, Basel, Switzerland) and diluted in DPB. The AUC-SV experiments were run at 60000 rpm and 20 °C, using 3 mm and 12 mm Ti centerpieces (Nanolytics Instruments, Potsdam, Germany) and an An-60 Ti rotor (Beckman Coulter, CA). The SE experiments were performed in a multi-speed mode (10, 15, and 20 krpm or 7, 10, and 14 krpm for the FA Bis C16 and FA C24 conjugates respectively), and the FA-siRNA conjugate concentrations were set at 6 and 25 μM. SE analysis used a 3 mm Ti centerpiece (Nanolytics Instruments, Potsdam, Germany) and an An-60 Ti rotor (Beckman Coulter, CA). The density and viscosity of the buffer were determined using a densitometer and viscometer (DMA 5000M and AMVn respectively, Anton Paar, Aarau, Switzerland). The interaction of the FA-siRNA conjugate with MSA was analyzed in SV mode using a fluorescence detection system (Aviv Biomedical, NJ), with absorbance (290 - 305 nm) and / or fluorescence detection.

[0490] Results

[0491] The results are shown in Figure 4 , which provides a biophysical analysis of different fatty acid-conjugated AHA-1 specific siRNAs (FA-siRNA). Column 3: The tendency of different fatty acid conjugates to exist in different oligomeric states at a concentration of 25 μM (final oligomeric state measured by AUC); Column 4: The monomer percentage of different FA-siRNAs when dissolved in 25 μM PBS; Column 5: The binding affinity to mouse serum albumin (MSA; determined by ITC); Column 6: The number of FA-siRNA conjugates bound to MSA.

[0492] Results showed that: (i) the binding strength of fatty acid-conjugated siRNA molecules can be modulated / modified by the length of the fatty acid; (ii) introduction of a double bond (e.g., stearic acid vs. oleic acid) does not affect the binding of the fatty acid to albumin; and (iii) albumin binding can be employed as a vehicle for fatty acid-conjugated siRNA molecules and the binding strength can be adjusted as needed.

[0493] Example 5:

[0494] New Zealand white rabbits were administered topically in both the left and right eyes three times a day at intervals of at least 4 h for 5 days with 20 μl of a 25 μg / μL solution (500 μg per dose) of naked or C16-, C22- or cholesterol-conjugated siRNA molecules (containing the sequence of SEQ ID NO:3 formulated in PBS). Three days after the last administration, the rabbits were sacrificed and samples of the upper and lower palpebral conjunctiva were collected from each eye (n = 2 eyes), and tissue sections (n = 3 - 8) were analyzed by in situ hybridization (ISH) for the distribution of siRNA molecules in the conjunctival tissue.

[0495] The conjunctival tissue was dissected and fixed overnight in 10% NBF, then subjected to standard dehydration and embedded in paraffin the next day. Transverse conjunctival tissue sections (5 μm) were stained using an automated Ventana Discovery ULTRA stainer (Roche Diagnostics). The automated protocol was designed to bake (60 °C for 20 minutes), dewax (69 °C for 24 minutes), and perform ISH protease 3 treatment (one drop, 32 minutes). The slides were treated with DISC inhibitor (one drop, 12 minutes) and washed with reaction buffer (1x). The DIG-labeled probe targeting SEQ ID NO:3 was diluted to a final concentration of 0.25 nM in miRCURY LNA miRNA ISH buffer (1x in RNase-free water) and manually applied to the slides. The slides were denatured at 90 °C for 8 minutes and then hybridized at 65 °C for 16 minutes. The slides were then washed with 0.1x SSC (5x at 54 °C for 4 minutes). DIG molecules were detected using anti-DIG-HRP (one drop, 4 °C, no heating), followed by the application of DISC AMP TSA BF and DISC AMP H2O2BF (one drop each, no heating, 4 minutes) and by DISC anti-BF HRP (one drop each, no heating, 4 minutes). The HRP was detected using a DAB (brown) detection kit. Before mounting in EcoMount medium, the slides were counterstained with hematoxylin II (no heating, 4 minutes) and then treated with a bluing reagent (no heating, 4 minutes). The slides were scanned at 20x magnification using an Olympus VS120 slide scanner to visualize the results. Positive staining was quantified using Halo, Indica Labs (V3.6.4134.166).

