Ocular delivery of oligonucleotides

Abstract

Provided herein are conjugated oligonucleotides characterized by efficient and specific intraocular distribution.

Description

[Technical Field]

[0001] Related Applications This invention claims the benefit of U.S. Provisional Patent Application No. 63 / 412,051, filed September 30, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present disclosure relates to oligonucleotide conjugates and branched oligonucleotides for delivery to the eye. [Background technology]

[0003] Eye diseases are caused by genetic and non-genetic risk factors. Some of these diseases have a clear underlying genetic etiology (mutation) that can be inherited in a dominant, recessive, or X-linked inheritance pattern. Such hereditary retinal dystrophies are caused by more than 250 genes. These include dominant mutations that cause dominant retinitis pigmentosa, such as the P23H mutation in the rhodopsin gene, which is the most common dominant mutation among individuals with dominant retinitis pigmentosa. Eye diseases of unknown etiology include age-related macular degeneration, diabetic retinopathy, and glaucoma. While some of these diseases are caused by environmental risk factors, many genetic risk factors that are thought to contribute to disease progression have also been identified. Interestingly, several non-coding RNA sequences have also been identified that contribute to various diseases. Therefore, there is an unmet need to efficiently regulate gene expression in the eye to treat various eye diseases. Described herein is a method for downregulating the expression of disease-causing genes in the eye that directly or indirectly contribute to pathology.

[0004] Oligonucleotides such as small interfering RNA (siRNA) molecules have been used to regulate gene expression levels across various organs. However, their implementation in the eye has been hindered by the low permeability of siRNA molecules into various cell types, the stability of siRNA, and the long-term persistence of knockdown effects. This is particularly important in the eye, where repeated injections on a biweekly or monthly basis are burdensome for patients and healthcare providers and increase the risk of ocular complications. This paper describes an oligonucleotide platform in which oligonucleotides (e.g., siRNA molecules) are chemically stabilized for long-term gene knockdown and modified in structure or binding to improve cellular entry into different cell types of the retina. Summary of the Invention

[0005] Provided herein are methods for delivering oligonucleotide conjugates and branched oligonucleotides to the eye, particularly to specific ocular cells. The oligonucleotide conjugates and branched oligonucleotides can achieve efficient gene knockdown in the eye. Several different functional moieties and branched oligonucleotides have demonstrated ocular cell-specific delivery upon administration.

[0006] The oligonucleotide conjugates and branched oligonucleotides described herein facilitate simple, efficient, and non-toxic delivery of oligonucleotides (e.g., siRNA) and potent silencing of therapeutic targets in various ocular cell types in vivo.

[0007] In one aspect, the disclosure provides a method for delivering an oligonucleotide conjugate to the eye of a subject, the method comprising administering to the subject an oligonucleotide conjugate comprising: i) an oligonucleotide comprising a 5' end and a 3' end and having complementarity to a target nucleic acid; and ii) a functional moiety linked to the oligonucleotide, the functional moiety comprising any one of a triple amine, retinoic acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), alpha-tocopheryl succinate, or lithocholic acid (LA).

[0008] In another aspect, the present disclosure provides a method for delivering a branched oligonucleotide to the eye of a subject, the method comprising administering a branched oligonucleotide to the subject, the branched oligonucleotide comprising two or more oligonucleotides, each oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid. In certain embodiments, one or more of the oligonucleotides of the branched oligonucleotide further comprise a functional moiety linked to the oligonucleotide, the functional moiety comprising any one of a triple amine, retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA.

[0009] In certain embodiments, two DHA functional moieties are linked to the oligonucleotide.

[0010] In certain embodiments, the oligonucleotide comprises an antisense oligonucleotide or an siRNA.

[0011] In certain embodiments, the siRNA comprises a sense strand and an antisense strand. In certain embodiments, the antisense strand comprises about 15 to 25 nucleotides in length. In certain embodiments, the sense strand comprises about 15 to 25 nucleotides in length. In certain embodiments, the antisense strand is 20, 21, or 22 nucleotides in length. In certain embodiments, the sense strand is 15, 16, 18, or 20 nucleotides in length.

[0012] In certain embodiments, the siRNA comprises a double-stranded region of 15 to 20 base pairs, 15, 16, 18, or 20 base pairs.

[0013] In certain embodiments, the siRNA comprises at least one blunt end.

[0014] In certain embodiments, the siRNA comprises at least one single-stranded nucleotide overhang. In certain embodiments, the siRNA comprises a single-stranded nucleotide overhang of about 2 to 5 nucleotides. In certain embodiments, the siRNA comprises a 2-nucleotide single-stranded nucleotide overhang or a 5-nucleotide single-stranded nucleotide overhang.

[0015] In certain embodiments, the siRNA comprises naturally occurring nucleotides.

[0016] In certain embodiments, siRNA comprises at least one modified nucleotide.In certain embodiments, modified nucleotide comprises 2'-O-methyl modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide comprising unnatural base, or mixture thereof.

[0017] In certain embodiments, the siRNA comprises at least one modified internucleotide linkage. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the siRNA comprises 4 to 16 phosphorothioate internucleotide linkages. In certain embodiments, the siRNA comprises 8 to 13 phosphorothioate internucleotide linkages.

[0018] In certain embodiments, the siRNA comprises at least 80% chemically modified nucleotides, hi certain embodiments, the siRNA is fully chemically modified.

[0019] In certain embodiments, the siRNA comprises at least 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises at least 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises 70%-90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 65% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0020] In certain embodiments, the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand.

[0021] In certain embodiments, the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate. In certain embodiments, the antisense strand comprises a 5' vinyl phosphonate.

[0022] In certain embodiments, the functional moiety is linked to the 5' and / or 3' end of the oligonucleotide.

[0023] In certain embodiments, the functional moiety is linked to the 5' and / or 3' end of the sense strand or the 5' and / or 3' end of the antisense strand.

[0024] In certain embodiments, the functional moiety is linked to the 3' end of the sense strand.

[0025] In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

[0026] In certain embodiments, the linker comprises a bivalent or trivalent linker.

[0027] In certain embodiments, the bivalent or trivalent linker is [ka] wherein n is 1, 2, 3, 4, or 5.

[0028] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0029] In certain embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative, in certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] where X is O, S or BH3.

[0030] In certain embodiments, the nucleotides 1 and 2 from the 3' end of the sense strand and the nucleotides 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate linkages.

[0031] In certain embodiments, two or more oligonucleotides in a branched oligonucleotide are linked to each other by one or more moieties independently selected from a linker, a spacer, and a branch point. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof. In certain embodiments, the branch point comprises a polyvalent organic species or a derivative thereof. In certain embodiments, the spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

[0032] In certain embodiments, the linker has the structure L1: [ka] Includes.

[0033] In certain embodiments, the linker has the structure L2: [ka] Includes.

[0034] In certain embodiments, the branched oligonucleotide consists of two oligonucleotides. In certain embodiments, the branched oligonucleotide consists of three oligonucleotides. In certain embodiments, the branched oligonucleotide consists of four oligonucleotides.

[0035] In certain embodiments, the oligonucleotides within the branched oligonucleotide are siRNAs.

[0036] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is administered by intravitreal injection.

[0037] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is delivered to ocular cells after administration to a subject.

[0038] In certain embodiments, the ocular cell is selected from the group consisting of a Müller glial cell, a rod photoreceptor cell, a cone photoreceptor cell, a ganglion cell, an amacrine cell, a bipolar cell, and a horizontal cell.

[0039] In certain embodiments, the ocular cell is selected from the group consisting of a glutamine synthetase (GS)-expressing ocular cell, a rhodopsin-expressing ocular cell, a cone arrestin (CA)-expressing ocular cell, a Vglut2-expressing ocular cell, a VGAT-expressing ocular cell, a protein kinase C alpha (PKCa)-expressing ocular cell, and a Lim1-expressing ocular cell.

[0040] In certain embodiments, the ocular cells are Müller glial cells, and i) the oligonucleotide conjugate comprises DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments, the DHA, α-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified DHA (PC-DHA), α-tocopheryl succinate (PC-TS), and LA (PC-LA).

[0041] In certain embodiments, the ocular cell is a rod photoreceptor cell, and i) the oligonucleotide conjugate comprises DCA, or ii) the branched oligonucleotide consists of three or four oligonucleotides.

[0042] In certain embodiments, the ocular cell is a cone photoreceptor cell, and i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments, the retinoic acid and α-tocopheryl succinate are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA) and α-tocopheryl succinate (PC-TS). In certain embodiments, the oligonucleotide conjugate comprises two DHA functional moieties. In certain embodiments, the ocular cell is a ganglion cell, and the oligonucleotide conjugate comprises α-tocopheryl succinate. In certain embodiments, the α-tocopheryl succinate is phosphatidylcholine (PC)-esterified α-tocopheryl succinate (PC-TS). In certain embodiments, the lithocholic acid (LA) is phosphatidylcholine (PC)-esterified lithocholic acid (PC-LA). In certain embodiments, the natural lithocholic acid (LA) is phosphatidylcholine (PC)-esterified natural lithocholic acid (PC-natural LA). In certain embodiments, the isomeric lithocholic acid (LA) is phosphatidylcholine (PC)-esterified isomeric lithocholic acid (PC-isomer LA).

[0043] In certain embodiments, the ocular cell is an amacrine cell, and i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides.

[0044] In certain embodiments, the retinoic acid, DHA, α-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), α-tocopheryl succinate (PC-TS), and LA (PC-LA). In certain embodiments, the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

[0045] In certain embodiments, the ocular cell is a bipolar cell, and i) the oligonucleotide conjugate comprises a triple amine, retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments, the retinoic acid, DHA, α-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), α-tocopheryl succinate (PC-TS), and LA (PC-LA). In certain embodiments, the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

[0046] In certain embodiments, the ocular cell is a horizontal cell, and i) the oligonucleotide conjugate comprises DCA, or ii) the branched oligonucleotide consists of two oligonucleotides.

[0047] In certain embodiments, the oligonucleotide conjugate has the structure: [ka] [ka] [ka] Includes.

[0048] In certain embodiments, the branched oligonucleotide has the structure: [ka] Includes.

[0049] In certain embodiments, expression of the target nucleic acid is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0050] In certain embodiments, the oligonucleotide conjugate has selective affinity for a retinal protein.

[0051] In certain embodiments, the subject has an ocular disorder. In certain embodiments, administration of the oligonucleotide conjugate or branched oligonucleotide treats the ocular disorder in the subject.

[0052] In certain embodiments, the ocular disorder is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, central cataract, normal tension glaucoma, macular edema, and glaucoma.

[0053] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; and ii) a di-docosahexaenoic acid (di-DHA) functional moiety linked to the oligonucleotide.

[0054] In certain embodiments, the di-DHA functional moiety is phosphatidylcholine (PC) esterified di-DHA (PC-di-DHA).

[0055] In certain embodiments, the oligonucleotide conjugate has the structure: [ka] Includes.

[0056] In certain embodiments, the oligonucleotide corresponds to an antisense oligonucleotide or siRNA. In certain embodiments, the siRNA comprises a sense strand and an antisense strand. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand.

[0057] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; and ii) a di-docosahexaenoic acid (di-DHA) functional moiety linked to the oligonucleotide.

[0058] In certain embodiments, the triple amine moiety is a phosphatidylcholine (PC) esterified triple amine (PC-triple amine).

[0059] In certain embodiments, the oligonucleotide conjugate has the structure: [ka] Includes.

[0060] In certain embodiments, the oligonucleotide corresponds to an antisense oligonucleotide or an siRNA. In certain embodiments, the siRNA comprises a sense strand and an antisense strand. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand.

[0061] In certain embodiments, the functional moiety is linked to the 3' end of the sense strand. [Brief explanation of the drawings]

[0062] [Figure 1] Retinal cross sections containing 12 different siRNA distributions are shown 3 days after injection of 0.3 nmoles of siRNA (left panel: retinoic acid (RA), docosahexaenoic acid (DHA), phosphocholine (PC), α-tocopheryl succinate (TS), docosanoic acid (DCA)). To the right: one example per group, whole retinal cross sections with distribution throughout the retina. All siRNAs are Cy3 labeled and shown in red. Glutamine synthetase (GS) expression is shown in green. Nuclear DAPI is shown in blue. To the right: one example per group, whole retinal cross sections with distribution throughout the retina.

[0063] [Figure 2] Figure 1 shows the enrichment of siRNAs in various retinal cell types, arranged by cell type. Bars indicate relative protein levels of cell type-specific markers calibrated to whole retinal extracts of uninjected mouse retinas.

[0064] [Figure 3] Figure 1 shows the enrichment of siRNAs in various retinal cell types, arranged by modification. Bars indicate relative protein levels of cell-type-specific markers calibrated to whole retinal extracts of uninjected mouse retinas. For each modification (i.e., monomer, dimer, trimer, etc.), each bar represents, from left to right, rhodopsin (rods), cone arrestin (CA) (cones), glutamine synthetase (GS) (Müller cells), Vglut2 (ganglion cells), VGAT (amacrine cells), protein kinase C alpha (PKCa) (bipolar cells), and Lim1 (horizontal cells).

[0065] [Figure 4] An example of siRNA distribution in a retinal cross section 3 days after injection of 0.3 nmoles of siRNA is shown. The siRNAs were labeled with Cy3 and shown in red. All siRNAs targeted the Huntington gene. A non-targeting control (NTC) is shown, along with PC-TS modifications, trimers, and tetramers. Cone segments are labeled with PNA (peanut agglutinin lectin) and highlighted in green; Müller glia are labeled with glutamine synthetase (GS) and shown in cyan; and nuclei are labeled with DAPI and shown in blue.

[0066] [Figure 5] An example of siRNA distribution in a retinal cross section 3 days after injection of 0.3 nmol of siRNA is shown without blue-green GS staining. Additionally, the outer nuclear layer (ONL) is shown at high magnification to highlight the distribution of siRNA in the photoreceptor layer for trimers and tetramers only. Half of the panels show only siRNA for better visualization of the signal.

[0067] [Figure 6] Figure 1 shows antibody staining of retinal cross-sections for HTT protein two weeks after injection of Htt-siRNA. The first column shows staining in control mice injected with NTC-siRNA. The second column shows HTT protein expression after knockdown with PC-RA-Htt siRNA. Shown are examples from two different mice, each injected with approximately 0.3 nmoles of Htt-siRNA.

[0068] [Figure 7] Quantification of total HTT protein by Western blotting 2 weeks after Htt-siRNA injection, same experimental setup as in Figure 6 (same injection batch, different mice), quantifying total HTT protein remaining from whole retinal extracts.

[0069] [Figure 8] Quantification by bDNA assay to quantify total Htt mRNA levels 2 weeks after injection of Htt-siRNA using 0.1 nmoles per injection of the indicated siRNA modifications is shown.

[0070] [Figure 9] Quantification by bDNA assay to quantify total Htt mRNA levels 3 days after injection of Htt-siRNA using the indicated siRNA modification at 0.3 nmoles per injection is shown. Each dot represents one retina.

[0071] [Figure 10] Quantification by bDNA assay to quantify total Htt mRNA levels 100 days after injection of Htt-siRNA using 0.3 nmoles per injection of the indicated siRNA modification is shown. Each dot represents one retina.

[0072] [Figure 11]Representative fundus images over time are shown for eyes injected with Cy3-labeled siRNA containing the indicated modifications. The exposure of the fluorescent signal is the same for all four siRNAs at any given time point, but not over time. This figure complements Figure 10 and shows fundus images of the mice used in Figure 10. All mice were intravitreally injected with 0.3 nmoles of siRNA.

[0073] [Figure 12] A dose-escalation study of HTT knockdown in the retina is shown. Mice were injected with the indicated amounts (1–60 micrograms [note: not nanomoles] of Cy3-labeled tetramer with Htt-siRNA) in a total volume of 2 microliters. Five mice were injected per siRNA dose. Two weeks after injection, tissues were harvested and retinal HTT protein was quantified by Western blotting. Injections of 15–30 micrograms roughly correspond to the same knockdown observed in previous experiments with approximately 0.3 nanomoles.

[0074] [Figure 13]

[0043] Figure 13 shows fundus images from the dose escalation study shown in Figure 12. Images were taken two weeks after injection, prior to euthanasia. Normal brightfield fundus images and Cy3 images are shown for each concentration.

[0075] [Figure 14] Retinal cross sections from eyes from the dose escalation study shown in Figures 12 and 13 are shown. The images show Cy3 distribution throughout the retinal section, indicating uniform uptake of the siRNA throughout the eye.

[0076] [Figure 15] Retinal cross sections from eyes from the dose-escalation study shown in Figure 14 were stained with Iba1 (green) to identify Iba1-positive cells migrating to the outer nuclear layer (ONL) where photoreceptors reside. Half of each panel (dotted line) shows only Iba1 signal to better visualize the signal, and blue indicates nuclear DAPI.

[0077] [Figure 16] Retinal cross sections from eyes from the dose-escalation study shown in Figure 14 stained for GFAP (red) to identify reactive gliosis in Müller glial cells. siRNA is not shown as the sections are from the same eyes shown in Figure 15. Blue indicates nuclear DAPI, and green indicates cone photoreceptor segments with peanut agglutinin lectin (PNA).

[0078] [Figure 17] Electroretinography (ERG) measurements of photoreceptor and retinal function under dark-adapted (0.01 cd.s / m² to 1 cd.s / m²) and light-adapted conditions (3 and 10 flashes) are shown. A- and b-waves are recorded at several injected doses.

[0079] [Figure 18] The fluorescence intensity of tetramer-Htt-Cy3 after intravitreal delivery in pig eyes is shown. The amount of siRNA delivered is indicated at the top of each panel (100-1500 micrograms of tetramer). The top panel shows Cy3 fluorescence in unfixed tissue immediately after opening the eye. The bottom panel shows a further enlargement of the area in the top panel.

[0080] [Figure 19]Figure 18 shows huntingtin protein knockdown in pigs, as measured by Western blot analysis of the eyes shown. Knockdown was compared to huntingtin protein levels in NTCs injected with 250 μg of tetramer-siRNA-Cy3. The top panel shows a bar graph of the knockdown observed in the four major quadrants of the retina (DT: dorsal-temporal, DN: dorsal-nasal, VT: temporal-nasal, VN: ventral-nasal). In the middle panel, knockdown is shown on a flat-mount cutout with the corresponding regional knockdown values ​​shown in the bar graph. Bottom panel: Average huntingtin protein knockdown across the entire retina, calculated by averaging the knockdown observed in each quadrant per retina. Data shown represent one biological sample for each amount of siRNA delivered. Error bars in the first panel were generated by technical replicates. Error bars in the last panel were generated by averaging four data points in each quadrant per retina.

[0081] [Figure 20] 19 shows antibody staining for huntingtin protein in sections of eyes injected with different amounts as shown in Figure 19. The area of ​​the sections is shown in the middle panel of Figure 19.

[0082] [Figure 21] Figures 18 and 19 show antibody staining for the expression of GFAP (glial fibrillary acidic protein) and Iba1 (ionized calcium binding adaptor protein 1) (as shown for mice in Figures 15 and 16) in retinal sections from eyes injected with different doses to determine dose-dependent toxicity. GFAP and Iba1 are both shown in green, as shown on the left side of each row. Red staining indicated siRNA distribution throughout the retinal section. Nuclei are labeled with intranuclear DAPI. Half of each panel shows only the signal of interest (siRNA, GFAP, or Iba1) for better signal visualization.

[0083] [Figure 22] 1 shows the initial in vitro knockdown efficiency of siRNA duplexes formed from the sense and antisense strands shown in Tables 3 and 4.

[0084] [Figure 23] FIG. 22 shows the dose response curves for duplexes 2, 3, 9, and 10.

[0085] [Figure 24] RNA-Scope in situ hybridization was performed on mouse retinal cross sections to detect siRNA tetramers against S6K1. The top row shows sections from three mice injected with tetrameric NTCs for S6K1. The middle row shows sections from three mice injected with tetrameric siRNA against S6K1 at 3 μg / eye. The bottom row shows sections from three mice injected with tetrameric siRNA against S6K1 at 6 μg / eye. siRNA was delivered intravitreally, and animals were euthanized two weeks after injection.

[0086] [Figure 25] Figure 1 shows knockdown of S6K1 in mice after intravitreal injection of 6 μg of tetrameric siRNA. A shows S6K1 protein levels detected by Western blot two weeks after injection. B shows the same data as the first graph two months after injection. Each dot in the graph represents one biological sample (retina) from one animal.

[0087] [Figure 26]This figure shows knockdown of S6K1 protein in non-human primates (NHPs). Western blot data are obtained using retinal protein extracts from the superior temporal (ST) region (also known as the dorsal-temporal region) of one NHP intravitreally injected with 225 μg of S6K1-tetramer (75 μL) and retinas from six untreated NHPs from the same region. The first set of bar graphs shows a comparison between the uninjected contralateral eye and the S6K1 siRNA-injected eye, allowing for direct intraanimal comparisons between eyes. The second bar graph shows a comparison between six untreated NHPs and the S6K1 siRNA-injected NHPs. NHP eyes were harvested one month after injection. Phosphorylation of the ribosomal protein S6, a canonical target of S6K1, is also shown. As with the S6K1 knockdown data, intraanimal comparisons are shown on the left, and comparisons across multiple NHPs are shown on the right.

[0088] [Figure 27] Figure 1 shows knockdown of S6K1 protein in non-human primate (NHP) retinal cross sections after siRNA treatment. Data were generated using one injected eye and an uninjected contralateral eye. Sections were taken from the central region as shown for the pig in Figure 19. Left: entire cross section encompassing the fovea. Right: higher magnification images of the temporal and nasal regions and the fovea. The top row shows an uninjected eye, and the bottom row shows an eye intravitreally injected with 225 μg of S6K1-tetramer (75 μL).

[0089] [Figure 28] Figure 2 shows the reduction of phosphorylated S6 protein (pS6) on non-human primate (NHP) retinal cross sections after siRNA treatment. The data are the same as those shown in Figure 27, except that the staining probes for pS6 expression (red signal). In each panel, the green and blue signals were removed in half of the panel (dotted lines) to better visualize pS6 knockdown. Blue indicates nuclear DAPI, and green indicates cone segments labeled with peanut agglutinin lectin (PNA).

[0090] [Figure 29]Figure 2 shows the expression of inflammatory markers in NHPs after siRNA treatment with S6K1 siRNA (75 microliters, 225 μg siRNA in tetramer configuration). The data are the same as those shown in Figures 27 and 28, except that staining probes for Iba1 (red signal, first set) and GFAP (red signal, second set) expression. The untreated contralateral eye is shown in the first row of each set, and the treated eye is shown in the second row. In each panel, the green and blue signals were removed (dotted lines) in half of the panel to better visualize the Iba1 and GFAP signals. Blue indicates nuclear DAPI, and green indicates cone segments labeled with peanut agglutinin lectin (PNA). DETAILED DESCRIPTION OF THE INVENTION

[0091] The present disclosure relates to oligonucleotide conjugates and branched oligonucleotides capable of efficient gene knockdown in the eye. Several different functional moieties and branched oligonucleotides have demonstrated ocular cell-specific delivery upon administration.

[0092] The oligonucleotide conjugates and branched oligonucleotides described herein facilitate simple, efficient, and non-toxic delivery of oligonucleotides (e.g., siRNA) and potent silencing of therapeutic targets in various ocular cell types in vivo.