[0496] 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), DISCChromoMap DAB RUO (05266645001), all purchased from Roche Diagnostics. miRCURY LNA miRNA ISH buffer (#1108512) and DIG-labeled probe targeting SEQ ID NO:3 (custom miRCURY LNA / 5DiGN / CGTGGCCTTAATGAA / 3DiG_N / ) were purchased from Qiagen.

[0497] The results were as Figure 5 shown: For naked siRNA molecules containing the sequence of SEQ ID NO:3, C16-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, C22-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, and cholesterol-conjugated siRNA molecules containing the sequence of SEQ ID NO:3, 0.3% to 0.4% conjunctival tissue ISH staining was observed, indicating that C16, C22, and cholesterol AHSA1 siRNAs have more excellent tissue staining compared to naked AHSA1 siRNA, and C16 and C22 AHAS1 siRNAs showed the most excellent results compared to naked and cholesterol AHSA1 siRNAs. In addition, the staining of naked AHSA1 siRNA was mainly located in the superficial layer of the conjunctiva, while the staining of C16, C22, and cholesterol AHSA1 siRNAs was also located in the stroma of the conjunctival tissue.

[0498] All publications mentioned in the above specification are incorporated herein by reference. Accordingly, various modifications and variations of the described methods and systems of the present 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 to the described manner of implementing the invention will be apparent to those skilled in the art of molecular biology or related fields and are intended to fall 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 is capable of binding to a target sequence, wherein 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, wherein the first strand comprises a continuous nucleotide sequence of at least 8 nucleotides that is complementary to the target sequence, and wherein the double-stranded RNA molecule is conjugated to at least one conjugate moiety.

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

3. The double-stranded RNA molecule according to claim 1 or claim 2, wherein the double-stranded RNA molecule is capable of inhibiting the expression of the target.

4. The double-stranded RNA molecule according to any one of claims 1 to 3, wherein the conjugate moiety 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 or claim 5, wherein the fatty acid molecule is C16.

7. The double-stranded RNA molecule according to claim 4 or claim 5, wherein the fatty acid molecule is C22.

8. The double-stranded RNA molecule according to any one of claims 4 to 7, wherein the fatty acid molecule is branched.

9. The double-stranded RNA molecule according to any one of claims 4 to 7, wherein the fatty acid molecule is unbranched.

10. The double-stranded RNA molecule according to any one of claims 4 to 9, wherein the fatty acid molecule is saturated.

11. The double-stranded RNA molecule according to any one of claims 4 to 9, wherein the fatty acid molecule is unsaturated.

12. The double-stranded RNA molecule according to claim 11, wherein the fatty acid molecule comprises 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-carbon double bonds.

13. The double-stranded RNA molecule according to claim 11 or claim 12, wherein the fatty acid molecule comprises 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 double-stranded RNA molecule according to any one of claims 11 to 13, wherein the fatty acid molecule is selected from the list consisting of: 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; 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; 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; C32:2; C32:3; C32:4; C32:5; 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; C35:14; 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; 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; C38:11; 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; 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 any one of claims 1 to 15, wherein the conjugate moiety (i) is located at the 5'-end or the 3'-end of the first strand; or (ii) is located at the 5'-end or the 3'-end of the second strand.

17. The double-stranded RNA molecule according to any one of claims 1 to 16, wherein the conjugate moiety is located at the 3'-end of the first strand.

18. The double-stranded RNA molecule according to any one of claims 1 to 17, wherein a linker is located between the double-stranded RNA molecule and the conjugate moiety.

19. The double-stranded RNA molecule according to any one of claims 1 to 18, wherein the linker is (i) C6; (ii) TEG; or (iii) a dinucleotide, optionally wherein the dinucleotide is CA.

20. The double-stranded RNA molecule according to any one of claims 1 to 19, wherein the length of 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.

21. The double-stranded RNA molecule according to any one of claims 1 to 20, wherein the length of the continuous nucleotide sequence is at least 20 nucleotides.

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

23. The double-stranded RNA molecule according to any one of claims 1 to 22, wherein the first strand consists of the continuous nucleotide sequence.