[0093] Unless otherwise specified, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein, and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The terminology and laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0094] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise indicated. The use of the term "including," as well as other forms such as "include" and "included," is non-limiting.

[0095] In order that this disclosure may be more readily understood, certain terms are first defined.

[0096] As used herein with respect to oligonucleotide sequences, "A" represents a nucleoside that includes the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside that includes the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside that includes the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside that includes the base adenine (e.g., cytidine or a chemically modified derivative thereof).

[0097] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also referred to as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety in an ester linkage. Exemplary nucleotides include nucleoside monophosphate, diphosphate, and triphosphate. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked together by phosphodiester or phosphorothioate linkages between the 5' and 3' carbon atoms.

[0098] The terms "RNA" or "RNA molecule" or "ribonucleic acid molecule" refer to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refer to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified post-transcriptionally. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

[0099] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNA") refers to an RNA (or RNA analog) containing about 10-50 nucleotides (or nucleotide analogs) and capable of directing or mediating RNA interference. An siRNA is a duplex formed by a sense strand and an antisense strand, which are sufficiently complementary to each other to form the duplex. In certain embodiments, an siRNA contains about 15-30 nucleotides or nucleotide analogs, or about 16-25 nucleotides (or nucleotide analogs), or about 18-23 nucleotides (or nucleotide analogs), or about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA containing about 21 nucleotides (or nucleotide analogs), e.g., 19, 20, 21, or 22 nucleotides. The term "long" siRNA refers to an siRNA containing about 24 to 25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. A short siRNA may contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, a long siRNA may contain more than 26 nucleotides, provided that the longer siRNA retains its ability to mediate RNAi without further processing, e.g., enzymatic processing, into short siRNAs.

[0100] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" or "chemically modified nucleotide" refers to a non-standard nucleotide, such as a non-natural ribonucleotide or deoxyribonucleotide. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide but retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, the 6-position, e.g., 6-(2-amino)propyluridine, and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine, O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or others known in the art) nucleotides, and other heterocyclic-modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0101] Nucleotide analogs may also contain modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, or the like. Other modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438. In certain embodiments, the nucleotide analog contains a 2'-O-methyl modification. In certain embodiments, the nucleotide analog contains a 2'-fluoro modification.

[0102] The phosphate group of a nucleotide can also be modified, for example, by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioate) or by other substitutions that allow the nucleotide to perform its intended function. For example, see Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Patent No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) reduce the hydrolysis rate of polynucleotides containing the analogs, for example, in vivo or in vitro.

[0103] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs. The term "oligonucleotide" includes, but is not limited to, antisense oligonucleotides (ASOs), siRNAs, and microRNAs.

[0104] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to the corresponding unmodified or unmodified RNA, but retains the same or similar properties or functions as the corresponding unmodified or unmodified RNA. As described above, oligonucleotides can be linked by linkages that result in a reduced hydrolysis rate of the RNA analog compared to RNA molecules with phosphodiester linkages. For example, the nucleotides of the analog can contain methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and / or phosphorothioate linkages. Some RNA analogs include sugar- and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include the addition of non-nucleotide material, such as to the end(s) or internal (one or more nucleotides of the RNA) of the RNA. The RNA analog need only be sufficiently similar to natural RNA to have the ability to mediate RNA interference.

[0105] The term " RNA interference " (" RNAi ") used herein refers to the selective intracellular degradation of RNA.RNAi occurs naturally in cells and removes foreign RNA (for example, viral RNA).Natural RNAi proceeds through the fragments cut from free dsRNA, and directs the degradation mechanism to other similar RNA sequences.Alternatively, RNAi can be initiated by human hands, for example, to silence the expression of target genes.

[0106] An RNAi agent, e.g., an RNA silencing agent, having a strand that is "sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has sufficient sequence to cause destruction of the target mRNA by the RNAi machinery or process.

[0107] As used herein, "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material or culture medium when produced by recombinant techniques, and that is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0108] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression), which result in the inhibition or "silencing" of expression of corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0109] The term "in vitro" has its art-recognized meaning, including, for example, purified reagents or extracts, e.g., cellular extracts. The term "in vivo" also has its art-recognized meaning, including, for example, living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0110] As used herein, "target" refers to a specific nucleic acid sequence (e.g., a gene, mRNA, miRNA, etc.) to which an oligonucleotide conjugate or branched oligonucleotide of the present disclosure binds and / or otherwise affects its expression. In certain embodiments, the target is expressed in the eye. In certain embodiments, the target is expressed in specific ocular cells. In other embodiments, the target is associated with a specific disease or disorder in a subject.

[0111] As used herein, the term "target gene" refers to a gene whose expression is substantially inhibited or "silenced." This silencing can be achieved, for example, by cleaving the target gene's mRNA or silencing RNA through translational repression of the target gene. The term "non-target gene" refers to a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNAs encoded by the target gene and the non-target gene) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene can be a homolog (e.g., an ortholog or paralog) of the target gene.

[0112] The term "RNA silencing agent" as used herein refers to an RNA that can inhibit or "silence" the expression of a target gene. In certain embodiments, an RNA silencing agent can prevent complete processing (e.g., complete translation and / or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include small (<50 b.p.), non-coding RNA molecules, such as RNA duplexes containing paired strands, as well as precursor RNAs that can generate such small non-coding RNAs. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In one embodiment, an RNA silencing agent can induce RNA interference. In another embodiment, an RNA silencing agent can mediate translational repression.

[0113] As used herein, the term "rare nucleotide" refers to a naturally occurring nucleotide that occurs infrequently, e.g., a naturally occurring deoxyribonucleotide or ribonucleotide that occurs infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0114] The term "engineered" indicates that the precursor or molecule is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans, such as an engineered RNA precursor or engineered nucleic acid molecule. Once created or selected, the sequence is replicated, translated, transcribed, or otherwise processed by machinery within the cell. Thus, an RNA precursor produced within a cell from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0115] As used herein, the term "microRNA" ("miRNA"), also known in the art as "small temporal RNA" ("stRNA"), refers to small (e.g., 10-50 nucleotide) RNAs that are genetically encoded (e.g., by viral, mammalian, or plant genomes) and can direct or mediate RNA silencing. "miRNA disorder" refers to a disease or disorder characterized by aberrant expression or activity of miRNA.

[0116] As used herein, the term "bifunctional oligonucleotide" refers to an RNA silencing agent having the formula TL-μ, where T is an mRNA targeting moiety, L is a linking moiety, and μ is an miRNA recruitment moiety. As used herein, the terms "mRNA targeting moiety," "targeting moiety," "mRNA targeting moiety," or "targeting moiety" refer to a domain, portion, or region of a bifunctional oligonucleotide that has sufficient size and complementarity to a portion or region of an mRNA selected or targeted for silencing (i.e., the portion has sufficient sequence to capture the target mRNA).

[0117] As used herein, the term "linking moiety" or "linking portion" refers to a domain, portion, or region of an RNA silencing agent that covalently joins or links an mRNA.

[0118] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA, refers to the strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides, of the mRNA of a gene targeted for silencing. The antisense strand, or first strand, has a sequence that is sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., sufficiently complementary to cause destruction of the desired target mRNA by the RNAi mechanism or process (RNAi interference) or sufficiently complementary to cause translational repression of the desired target mRNA.

[0119] The term "sense strand" or "second strand" of an RNA silencing agent, such as an siRNA or RNA silencing agent, refers to the strand complementary to the antisense strand or first strand. The antisense strand and the sense strand can also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and the second strand or first strand, respectively, is complementary to the first strand or second strand. The miRNA duplex intermediate or siRNA-like duplex includes an miRNA strand that is sufficiently complementary to a section of approximately 10-50 nucleotides of the mRNA of the gene targeted for silencing, and an miRNA* strand that is sufficiently complementary to form a duplex with the miRNA strand.

[0120] The term "guide strand" as used herein refers to the strand of an RNA silencing agent, e.g., the antisense strand of an siRNA duplex or siRNA sequence, that enters the RISC complex and directs cleavage of the target mRNA.

[0121] The term " asymmetry " used herein refers to the unequal binding strength or base pair strength between the ends of an RNA silencing agent (for example, between the terminal nucleotide on the first strand or stem portion and the terminal nucleotide on the opposite second strand or stem portion), such as in the asymmetry of the double-stranded region of an RNA silencing agent (for example, the stem of shRNA).This causes the 5'-end of one strand of the double strand to be in a transient unpaired state, for example, a single-stranded state, more frequently than the 5'-end of the complementary strand.This structural difference determines that one strand of the double strand will be preferentially incorporated into RISC complex.The strand whose 5'-end is less tightly paired with the complementary strand will be preferentially incorporated into RISC and mediate RNAi.

[0122] As used herein, the term "binding strength" or "base pair strength" refers primarily to the strength of interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex) and between these nucleotides (or nucleotide analogs) through H-bonding, van der Waals interactions, etc.

[0123] As used herein, "5' end" refers to the 5'-terminal nucleotide, e.g., between 1 and about 5 nucleotides from the 5' end of the antisense strand, as in the 5' end of the antisense strand. As used herein, "3' end" refers to the region complementary to the 5'-terminal nucleotide of the complementary antisense strand, as in the 3' end of the sense strand, as in the 1' end of the sense strand, as in the 5' end of the sense strand.

[0124] As used herein, the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analog that can base pair with a second nucleotide or nucleotide analog such that the base pair has a lower binding strength than conventional base pairs (i.e., Watson-Crick base pairs). In certain embodiments, the destabilizing nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide can form a wobble base pair with the second nucleotide. In still other embodiments, the destabilizing nucleotide can form an ambiguous base pair with the second nucleotide.

[0125] As used herein, the term "base pair" refers to an interaction between a pair of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), primarily through H-bonding, van der Waals interactions, etc. between the nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of the base pair.

[0126] As used herein, the term "mismatch base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair, e.g., not a normal complementary G:C, A:T, A:U base pair. As used herein, the term "ambiguous base pair" (also known as a non-discriminating base pair) refers to a base pair formed by universal nucleotides.

[0127] As used herein, the term "universal nucleotide" (also known as "neutral nucleotide") includes nucleotides (e.g., certain destabilizing nucleotides) that have bases ("universal bases" or "neutral bases") that do not significantly discriminate between bases on a complementary polynucleotide when base-pairing. Universal nucleotides are primarily hydrophobic molecules that can efficiently pack into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0128] As used herein, the term "sufficient complementarity" or "sufficient degree of complementarity" means that the RNA silencing agent has sufficient sequence (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruitment portion) to bind to the desired target and induce RNA silencing of the target mRNA, respectively.

[0129] The term " translational repression " used herein refers to the selective inhibition of mRNA translation.Natural translational repression proceeds through the miRNA cleaved from shRNA precursor.Both RNAi and translational repression are mediated by RISC.Both RNAi and translational repression can occur naturally or be initiated by human hands, for example, to silence the expression of target gene.

[0130] Various methodologies of the present disclosure include a step that involves comparing a value, level, characteristic, property, or the like to a "suitable control," referred to interchangeably herein as a "suitable control." A "suitable control" or "suitable control" is any control or standard useful for comparison purposes and familiar to those of skill in the art. In one embodiment, a "suitable control" or "suitable control" is a value, level, characteristic, property, or the like that is determined prior to performing an RNAi methodology, as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, or the like, can be determined prior to introducing an RNA silencing agent of the present disclosure into a cell or organism. In another embodiment, a "suitable control" or "suitable control" is a value, level, characteristic, property, or the like that is determined in a cell or organism, e.g., a control or, e.g., a normal cell or organism exhibiting a normal trait. In yet another embodiment, a "suitable control" or "suitable control" is a predefined value, level, characteristic, property, or the like.

[0131] Oligonucleotide Conjugates The oligonucleotide conjugates described herein comprise an oligonucleotide linked to a functional moiety that enhances ocular delivery of the oligonucleotide, including ocular cell-specific delivery.

[0132] In one aspect, the present disclosure provides a method for delivering an oligonucleotide conjugate to the eye of a subject, the method comprising administering to the subject an oligonucleotide conjugate comprising: i) an oligonucleotide comprising a 5' end and a 3' end and having complementarity to a target nucleic acid (e.g., a target gene or a target mRNA), and ii) a functional moiety linked to the oligonucleotide, the functional moiety comprising any one of a triple amine, retinoic acid (RA), docosahexaenoic acid (DHA), docosanoic acid (DCA), alpha-tocopheryl succinate (TS), or lithocholic acid (LA).

[0133] Each of the above functional moieties is structurally represented below: Functional moieties may have different isomeric configurations than those presented in this disclosure.

[0134] [ka]

[0135] [ka]

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] In certain embodiments, two DHA functional moieties are linked to the oligonucleotide.

[0142] In certain embodiments, the oligonucleotide comprises an antisense oligonucleotide or an siRNA.

[0143] In certain embodiments, the siRNA comprises a sense strand and an antisense strand. In certain embodiments, the antisense strand is about 15 to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In certain embodiments, the antisense strand is 20, 21, or 22 nucleotides in length. In certain embodiments, the sense strand is about 15 to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In certain embodiments, the sense strand is 15, 16, 18, or 20 nucleotides in length.

[0144] In certain embodiments, the siRNA comprises a double-stranded region of 15 to 20 base pairs (e.g., 15, 16, 17, 18, 19, or 20 base pairs). In certain embodiments, the siRNA comprises a double-stranded region of 15, 16, 18, or 20 base pairs.

[0145] In certain embodiments, the siRNA comprises at least one blunt end. In certain embodiments, the siRNA comprises two blunt ends.

[0146] In certain embodiments, the siRNA comprises at least one single-stranded nucleotide overhang (also referred to herein as a "single-stranded tail"). In certain embodiments, the siRNA comprises two single-stranded nucleotide overhangs. In certain embodiments, the siRNA comprises about 2-5 nucleotide single-stranded nucleotide overhangs (e.g., 2, 3, 4, or 5 nucleotide overhangs). In certain embodiments, the siRNA comprises 2-nucleotide single-stranded nucleotide overhangs or 5-nucleotide single-stranded nucleotide overhangs.

[0147] In certain embodiments, the siRNA comprises natural nucleotides (ie, unmodified ribonucleotides).

[0148] In certain embodiments, siRNA comprises at least one modified nucleotide.In certain embodiments, modified nucleotide comprises 2'-O-methyl modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide comprising unnatural base, or mixture thereof.

[0149] In certain embodiments, the siRNA comprises at least one modified internucleotide linkage. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the siRNA comprises 4 to 16 phosphorothioate internucleotide linkages. In certain embodiments, the siRNA comprises 8 to 13 phosphorothioate internucleotide linkages.

[0150] In certain embodiments, the siRNA comprises at least 80% chemically modified nucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides). In certain embodiments, the siRNA is completely chemically modified.

[0151] In certain embodiments, the siRNA comprises at least 70% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl nucleotide modifications). In certain embodiments, the antisense strand comprises at least 70% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl nucleotide modifications). In certain embodiments, the antisense strand comprises 70% to 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least 65% 2'-O-methyl nucleotide modifications (e.g., 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl nucleotide modifications). In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0152] In certain embodiments, the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand.

[0153] In certain embodiments, the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate. In certain embodiments, the antisense strand comprises a 5' vinyl phosphonate.

[0154] In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the oligonucleotide. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand.

[0155] In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

[0156] In certain embodiments, the linker comprises a bivalent or trivalent linker.

[0157] In certain embodiments, the bivalent or trivalent linker is [ka] wherein n is 1, 2, 3, 4, or 5.

[0158] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0159] In certain embodiments, when the linker is a trivalent linker, the linker further connects phosphodiesters or phosphodiester derivatives.

[0160] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] where X is O, S or BH3.

[0161] The moiety Zc1 is phosphatidylcholine (PC). Any one of the functional moieties described herein may include phosphatidylcholine (PC) esterified derivatives, i.e., phosphatidylcholine (PC) esterified triple amine (PC-tripleamine), phosphatidylcholine (PC) esterified retinoic acid (PC-RA), phosphatidylcholine (PC) esterified docosahexaenoic acid (PC-DHA), phosphatidylcholine (PC) esterified docosanoic acid (PC-DCA), phosphatidylcholine (PC) esterified α-tocopheryl succinate (PC-TS), and phosphatidylcholine (PC) esterified lithocholic acid (PC-TS).

[0162] In certain embodiments, the nucleotides 1 and 2 from the 3' end of the sense strand and the nucleotides 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate linkages.

[0163] In certain embodiments, the oligonucleotide conjugate has the structure:

[0164] [ka]

[0165] [ka]

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169]

change

[0170]

change

[0171]

change

[0172]

change

[0173]

change

[0174]

change

[0175]

change

[0176]

change

[0177]

change

[0178]

change

[0179]

change

[0180] For any of the above listed structures, the term "oligonucleotide" corresponds to any of the oligonucleotides listed herein, such as ASO or siRNA.In certain embodiments, the term "oligonucleotide" in the above structure corresponds to the sense strand of siRNA.In certain embodiments, the oxygen atom immediately adjacent to the term "oligonucleotide" in the structure is linked to the 3' end of the sense strand of siRNA.

[0181] Di-DHA oligonucleotide conjugate

[0182] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; and ii) a di-docosahexaenoic acid (di-DHA) functional moiety linked to the oligonucleotide.

[0183] In certain embodiments, the di-DHA functional moiety is phosphatidylcholine (PC) esterified di-DHA (PC-di-DHA).

[0184] In certain embodiments, the oligonucleotide conjugate has the structure: [ka] Includes.

[0185] In certain embodiments, the oligonucleotide corresponds to an antisense oligonucleotide or an siRNA.

[0186] In certain embodiments, the siRNA comprises a sense strand and an antisense strand.

[0187] In certain embodiments, the functional moiety (i.e., di-DHA or PC-di-DHA) is linked to the 5' and / or 3' end of the sense strand or the 5' and / or 3' end of the antisense strand. In certain embodiments, the functional moiety is linked to the 3' end of the sense strand.

[0188] Triple Amine Oligonucleotide Conjugates

[0189] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; and ii) a triple amine functionality linked to the oligonucleotide.

[0190] In certain embodiments, the triple amine moiety is a phosphatidylcholine (PC) esterified triple amine (PC-triple amine).

[0191] In certain embodiments, the oligonucleotide conjugate has the structure: [ka] Includes.

[0192] In certain embodiments, the oligonucleotide corresponds to an antisense oligonucleotide or an siRNA.

[0193] In certain embodiments, the siRNA comprises a sense strand and an antisense strand.

[0194] In certain embodiments, the functional moiety (i.e., triple amine or PC-triple amine) is linked to the 5' and / or 3' end of the sense strand or the 5' and / or 3' end of the antisense strand. In certain embodiments, the functional moiety is linked to the 3' end of the sense strand.

[0195] Branched Oligonucleotides The branched oligonucleotides described herein comprise two or more oligonucleotides linked together. Different branched oligonucleotides described herein (e.g., branched oligonucleotides comprising two, three, or four oligonucleotides) have enhanced ocular delivery of oligonucleotides, including ocular cell-specific delivery.

[0196] In one aspect, the disclosure provides a method for delivering a branched oligonucleotide to the eye of a subject, the method comprising administering a branched oligonucleotide to the subject, the branched oligonucleotide comprising two or more oligonucleotides, each oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid.

[0197] In certain embodiments, one or more of the oligonucleotides of the branched oligonucleotide further comprises a functional moiety linked to the oligonucleotide, and this functional moiety comprises any one of triple amine, retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA. The functional moieties described above in the section on oligonucleotide conjugates can also be applied to the oligonucleotides of the branched oligonucleotides. Similarly, the oligonucleotides described above in the section on oligonucleotide conjugates can function as the oligonucleotides of the branched oligonucleotides, including the type (ASO or siRNA), chain length, and chemical modification.

[0198] In certain embodiments, the two or more oligonucleotides in the branched oligonucleotide are linked to one another by one or more moieties independently selected from a linker, a spacer, and a branch point.

[0199] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

[0200] In certain embodiments, the branch points comprise a polyvalent organic species or derivatives thereof.

[0201] In another embodiment, the branch point is an amino acid derivative. Another embodiment of the branch point has the following formula: [ka] is selected from.

[0202] Polyvalent organic species are moieties that contain carbon and three or more valencies (i.e., points of attachment to moieties such as S, L, or N, as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol, etc.), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, etc.), tetracarboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, etc.), tertiary amines (e.g., tripropargylamine, triethanolamine, etc.), triamines (e.g., diethylenetriamine, etc.), tetramines, and species that contain combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, etc.).

[0203] In certain embodiments, the spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

[0204] In certain embodiments, the linker has the structure L1:

[0205] [ka] Includes.

[0206] In certain embodiments, the linker has the structure L2:

[0207] [ka] Includes.

[0208] In certain embodiments, the branched oligonucleotide consists of two oligonucleotides. In certain embodiments, the branched oligonucleotide consists of three oligonucleotides. In certain embodiments, the branched oligonucleotide consists of four oligonucleotides. In certain embodiments, the oligonucleotide is an siRNA.

[0209] In certain embodiments, the branched oligonucleotide has the structure: [ka] Includes.

[0210] For any of the above listed structures, the term "oligonucleotide" corresponds to any of the oligonucleotides listed herein, such as ASO or siRNA.In certain embodiments, the term "oligonucleotide" in the above structure corresponds to the sense strand of siRNA.In certain embodiments, the oxygen atom immediately adjacent to the term "oligonucleotide" in the structure is linked to the 3' end of the sense strand of siRNA.

[0211] Branched oligonucleotides, including their synthesis and methods of use, are described in further detail in WO2017 / 132669, which is incorporated herein by reference. Further details regarding synthesis are provided in the Materials and Methods section of the Examples. Methods of delivery to ocular cells

[0212] The oligonucleotide conjugates and branched oligonucleotides described herein can be delivered specifically to ocular cells and effectively silence target genes. Any given oligonucleotide conjugate and branched oligonucleotide can be effective for delivery to multiple types of ocular cells.

[0213] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is administered by intravitreal injection.

[0214] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is delivered to ocular cells after administration to a subject.

[0215] In certain embodiments, the ocular cell is selected from the group consisting of a Müller glial cell, a rod photoreceptor cell, a cone photoreceptor cell, a ganglion cell, an amacrine cell, a bipolar cell, and a horizontal cell.

[0216] In certain embodiments, the ocular cell is selected from the group consisting of a glutamine synthetase (GS)-expressing ocular cell, a rhodopsin-expressing ocular cell, a cone arrestin (CA)-expressing ocular cell, a Vglut2-expressing ocular cell, a VGAT-expressing ocular cell, a protein kinase C alpha (PKCa)-expressing ocular cell, and a Lim1-expressing ocular cell.

[0217] In certain embodiments, the oligonucleotide conjugate has selective affinity for a retinal protein.

[0218] In certain embodiments, the ocular cells are Müller glial cells, and i) the oligonucleotide conjugate comprises DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments for Müller glial cell delivery, the DHA, α-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified DHA (PC-DHA), α-tocopheryl succinate (PC-TS), and LA (PC-LA).

[0219] In certain embodiments, the ocular cell is a rod photoreceptor cell, and i) the oligonucleotide conjugate comprises DCA, or ii) the branched oligonucleotide consists of three or four oligonucleotides.