24. The double-stranded RNA molecule according to any one of claims 1 to 23, wherein the double-stranded RNA molecule is for administration to the anterior part of the eye.

25. The double-stranded RNA molecule according to any one of claims 1 to 24, wherein the double-stranded RNA molecule is for administration to the conjunctiva of the eye or the 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 fornix conjunctiva.

27. The double-stranded RNA molecule according to any one of claims 1 to 26, wherein the continuous nucleotide sequence is at least 75% complementary to the target sequence.

28. The double-stranded RNA molecule according to claim 27, wherein the continuous 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 any one of claims 1 to 28, wherein the continuous nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8 or more mismatches with the target sequence.

30. The double-stranded RNA molecule according to any one of claims 2 to 29, wherein the target is AHA-1.

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

2.

32. The double-stranded RNA molecule according to any one of claims 1 to 31, 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 continuous nucleotide sequence comprises a nucleotide sequence complementary to SEQ ID NO:1 or SEQ ID NO:

2.

34. The double-stranded RNA molecule according to any one of claims 1 to 33, wherein the continuous nucleotide sequence comprises the following or consists of: SEQ ID NO:

3.

35. The double-stranded RNA molecule according to any one of claims 2 to 34, 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 any one of claims 1 to 35, 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 independently 2'-sugar modified nucleosides 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.

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

39. The double-stranded RNA molecule according to any one of claims 1 to 38, wherein one or more of the internucleoside linkages between the nucleosides located 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 linkages between the nucleosides located on the continuous nucleotide sequence are modified.

41. The double-stranded RNA molecule according to claim 39 or claim 40, wherein one or more or all of the modified internucleoside linkages comprise phosphorothioate linkages.

42. The double-stranded RNA molecule according to claim 41, wherein all of the internucleoside linkages present in the double-stranded RNA molecule are phosphorothioate internucleoside linkages.

43. The double-stranded RNA molecule according to any one of claims 1 to 42, 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 the double-stranded RNA molecule according to any one of claims 1 to 44 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

46. A method for treating or preventing a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the double-stranded RNA molecule according to any one of claims 1 to 44 or the pharmaceutical composition according to claim 45.

47. The double-stranded RNA molecule according to any one of claims 1 to 44 or the pharmaceutical composition according to claim 45 for use as a medicament in the treatment of a disease.

48. Use of the double-stranded RNA molecule according to any one of claims 1 to 44 or the pharmaceutical composition according to claim 45 for the preparation of a medicament for the treatment or prevention of a disease.

49. The method according to claim 46; the double-stranded RNA molecule or pharmaceutical composition used according to claim 47; or the use according to claim 48; wherein the disease is conjunctivitis, dry eye or inflammation.

50. The method according to claim 46; The double-stranded RNA molecule or pharmaceutical composition used according to claim 47; The use according to claim 48; or the method, the double-stranded RNA molecule or pharmaceutical composition used, or the use according to claim 49; wherein the double-stranded RNA molecule is administered to the eye once a day, twice a day, three times a day or more than three times a day; optionally wherein the double-stranded RNA molecule is administered to both eyes.

51. The method according to claim 46; The double-stranded RNA molecule or pharmaceutical composition used according to claim 47; The use according to claim 48; or the method, double-stranded RNA molecule or pharmaceutical composition used, or the use according to claim 49 or claim 50; wherein the double-stranded RNA molecule is administered for a period of less than one day, or for one, two, three, four, five, six, seven or more than seven days; optionally wherein the double-stranded RNA molecule is administered to both eyes.

52. The method according to claim 46; The double-stranded RNA molecule or pharmaceutical composition used according to claim 47; The use according to claim 48; or the method, double-stranded RNA molecule or pharmaceutical composition used, or the use according to claim 49 or claim 50; wherein the double-stranded RNA molecule is administered for the following periods: (i) one, two, three, four, five, six or more than six weeks, or (ii) one, two, three, four, five, six or more than six months, or (iii) one, two, three, four, five or more than five years; optionally wherein the double-stranded RNA molecule is administered to both eyes.

53. An in vitro method for modulating the expression of a target in a cell, the method comprising administering to the cell, in an effective amount, the double-stranded RNA molecule according to any one of claims 1 to 44 or the pharmaceutical composition according to claim 45.

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