[0220] In certain embodiments, the ocular cells are cone photoreceptor cells, and i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments for cone photoreceptor cell delivery, the retinoic acid and α-tocopheryl succinate are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA) and α-tocopheryl succinate (PC-TS). In certain embodiments for cone photoreceptor cell delivery, the oligonucleotide conjugate comprises two DHA functional moieties.

[0221] In certain embodiments, the ocular cell is a ganglion cell and the oligonucleotide conjugate comprises alpha-tocopheryl succinate. In certain embodiments for ganglion cell delivery, the alpha-tocopheryl succinate is phosphatidylcholine (PC)-esterified alpha-tocopheryl succinate (PC-TS).

[0222] In certain embodiments, the ocular cells are amacrine cells, and i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments for amacrine cell delivery, the retinoic acid, DHA, α-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), α-tocopheryl succinate (PC-TS), and LA (PC-LA). In certain embodiments for amacrine cell delivery, the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

[0223] In certain embodiments, the ocular cell is a bipolar cell, and i) the oligonucleotide conjugate comprises a triple amine, retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA, or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments for bipolar cell delivery, the retinoic acid, DHA, α-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), α-tocopheryl succinate (PC-TS), and LA (PC-LA). In certain embodiments for bipolar cell delivery, the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

[0224] In certain embodiments, the ocular cell is a horizontal cell, and i) the oligonucleotide conjugate comprises DCA, or ii) the branched oligonucleotide consists of two oligonucleotides.

[0225] Gene silencing / therapeutic methods The oligonucleotide conjugates and branched oligonucleotides described herein are capable of silencing target genes (ie, target nucleic acids) in the eye and in specific ocular cells.

[0226] In certain embodiments, expression of the target nucleic acid in the ocular cells is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0227] In certain embodiments, expression of the target nucleic acid in ocular cells selected from the group consisting of Müller glial cells, rod photoreceptor cells, cone photoreceptor cells, ganglion cells, amacrine cells, bipolar cells, and horizontal cells is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0228] In certain embodiments, expression of a target nucleic acid in an ocular cell selected from the group consisting of a glutamine synthetase (GS)-expressing ocular cell, a rhodopsin-expressing ocular cell, a cone arrestin (CA)-expressing ocular cell, a Vglut2-expressing ocular cell, a VGAT-expressing ocular cell, a protein kinase C alpha (PKCa)-expressing ocular cell, and a Lim1-expressing ocular cell is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0229] In one aspect, the present disclosure provides a method of treating an ocular disorder in a subject in need thereof, the method comprising administering to the subject an oligonucleotide conjugate and / or branched oligonucleotide described herein, thereby treating the ocular disorder.

[0230] In certain embodiments, administration of the oligonucleotide conjugate or branched oligonucleotide treats an ocular disorder in a subject.

[0231] In certain embodiments, administration of the oligonucleotide conjugate or branched oligonucleotide reduces gene expression from a target nucleic acid associated with an ocular disorder in a subject.

[0232] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is administered by intravitreal injection.

[0233] In certain embodiments, the ocular disorder is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, central cataract, normal tension glaucoma, macular edema, and glaucoma. [Example]

[0234] Materials and Methods Synthesis of lipid-functionalized solid supports

[0235] Non-phosphocholine (PC) lipid moieties (except α-tocopheryl succinate) were directly conjugated via peptide bonds to controlled pore glass (CPG) functionalized with a C7 linker, as previously described (Nikan M, Osborn MF, Coles AH, et al. Docosahexaenoic acid conjugation enhances distribution and safety of siRNA upon local administration in mouse brain. Mol. Ther. Nucleic Acids. 2016;5:e344). To synthesize PC derivatives, amino-C7 CPG was first functionalized with phosphocholine (Nikan M, Osborn MF, Coles AH, et al. Synthesis and evaluation of parenchymal retention and efficacy of a metabolically stable O-phosphocholine-N-docosahexaenoyl-l-serine siRNA conjugate in mouse brain. Bioconjug. Chem. 2017;28:758-1766). Briefly, Fmoc-L-serine tert-butyl ester (TCI America) was phosphitylated using 2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (ChemGenes). The resulting phosphoramidite was coupled to choline p-toluenesulfonate (Alfa Aesar) using 5-(ethylthio)-1H-tetrazole (ETT) as an activator. The phosphine ester was then oxidized, and the carboxylic acid and phosphate ester groups were deprotected (i.e., the tert-butyl and cyanoethyl groups were removed). The resulting intermediate was conjugated to amino C7 CPG via a peptide bond to form a phosphocholine-functionalized CPG. The Fmoc group was removed, and the selected lipid moiety was attached to the CPG via a peptide bond. All lipid-functionalized solid supports were obtained with a loading of 55 μmol / g.

[0236] Synthesis of α-tocopheryl succinate-conjugated oligonucleotides

[0237] Alpha-tocopheryl succinate was conjugated to the amino group at the 3' end of oligonucleotides synthesized and purified on amino C7 CPG or phosphocholine-functionalized amino C7 CPG. Alpha-tocopheryl N-hydroxysuccinimide and purified oligonucleotides were mixed in 0.1 M sodium bicarbonate, 20% (v / v) dimethylformamide solution and incubated overnight at room temperature. One-tenth volume of 3 M sodium acetate (pH 5.2) was added to obtain a final concentration of 0.3 M sodium acetate. Three volumes of 95% (v / v) ethanol were added, and the mixture was vortexed and then placed at -80°C for 1 hour. The solution was pelleted by centrifugation at 5200 x g for 30 minutes. The pellet, containing the sense strand of the lipid-conjugated siRNA, was dissolved in water, purified, and desalted as described below.

[0238] Oligonucleotide synthesis

[0239] Oligonucleotides were synthesized by phosphoramidite solid-phase synthesis on a Dr Oligo 48 (Biolytic, Fremont, CA) or MerMade 12 (Biosearch Technologies, Novato, CA) using 2'-F or 2'-O-Me modified phosphoramidites with standard protecting groups. 5'-(E)-vinyltetraphosphonate (pivaloyloxymethyl) 2'-O-methyl-uridine 3'-CE phosphoramidite (VP) for in vivo unconjugated oligonucleotides was purchased from Hongene Biotech, USA, and Quasar 570 CE phosphoramidite (Cy3) was purchased from GenePharma, Shanghai, China. Bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite (5'P) for in vitro unconjugated oligonucleotides and all other phosphoramidites used were purchased from ChemGenes, Wilmington, MA. Phosphoramidites were prepared at 0.1 M in anhydrous acetonitrile (ACN), except for 2'-O-methyluridine phosphoramidite, which was dissolved in anhydrous ACN containing 15% dimethylformamide. 5-(benzylthio)-1H-tetrazole (BTT) was used as the activator at 0.25 M, and the coupling time for all phosphoramidites was 4 min using 10 equivalents. Detritylation was performed using 3% trichloroacetic acid in dichloromethane. The capping reagents used were CAP A (20% n-methylimidazole in ACN) and CAP B (20% acetic anhydride and 30% 2,6-lutidine in ACN). Reagents for capping and detritylation were purchased from AIC, Framingham, MA. Phosphorous oxidation to convert to phosphate or phosphorothioate was carried out for 4 min using 0.05 M iodine in pyridine-HO (9:1, v / v) or a 0.1 M solution of 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine (ChemGenes).Unconjugated oligonucleotides were synthesized on 500 Å long-chain alkylamine (LCAA) controlled pore glass (CPG) functionalized with Unylinker terminus (ChemGenes). Cholesterol-conjugated oligonucleotides were synthesized on 500 Å LCAA-CPG supports, in which the cholesterol moiety was attached to tetraethyl ene glycol via a succinic acid linker (ChemGenes, Wilmington, MA). Lipid-conjugated oligonucleotides were synthesized on modified solid supports (synthesis described above). Bivalent oligonucleotides (dimers) were synthesized on modified solid supports using a previously described synthesis method (Alterman JF, Godinho BMDC, Hassler MR, et al. A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nat Biotechnol 37, 884-894 (2019)).

[0240] Synthesis of branched oligonucleotides

[0241] Branched oligonucleotide synthesis was carried out by phosphoramidite solid phase on an AKTA Oligoprilot 10 (Cytiva, Marlborough, MA) using the parameters described above or specified herein. Trimeric and tetrameric branched oligonucleotides were prepared using commercially available trebler and doubler phosphoramidites, respectively, purchased from Glen Research, Sterling, VA. Trimeric linkers were generated in two steps as follows: 10 equivalents of DMT-tetraethyloxy-glycol CED phosphoramidite (ChemGenes) were first coupled onto 1000 Å thymidine 3'-LCAA-CPG (ChemGenes) for 8 minutes, followed by 10 equivalents of trebler phosphoramidite for 8 minutes. Trivalent oligonucleotides were then grown on this linker using 30 equivalents. The tetrameric linker was generated in three steps as follows: first, onto the 1000 Å thymidine 3'-LCAA-CPG, DMT-tetraethyloxy-glycol CED phosphoramidite was coupled using 10 equivalents for 8 minutes, second, a doubler phosphoramidite was coupled using 10 equivalents for 8 minutes, and third, a doubler phosphoramidite was subsequently coupled using 20 equivalents for 8 minutes, after which the tetravalent oligonucleotide was grown using 40 equivalents.

[0242] Deprotection and purification of oligonucleotides for sequence screening

[0243] Prior to deprotection, the synthesis column containing the oligonucleotides was treated with 10% diethylamine (DEA) in ACN to deprotect the cyanoethyl groups. Then, both the unconjugated and cholesterol-conjugated oligonucleotides on the solid support were deprotected using methylamine gas (Airgas) at room temperature for 1 hour in the synthesis column. The deprotected oligonucleotides released from the solid support were precipitated onto the support by passing a mixture of (i) 0.1 M sodium acetate in 85% ethanol, followed by (ii) a solution of 85% ethanol through the synthesis column. The excess ethanol on the solid support was dried using an air stream, and the oligonucleotides were flushed out by passing water through the column. This procedure yielded highly pure oligonucleotides for use in in vitro experiments.

[0244] Deprotection and purification of oligonucleotides for in vivo experiments

[0245] Prior to deprotection, the synthesis column containing the oligonucleotides was treated with 10% diethylamine (DEA) in ACN to deprotect the cyanoethyl groups. Cy3-labeled lipid-conjugated oligonucleotides were cleaved and deprotected in 28–30% ammonium hydroxide, 40% methylamine (1:1, v / v) (AMA) at room temperature for 2 hours. Cy3-labeled and unlabeled unconjugated divalent, trivalent, and tetravalent oligonucleotides were cleaved and deprotected by AMA treatment at 45°C for 2 hours. VP-containing oligonucleotides were cleaved and deprotected as previously described without post-synthesis DEA pretreatment (O'Shea J, Theile CS, Das R, et al., An efficient deprotection method for 5'-[O,O-bis(pivaloyloxymethyl)]-(E)-vinylphosphonate containing oligonucleotides. Tetrahedron 74, 6182–6186 (2018)). Briefly, CPG bearing VP oligonucleotides was treated with a solution of 3% DEA in 28-30% ammonium hydroxide at 35°C for 20 h.

[0246] All solutions containing cleaved oligonucleotides were filtered to remove CPG and dried under vacuum. The resulting pellet was resuspended in 5% aqueous ACN. Purification was performed on an Agilent 1290 Infinity II HPLC system. VP and unlabeled, unconjugated, divalent, trivalent, and tetravalent oligonucleotides were purified using a custom 25 x 150 mm column packed with Source 15Q anion exchange resin (Cytiva, Marlborough, MA). Run conditions were: Eluent A, 10 mM Tris-HCl buffer (pH 9) in 7.5% aqueous ACN; Eluent B, 1 M sodium perchlorate in 10 mM Tris-HCl buffer (pH 9) in 7.5% aqueous ACN; linear gradient, 12–35% B in 40 min, 50°C. Lipid-conjugated, Cy3-labeled oligonucleotides were purified using a 21.2 x 150 mm PRP-C18 column (Hamilton Co., Reno, NV); run conditions were: eluent A, 50 mM sodium acetate (pH 6) in 5% aqueous ACN; eluent B, 100% ACN; linear gradient, 15 to 60% B in 40 min, 60 °C. For both methods, the flow rate was 40 mL / min, and peaks were monitored at 260 nm for unlabeled oligonucleotides and 550 nm for labeled oligonucleotides. To avoid cross-contamination, a separate column was used for Cy3-labeled oligonucleotides. Fractions were analyzed by liquid chromatography-mass spectrometry (LC-MS), and pure fractions were combined and dried under vacuum. The oligonucleotides were resuspended in 5% ACN and desalted by size exclusion on a 25 × 250 mm custom column packed with Sephadex G-25 medium (Cytiva, Marlborough, MA) using an isocratic method with HPLC-grade water (Honeywell Chemicals, Charlotte, NC), and finally the oligonucleotides were lyophilized.

[0247] LC-MS analysis of oligonucleotides

[0248] The identity of the oligonucleotides was verified by LC-MS analysis on an Agilent 6530 accurate-mass Q-TOF using the following conditions: Buffer A: 100 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 9 mM triethylamine (TEA) in LC-MS-grade water; Buffer B: 100 mM HFIP and 9 mM TEA in LC-MS-grade methanol; column: Agilent AdvanceBio Oligonucleotide C18; linear gradient 0–40% B in 5 min (unconjugated, bivalent, trivalent, and tetravalent oligonucleotides); linear gradient 50–100% B in 5 min (lipid-conjugated, Cy3-labeled oligonucleotides); temperature: 60°C; flow rate: 0.85 ml / min. LC peaks were monitored at 260 nm and 550 nm for labeled oligonucleotides. MS parameters: Source, electrospray ionization; ion polarity, negative mode; range, 100–3,200 m / z; scan rate, 2 spectra / sec; capillary voltage, 4,000; fragmentor, 200 V; gas temperature, 325°C.

[0249] In vivo experiments

[0250] Intravitreal injections into adult mice were performed as previously described (Venkatesh A, Ma S, Langellotto F, et al. Retinal gene delivery by rAAV and DNA electroporation. Curr Protoc Microbio: 2013; Chapter 14: Unit 14D 14). Approximately 2 mL of fluid was delivered into the vitreous using an Eppendorf FemtoJet with a glass needle (Clunbury Scientific LLC; Catalog No. B100-58-50) at a constant pressure of 300 psi and an injection time of 1.5 seconds, respectively. All concentrations were adjusted so that a 2 mL injection volume was used for the desired amount of siRNA. For intravitreal injections into adult pigs, 100 mL of siRNA was injected approximately 2–3 mm into the vitreous via the temporal limbus using an insulin injection needle. Anesthesia and euthanasia of the pigs were performed by a veterinary pharmacy according to standard procedures. Corneas were treated with proparacaine and ophthalmic Betadine before siRNA injection. After injection, eyes were rinsed with saline eye wash. Enucleated pig and mouse eyes were processed as described (Venkatesh A, Ma S, Langellotto F, et al. Retinal gene delivery by rAAV and DNA electroporation. Curr Protoc Microbio:2013;Chapter 14:Unit 14D 14).

[0251] Example 1: Delivery of siRNA for ocular diseases. An initial screen of various siRNAs, all targeting the Htt gene with the same sequence, was performed to test their distribution and efficiency for knockdown within the eye.

[0252] It is difficult to ascertain cellular distribution from Cy3 labeling alone. Therefore, a lower dose of siRNA compound (0.1 nmol) was injected (Cy3-labeled siRNA is shown in red in Figure 1). After 3 days, tissues were dissociated, Cy3-positive cells were FACS-sorted, and cell-type-specific antibodies were used to determine which cell types were enriched for which siRNA modifications.

[0253] Figure 1 shows that all siRNAs were labeled with Cy3, which is shown in red, while Müller glial cell-specific glutamine synthetase (GS) expression is shown in green and nuclear DAPI is shown in blue. All siRNAs were observed throughout the retinal cross section, with slightly different cellular distributions. The right side of Figure 1 shows one example per group, showing whole retinal cross sections with distribution throughout the retina. Half of the cross sections show nuclear DAPI, GS, and siRNA, while the other half show only siRNA.

[0254] The overall goal was to A) determine cellular distribution and B) identify modifications that would allow optimal cellular entry into cone and rod photoreceptors, as well as into Müller glial cells, since these three cell types are the most important cell types targeted in many retinal diseases. Figures 2 and 3 show the results of siRNA enrichment in different retinal cell types, arranged by cell type (Figure 2) and modification (Figure 3). These findings suggest that the monomeric configuration is suitable for cone photoreceptors, the tetramer for rod photoreceptors, and the dimer for Müller glial cells. Other favorable Müller glial cell compounds are PC-TS, PC-DHA, and DCA.

[0255] Repeated injections of a subset of these compounds and higher magnifications show that PC-TS accumulates substantially in Müller glial cells, and trimers and tetramers accumulate substantially in rod photoreceptors (Fig. 4 and 5 ).

[0256] To determine whether this tetramer could knockdown HTT protein in photoreceptors better than other constructs, antibody staining for HTT protein was performed 2 weeks after intravitreal injection of 0.3 nmoles of siRNA, as shown in Figure 6. The first column in Figure 6 shows antibody staining in control mice injected with NTC-siRNA. HTT protein has pan-retinal expression, with particularly high expression in the photoreceptor inner segments (IS), the outer plexiform layer (OPL) where photoreceptors form synaptic connections with bipolar and horizontal cells, moderate expression in the inner nuclear layer (INL) where bipolar, amacrine, and horizontal cells, and Müller glial cell bodies are present, and strong expression in the inner plexiform layer (IPL) where synaptic connections with amacrine, bipolar, and ganglion cells are present. The second column shows HTT protein expression after knockdown with PC-RA-Htt siRNA. This siRNA tends to preferentially accumulate in bipolar and amacrine cells, as shown in Figures 2 and 3. Therefore, expression in the OPL and IPL is more efficiently reduced with this siRNA. The third column shows HTT protein expression in two different mice, each injected with approximately 0.3 nmoles of Htt-siRNA, after knockdown with tetramer-Htt siRNA. This configuration tends to efficiently accumulate in rod photoreceptor cells and bipolar cells. Consistently, expression in the photoreceptor IS is much lower than with PC-RA-Htt siRNA.

[0257] Two weeks after Htt-siRNA injection, we also performed Western blot quantification of remaining total HTT protein in whole retinal extracts using the same experimental setup as in Figure 6, using different mice from the same injection batch. Note that when quantifying total HTT protein, knockdown is approximately 50% relative expression two weeks after injection. The two constructs targeted different cell populations in the retina with different efficiencies. Nevertheless, due to the ubiquitous expression of HTT within the retina, overall knockdown is similar.

[0258] Example 2: Evaluation of Htt mRNA knockdown by bDNA assay. Using 0.1 nmoles of the described siRNA modification per injection, we quantified total Htt mRNA levels using a bDNA assay 2 weeks after Htt-siRNA injection. As shown in Figure 8, quantification of Htt mRNA levels revealed approximately 20%-30% knockdown 2 weeks after injection with 0.1 nmoles of the described siRNA modification.

[0259] Using 0.3 nmoles of the described siRNA modification per injection, we quantified total Htt mRNA levels using a bDNA assay 3 days after Htt-siRNA injection. Note that quantification of Htt mRNA levels yields approximately 30%–60% knockdown 3 days after injection at 0.3 nmoles. Furthermore, PC-RA demonstrated a similar knockdown rate when compared to total protein measurements 2 weeks after injection (Figure 7: 60% knockdown), demonstrating that both quantification methods are similar for the retinal Htt gene and that there is a direct correlation between mRNA and protein levels of this gene (each dot represents one retina).

[0260] Example 3: Long-term evaluation of Htt mRNA knockdown by bDNA assay. Figure 10 shows the results of a bDNA assay performed to quantify total Htt mRNA levels 100 days after injection of Htt-siRNA using 0.3 nmoles per injection of the indicated siRNA modifications. Note that, compared to Figure 9, the knockdown effect only changed by approximately 10% (from 60% to 50% knockdown) over a time window of approximately 100 days, indicating that the knockdown was very stable (each dot in Figure 10 represents one retina). Figure 11 shows representative fundus images over time of eyes injected with Cy3-labeled siRNA containing the modifications indicated. The exposure of the fluorescent signal is the same for all four siRNAs at any given time point, but not over time. Figure 11 complements Figure 10 and shows fundus images of the mice used in Figure 10. All mice were intravitreally injected with 0.3 nmoles of siRNA.

[0261] Example 4: Dose-escalating study of HTT knockdown in mice in a tetrameric configuration Figure 12 shows the results of a dose-escalation study of HTT knockdown in the retina. Mice were injected with the indicated amounts (Cy3-labeled tetramer 1–60 with Htt-siRNA) in a total volume of 2 microliters. Five mice were injected per siRNA dose. Two weeks after injection, tissues were harvested for quantification of remaining HTT protein in the retina by Western blotting. Injections of 15–30 micrograms roughly correspond to the same knockdown observed in previous experiments with approximately 0.3 nanomoles.

[0262] Figure 14 shows retinal cross sections from eyes from a dose-escalation study of HTT knockdown in mice in a tetrameric configuration (the results of which are shown in Figures 12 and 13). The image shows Cy3 distribution throughout the retinal section, indicating uniform uptake of the siRNA throughout the eye.

[0263] To determine toxicity, antibody staining was performed on retinal sections from the eyes shown in Figure 14 to identify Iba1-positive cells and changes in GFAP. Figure 15 shows retinal sections from these eyes stained with Iba1 (green) to identify Iba1-positive cells migrating to the outer nuclear layer (ONL), where photoreceptors reside. Iba1-positive cells in the ONL were observed at 60 micrograms per injection and occasionally at 30 micrograms, indicating an inflammatory response at 60 micrograms and a mild response at 30 micrograms. Half of each panel (dotted lines in Figure 15) shows only Iba1 signal to better visualize the signal, and blue indicates nuclear DAPI. Figure 16 shows retinal sections from the dose-escalation study eyes shown in Figure 14 stained with GFAP (red) to identify reactive gliosis in Müller glial cells. A slight increase in GFAP expression is observed at the level of the ganglion cell layer (GCL), where astrocytes reside, but the expression does not extend upward into Müller glial cells. GFAP expression in astrocytes is normal. This expression is increased at 60 micrograms, consistent with the results observed with Iba1. However, the absence of reactive gliosis indicates the absence of severe retinal degenerative events induced by siRNA. siRNA is not shown because these sections are from the same eyes as those shown in Figure 15. In Figure 16, blue indicates nuclear DAPI, and green indicates cone photoreceptor segments with peanut agglutinin lectin (PNA). Figure 17 shows measurements of photoreceptor and retinal function by electroretinography under dark-adapted (0.01 cd.s / m² to 1 cd.s / m²) and light-adapted conditions (3 and 10 flashes). Recordings of the a-wave and b-wave, respectively, indicate normal photoreceptor and inner retinal function for all injected doses. As can be seen in the top two graphs, there are no statistically significant differences between recordings (n = 5 mice per siRNA dose). The implicit times of the a-wave and b-wave (bottom two graphs) are also not significantly different between the different groups of injected mice.

[0264] Example 5: Large Animal Model: siRNA in the Pig Eye (All data presented below are generated using tetramer-Htt-siRNA-Cy3 and its NTC in pigs) For translational purposes, the suitability of this siRNA technology in a large animal model was tested to determine distribution, knockdown efficiency, and toxicity. For this purpose, a pig model was chosen (35 kg pigs were used) because the size of the pig's eye is similar to that of humans. The only difference is the absence of a fovea. As an initial test run, three pigs were injected with five different amounts of siRNA of the same chemical composition, keeping the injection volume constant at 100 microliters. The following data summarizes the injection of siRNA against HTT in a tetrameric configuration in pigs. All siRNA molecules were also labeled with Cy3. The pigs were euthanized 10 days after intravitreal injection. Figure 18 shows the fluorescence intensity of tetramer-Htt-Cy3 after intravitreal delivery in pig eyes. The amount of siRNA delivered is indicated at the top of each panel in Figure 18 (100–1500 micrograms of tetramer). The fluorescence intensity is well distributed throughout the eye. The top panel of Figure 18 shows Cy3 fluorescence in unfixed tissue immediately after eye opening, while the bottom panel is a magnified area of ​​the top panel.

[0265] Huntington protein knockdown in pigs was measured by Western blot analysis from the pig eyes shown in Figure 18. Knockdown was compared to huntingtin protein levels in NTCs injected with 250 μg of tetramer-siRNA-Cy3. In Figure 19, the upper panel shows bar graphs of knockdown observed in the four major quadrants of the retina (DT: dorsal-temporal, DN: dorsal-nasal, VT: temporal-nasal, VN: ventral-nasal), with error bars representing technical replicates. Knockdown efficiency in each quadrant depends on the needle position and insertion angle. The needle was typically inserted from the temporal side and aimed toward the center of the eye. In the center panel of Figure 19, knockdown is shown on a flat-mount cutout with the corresponding regional knockdown values ​​indicated in the bar graph. The bottom panel of Figure 19 shows the average knockdown of Huntington protein across the entire retina, calculated by averaging the knockdown observed in each quadrant per retina, with error bars generated by averaging four data points in each quadrant per retina. The data shown in the bottom panel of Figure 19 represent one biological sample for each amount of siRNA delivered.

[0266] Figure 20 shows antibody staining for Huntington protein in sections from eyes injected with different amounts than those shown in Figure 19. The areas of the eye sections are shown in the center panel of Figure 19. Huntington's disease knockdown is observed throughout all layers of the retina, particularly in the inner and outer retinal layers (IPL, OPL) and where the photoreceptor segments (PS) are located.

[0267] Figure 21 shows antibody staining for GFAP (glial fibrillary acidic protein) and Iba1 (ionized calcium binding adaptor protein 1) expression (as shown for mice in Figures 15 and 16) in retinal sections from eyes injected with different doses as shown in Figures 18 and 19 to determine dose-dependent toxicity. GFAP and Iba1 are both shown in green in Figure 21, as shown on the left side of each row. Red staining in Figure 21 indicates siRNA distribution throughout the retinal section, while nuclei are labeled with intranuclear DAPI. GFAP and Iba1 expression clearly increases in a dose-dependent manner. Up to 500 μg, Iba1 and GFAP siRNA expression rarely progresses into the outer nuclear layer (ONL), where photoreceptors reside. At 1000 μg and 1500 μg, GFAP and Iba1 expression clearly increases in the ONL. Furthermore, much of the siRNA appears to be taken up by Iba1-positive cells, which may reflect macrophages taking up excess extracellular material. Half of each panel in Figure 21 shows only the signal of interest (siRNA, GFAP, or Iba1) for better visualization of the signal.

[0268] Summary of pig data: Tetramer-Htt-siRNA distributes well throughout the retina after a single intravitreal delivery in large eyes such as pigs. This is particularly important because pig eyes are similar in size to human eyes. In addition to the lack of a fovea, pig eyes are the animal model that most closely resembles the human eye. Their similar size compared to most experimental NHPs makes pig eyes more suitable for distribution studies. The dose-response in Figure 19 and toxicity in Figure 21 indicate that this particular compound may be used at doses ranging from 100 to 500 μg for further testing. This should result in approximately 50% knockdown of HTT, similar to that observed in mice using the tetramer. Toxicity may be reduced by removing the Cy3 molecules still attached in this study.

[0269] Example 6: Development of siRNA against S6K1 (RPS6KB1: ribosomal protein S6 kinase B1) An initial bioinformatics screen was performed to identify potential siRNA sequences against S6K1. The lists of sequences identified in the initial bioinformatics screen are shown in Tables 1 and 2 below. [Table 1]

[0270] For the above-mentioned 45 nucleotide gene region, their sequences correspond to the DNA gene sequence, but the mRNA encoded by the S6K1 gene has the same sequence, with T nucleotides replaced with U nucleotides. Thus, for example, siRNA with an antisense strand targeting SEQ ID NO: 1 targets the mRNA sequence corresponding to the gene region of SEQ ID NO: 1. [Table 2] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0271] For the sense and antisense sequences in Tables 3 and 3, "m" corresponds to a 2'-O-methyl modified nucleotide, "f" corresponds to a 2'-fluoro modified nucleotide, "#" corresponds to a phosphorothioate internucleotide linkage, "P" corresponds to a 5' phosphate, and "TegChol" corresponds to a cholesterol moiety linked with triethylene glycol or tetraethylene glycol.

[0272] Figure 22 shows the initial in vitro knockdown efficiency of duplexes formed from the sense and antisense strands shown in Tables 3 and 4. The candidates that yielded the best knockdown results were duplexes 2, 3, 7, 9, 10, and 19. Figure 23 shows the dose-response curves for the four sequences highlighted in red in Figure 22. Duplex 2 (Rps6k1b_459) showed the most consistent response and was therefore selected for further testing in vivo.

[0273] The primary purpose of siRNA against S6K1 is to knock down S6K1 in photoreceptors to treat AMD. Based on data generated by various HTT-siRNA complexes, we first developed a tetrameric siRNA without Cy3 labeling to reduce toxicity. We then generated in vivo data from mice and NHPs based on the tetrameric configuration of duplex 2 (Rps6k1b_459).

[0274] Figure 24 shows RNA-Scope in situ hybridization performed on mouse retinal cross sections to detect siRNA tetramers against S6K1. The top row of Figure 24 shows sections from three mice injected with tetrameric NTCs against S6K1, the middle row shows sections from three mice injected with tetrameric siRNA against S6K1 at 3 μg / eye, and the bottom row shows sections from three mice injected with tetrameric siRNA against S6K1 at 6 μg / eye. The siRNA was delivered intravitreally, and the animals were euthanized two weeks after injection.

[0275] Figures 25A and 25B show knockdown of S6K1 in mice after intravitreal injection of 6 μg of tetrameric siRNA. Both graphs in Figure 25 use rodTSC1- / - mice, which have been shown to develop age-related macular degeneration-like pathology. The rodTSC1+ / + mice provided are Cre-negative littermate controls that do not develop pathology. Figure 25A shows S6K1 protein levels detected by Western blot 2 weeks after injection. There is a slight trend toward decreased S6K1 protein compared to uninjected littermates or NTC mice. Figure 25B shows similar data 2 months after injection. Strong knockdown is observed with 6 μg of siRNA (40-45%). Each dot in the graphs in Figure 25 represents one biological sample (retina) from one animal.

[0276] Figure 26 shows knockdown of S6K1 protein in non-human primates (NHPs). Western blot data are shown for retinal protein extracts from the superior temporal (ST) region (also known as the dorsal-temporal region) of one NHP intravitreally injected with 225 μg of S6K1-tetramer (75 μL) and for six untreated NHP retinas from the same region. The first set of bar graphs shows a comparison between the uninjected contralateral eye and the S6K1 siRNA-injected eye, allowing for direct intraanimal comparison between the two eyes. The second set of bar graphs shows a comparison between six untreated NHPs and the S6K1 siRNA-injected NHPs. S6K1 knockdown efficiency appears to be approximately 50% in both cases. NHP eyes were harvested one month after injection. Also shown is a decrease in phosphorylation of the ribosomal protein S6, a canonical target of S6K1. As with the S6K1 knockdown data, within-animal comparisons are shown on the left and comparisons with multiple NHPs are shown on the right.

[0277] Figure 27 shows knockdown of S6K1 protein in retinal cross sections from non-human primates (NHPs) after siRNA treatment. Data were generated using one injected eye (see also Figure 26) and an uninjected contralateral eye. Sections were taken from the central region as shown for the pig in Figure 19. A general cross section including the fovea is shown on the left side of Figure 27. High-magnification images of the temporal and nasal regions and the fovea are shown on the right side of Figure 27. The top row of Figure 27 shows an uninjected eye, and the bottom row shows an eye intravitreally injected with 225 μg of S6K1-tetramer (75 μL). Consistent with the Western blot data generated using the superior temporal region of the same eye shown in Figure 26, there is a clear reduction in the signal for S6K1 protein expression. This reduction is also evident in the fovea, which contains only cones, indicating that knockdown in cones is as efficient as in rods.

[0278] Figure 28 shows the reduction of phosphorylated S6 protein (pS6) in non-human primate (NHP) retinal cross-sections after siRNA treatment. The data in Figure 28 are the same as those shown in Figure 27, except that the staining probes for pS6 expression (red signal). A clear reduction of pS6 is observed throughout the retina, particularly in photoreceptors such as foveal cones. In each panel of Figure 28, the green and blue signals were removed (dotted lines) in half of the panel to better visualize pS6 knockdown. Blue in Figure 28 indicates nuclear DAPI, while green indicates cone segments labeled with peanut agglutinin lectin (PNA).

[0279] Figure 29 shows the expression of inflammatory markers in NHPs after siRNA treatment with S6K1 siRNA (75 μL, 225 μg siRNA in tetramer configuration). The data in Figure 29 are the same as those shown in Figures 27 and 28, except that the staining probes for Iba1 (red signal, first set) and GFAP (red signal, second set) expression. The untreated contralateral eye is shown in the first row of each set, and the treated eye is shown in the second row. There is a slight increase in Iba1-positive cells migrating toward the photoreceptor layer in the siRNA-treated eyes. However, there is no reactive gliosis, as seen with GFAP staining. There is only a slight increase in staining in areas where astrocytes are present within the ganglion cell layer. There is no increase in the fovea. The data shown in Figure 29 suggest that there are no severe adverse reactions to siRNA treatment for S6K1 knockdown. In each panel of Figure 29, the green and blue signals have been removed from half of the panel (dotted lines) to better visualize the Iba1 and GFAP signals. In Figure 29, blue indicates intranuclear DAPI, while green indicates cone segments labeled with peanut agglutinin lectin (PNA).

[0280] Summary of S6K1-siRNA Data. Tetramer-S6K1-siRNA distributes well throughout the retina after a single intravitreal delivery of 6 μg into mouse eyes. Knockdown efficiency appears slow initially but becomes very potent over time. Therapeutic targeting requires approximately 50% knockdown of S6K1 protein in photoreceptors, which appears achievable. Duplex 2, selected from initial screening, functioned very efficiently in vivo, demonstrating that the S6K1 target site in SEQ ID NO: 1 is a useful target for S6K1 knockdown. Based on the dose-response curve of HTT-tetramer in mice, a subset of mice will be injected with 25 μg / eye in the near future and analyzed for S6K1 knockdown and age-related macular degeneration markers to determine whether disease progression is mitigated. Injections into NHPs confirmed that knockdown works equally efficiently in large eyes, distribution is widespread, therapeutic coverage is achievable, and there is no significant inflammatory response to treatment. Overall, the data indicate that gene knockdown in humans is feasible for the treatment of a variety of retinal diseases.

Claims

1. 1. A method for delivering an oligonucleotide conjugate to the eye of a subject, the method comprising administering the oligonucleotide conjugate to the subject, the oligonucleotide conjugate comprising: i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; ii) the delivery method comprises a functional moiety linked to the oligonucleotide, wherein the functional moiety comprises any one of a triple amine, retinoic acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), alpha-tocopheryl succinate, or lithocholic acid (LA).

2. A method for delivering a branched oligonucleotide to the eye of a subject, the method comprising administering the branched oligonucleotide to the subject, the branched oligonucleotide comprising two or more oligonucleotides, each oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid.

3. 3. The method of claim 2, wherein one or more of the oligonucleotides of the branched oligonucleotides further comprises a functional moiety linked to the oligonucleotide, wherein the functional moiety comprises any one of a triple amine, retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA.

4. The method according to any one of claims 1 to 3, wherein two DHA functional moieties are linked to the oligonucleotide.

5. The method of any one of claims 1 to 4, wherein the oligonucleotide comprises an antisense oligonucleotide or an siRNA.

6. The method of claim 5 , wherein the siRNA comprises a sense strand and an antisense strand.

7. The method of claim 6, wherein the length of the antisense strand is about 15 to 25 nucleotides.

8. The method of claim 6 or 7, wherein the sense strand has a length of about 15 to 25 nucleotides.

9. The method of any one of claims 6 to 8, wherein the antisense strand is 20, 21, or 22 nucleotides in length.

10. The method of any one of claims 6 to 8, wherein the sense strand is 15, 16, 18, or 20 nucleotides in length.

11. The method of any one of claims 5 to 10, wherein the siRNA comprises a double-stranded region of 15 to 20 base pairs.

12. 11. The method of claim 10, wherein the siRNA comprises a double-stranded region of 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

13. The method of any one of claims 5 to 12, wherein the siRNA comprises at least one blunt end.

14. The method of any one of claims 5 to 13, wherein the siRNA comprises at least one single-stranded nucleotide overhang.

15. 15. The method of claim 14, wherein the siRNA comprises a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

16. 16. The method of claim 15, wherein the siRNA comprises a single-stranded nucleotide overhang of 2 nucleotides or a single-stranded nucleotide overhang of 5 nucleotides.

17. The method of any one of claims 5 to 16, wherein the siRNA comprises natural nucleotides.

18. The method of any one of claims 5 to 17, wherein the siRNA comprises at least one modified nucleotide.

19. 19. The method of claim 18, wherein the modified nucleotides comprise 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, or mixtures thereof.

20. The method of any one of claims 5 to 19, wherein the siRNA comprises at least one modified internucleotide linkage.

21. 21. The method of claim 20, wherein the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage.

22. The method of any one of claims 5 to 21, wherein the siRNA comprises 4 to 16 phosphorothioate internucleotide linkages.

23. The method of any one of claims 5 to 22, wherein the siRNA comprises 8 to 13 phosphorothioate internucleotide linkages.

24. The method of any one of claims 5 to 23, wherein the siRNA comprises at least 80% chemically modified nucleotides.

25. The method of any one of claims 5 to 24, wherein the siRNA is fully chemically modified.

26. The method of any one of claims 5 to 25, wherein the siRNA comprises at least 70% 2'-O-methyl nucleotide modifications.

27. The method of any one of claims 6 to 26, wherein the antisense strand comprises at least 70% 2'-O-methyl nucleotide modifications.

28. 28. The method of claim 27, wherein the antisense strand comprises about 70% to 90% 2'-O-methyl nucleotide modifications.

29. The method of any one of claims 6 to 28, wherein the sense strand comprises at least 65% 2'-O-methyl nucleotide modifications.

30. 30. The method of claim 29, wherein the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

31. The method of any one of claims 6 to 30, wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

32. The method of any one of claims 6 to 30, wherein the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate.

33. 33. The method of claim 32, wherein the antisense strand comprises a 5' vinyl phosphonate.

34. The method of any one of claims 1 to 33, wherein the functional moiety is linked to the 5' end and / or the 3' end of the oligonucleotide.

35. The method according to any one of claims 6 to 34, wherein the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand.

36. The method of any one of claims 6 to 34, wherein the functional moiety is linked to the 3' end of the sense strand.

37. The method according to any one of claims 6 to 34, wherein the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

38. 38. The method of claim 37, wherein the linker comprises a bivalent or trivalent linker.

39. The bivalent or trivalent linker is 【Chemical 1】 is selected from the group consisting of 39. The method of claim 38, wherein n is 1, 2, 3, 4, or 5.

40. 40. The method of any one of claims 37 to 39, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

41. 40. The method of claim 38 or 39, wherein when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

42. The phosphodiester or phosphodiester derivative is 【Chemistry 2】 wherein X is O, S, or BH ３ 42. The method of claim 41, wherein:

43. 43. The method of any one of claims 6 to 42, wherein the nucleotides at positions 1 and 2 from the 3' end of the sense strand and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate linkages.

44. 44. The method of any one of claims 2 to 43, wherein the two or more oligonucleotides in the branched oligonucleotide are linked to each other by one or more moieties independently selected from a linker, a spacer, and a branch point.

45. 45. The method of claim 44, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

46. 45. The method of claim 44, wherein the branch point comprises a polyvalent organic species or a derivative thereof.

47. 45. The method of claim 44, wherein the spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

48. The linker has the structure L1: 【Chemistry 3】 45. The method of claim 44, comprising:

49. The linker has the structure L2: 【Chemistry 4】 45. The method of claim 44, comprising:

50. The method of any one of claims 2 to 49, wherein the branched oligonucleotide consists of two oligonucleotides.

51. The method of any one of claims 2 to 49, wherein the branched oligonucleotide consists of three oligonucleotides.

52. The method of any one of claims 2 to 49, wherein the branched oligonucleotide consists of four oligonucleotides.

53. The method of any one of claims 50 to 52, wherein the oligonucleotide is an siRNA.

54. 54. The method of any one of claims 1 to 53, wherein the oligonucleotide conjugate or branched oligonucleotide is administered by intravitreal injection.

55. 54. The method of any one of claims 1 to 53, wherein the oligonucleotide conjugate or branched oligonucleotide is delivered to an ocular cell after administration to the subject.

56. 56. The method of claim 55, wherein the ocular cell is selected from the group consisting of a Muller glial cell, a rod photoreceptor cell, a cone photoreceptor cell, a ganglion cell, an amacrine cell, a bipolar cell, and a horizontal cell.

57. 56. The method of claim 55, wherein the ocular cell is selected from the group consisting of a glutamine synthetase (GS)-expressing ocular cell, a rhodopsin-expressing ocular cell, a cone arrestin (CA)-expressing ocular cell, a Vglut2-expressing ocular cell, a VGAT-expressing ocular cell, a protein kinase C alpha (PKCa)-expressing ocular cell, and a Lim1-expressing ocular cell.

58. the ocular cells are Müller glial cells; i) the oligonucleotide complex comprises DHA, DCA, α-tocopheryl succinate, or LA; or ii) The method of any one of claims 55 to 57, wherein the branched oligonucleotide consists of two, three, or four oligonucleotides.

59. 59. The method of claim 58, wherein the DHA, alpha-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified DHA (PC-DHA), alpha-tocopheryl succinate (PC-TS), and LA (PC-LA).

60. the eye cells are rod photoreceptor cells; i) the oligonucleotide conjugate comprises DCA, or ii) The method of any one of claims 55 to 57, wherein the branched oligonucleotide consists of three or four oligonucleotides.

61. the eye cells are cone photoreceptor cells, i) the oligonucleotide complex comprises retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA; or ii) The method of any one of claims 55 to 57, wherein the branched oligonucleotide consists of two, three, or four oligonucleotides.

62. 62. The method of claim 61, wherein the retinoic acid and alpha-tocopheryl succinate are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA) and alpha-tocopheryl succinate (PC-TS).

63. 62. The method of claim 61, wherein the oligonucleotide complex comprises two DHA functional moieties.

64. 58. The method of any one of claims 55 to 57, wherein the ocular cell is a ganglion cell and the oligonucleotide conjugate comprises alpha-tocopheryl succinate.

65. 65. The method of claim 64, wherein the alpha-tocopheryl succinate is phosphatidylcholine (PC)-esterified alpha-tocopheryl succinate (PC-TS).

66. the eye cells are amacrine cells, i) the oligonucleotide complex comprises retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA; or ii) The method of any one of claims 55 to 57, wherein the branched oligonucleotide consists of two, three, or four oligonucleotides.

67. 67. The method of claim 66, wherein the retinoic acid, DHA, and alpha-tocopheryl succinate are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), and alpha-tocopheryl succinate (PC-TS).

68. 67. The method of claim 66, wherein the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

69. the eye cells are bipolar cells, i) the oligonucleotide conjugate comprises a triple amine, retinoic acid, DHA, DCA, α-tocopheryl succinate, or LA; or ii) The method of any one of claims 55 to 57, wherein the branched oligonucleotide consists of two, three, or four oligonucleotides.

70. 70. The method of claim 69, wherein the retinoic acid, DHA, alpha-tocopheryl succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), alpha-tocopheryl succinate (PC-TS), and LA (PC-LA).

71. 70. The method of claim 69, wherein the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

72. the eye cells are horizontal cells, i) the oligonucleotide conjugate comprises DCA, or ii) The method of any one of claims 55 to 57, wherein the branched oligonucleotide consists of two oligonucleotides.

73. The oligonucleotide conjugate has the structure: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 73. The method of any one of claims 1 to 72, comprising:

74. The branched oligonucleotide has the structure: 【Chemistry 6】 73. The method of any one of claims 1 to 72, comprising:

75. 75. The method of any one of claims 1 to 74, wherein expression of the target nucleic acid is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

76. 76. The method of any one of claims 1 to 75, wherein the oligonucleotide conjugate has selective affinity for a retinal protein.

77. 77. The method of any one of claims 1 to 76, wherein the subject has an eye disorder.

78. 78. The method of any one of claims 1 to 77, wherein administering said oligonucleotide conjugate or said branched oligonucleotide treats an ocular disorder in said subject.

79. 79. The method of claim 77 or 78, wherein the ocular disorder is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, central axial cataract, normal tension glaucoma, macular edema, and glaucoma.

80. i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; ii) a di-docosahexaenoic acid (di-DHA) functional moiety linked to the oligonucleotide. An oligonucleotide conjugate comprising:

81. 81. The oligonucleotide conjugate of claim 80, wherein the di-DHA functional moiety is phosphatidylcholine (PC)-esterified di-DHA (PC-di-DHA).

82. structure: 【Chemistry 7】 81. The oligonucleotide conjugate of claim 80, comprising:

83. 83. The oligonucleotide conjugate of claim 82, wherein the oligonucleotide corresponds to an antisense oligonucleotide or an siRNA.

84. 84. The oligonucleotide conjugate of claim 83, wherein the siRNA comprises a sense strand and an antisense strand.

85. 85. The oligonucleotide complex of claim 84, wherein the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand.

86. 85. The oligonucleotide complex of claim 84, wherein the functional moiety is linked to the 3' end of the sense strand.

87. i) an oligonucleotide having a 5' end and a 3' end and complementarity to a target nucleic acid; ii) a triple amine function linked to said oligonucleotide An oligonucleotide conjugate comprising:

88. 88. The oligonucleotide conjugate of claim 87, wherein the triple amine moiety is a phosphatidylcholine (PC) esterified triple amine (PC-triple amine).

89. structure: 【Chemistry 8】 88. The oligonucleotide complex of claim 87, comprising:

90. 90. The oligonucleotide conjugate of claim 89, wherein the oligonucleotide corresponds to an antisense oligonucleotide or an siRNA.

91. 91. The oligonucleotide conjugate of claim 90, wherein the siRNA comprises a sense strand and an antisense strand.

92. 92. The oligonucleotide complex of claim 91, wherein the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand.

93. 92. The oligonucleotide complex of claim 91, wherein the functional moiety is linked to the 3' end of the sense strand.

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