Ophthalmic delivery of oligonucleotides

Abstract

Provided herein are conjugated oligonucleotides characterized by an effective and specific ocular distribution.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 412,051, filed Sep. 30, 2022, the content of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] The present disclosure relates to oligonucleotide conjugates and branched oligonucleotides for delivery to the eye. Background Art

[0004] Eye diseases are caused by genetic and non-genetic risk factors. Some of these diseases have a well-defined underlying genetic etiology (mutation), which can be transmitted in dominant, recessive, or X-linked inheritance patterns. Such hereditary retinal dystrophies are caused by more than 250 genes. These include, for example, dominant mutations that cause dominant retinitis pigmentosa, such as the P23H mutation in the rhodopsin gene, which is the most common dominant mutation in individuals with dominant retinitis pigmentosa. Eye diseases of unknown etiology include diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. While some of these diseases are caused by environmental risk factors, many genetic risk factors have also been shown to contribute to disease progression. Interestingly, several non-coding RNA sequences have been shown to contribute to various diseases. Thus, the need for effective regulation of gene expression in the eye to treat various eye diseases has not been met. Described herein is a method for downregulating the expression of pathogenic genes that directly or indirectly cause pathology in the eye.

[0005] Oligonucleotides such as small interfering RNA (siRNA) molecules have been used to regulate gene expression levels in different organs. However, their application in the eye has been hampered by the low permeability of siRNA molecules to various cell types, the stability of siRNA, and the persistence of the knockdown effect. This is particularly important for the eye because repeated injections every two weeks or monthly impose a burden on the patient and the caregiver and increase the risk of eye complications. Described herein is an oligonucleotide platform in which oligonucleotides (e.g., siRNA molecules) have been chemically stabilized to extend the gene knockdown time, and whose configuration or attachment has been modified to improve cellular entry into different cell types of the retina. Summary of the Invention

[0006] Provided herein are methods for delivering oligonucleotide conjugates and branched oligonucleotides to the eye and particularly to specific eye cells. The oligonucleotide conjugates and branched oligonucleotides are capable of effective gene knockdown in the eye. Several different functional moieties and branched oligonucleotides exhibit eye cell-specific delivery upon administration.

[0007] The oligonucleotide conjugates and branched oligonucleotides described herein facilitate simple, effective, and non-toxic delivery of oligonucleotides (e.g., siRNA) and promote efficient silencing of therapeutic targets in multiple ocular cell types in vivo.

[0008] In one aspect, the present disclosure provides a method for delivering an oligonucleotide conjugate to the eye of a subject, the method comprising administering the oligonucleotide conjugate to the subject, wherein the oligonucleotide conjugate comprises: i) an oligonucleotide having 5' and 3' termini and complementary to a target nucleic acid; and ii) a functional moiety linked to the oligonucleotide, wherein the functional moiety comprises any one of triamine, retinoic acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), α-tocopherol succinate, or lithocholic acid (LA).

[0009] In another aspect, the present disclosure provides a method for delivering a branched oligonucleotide to the eye of a subject, the method comprising administering the branched oligonucleotide to the subject, wherein the branched oligonucleotide comprises two or more oligonucleotides, each oligonucleotide having 5' and 3' termini and complementary to a target nucleic acid. In certain embodiments, one or more of the oligonucleotides in the branched oligonucleotide further comprise a functional moiety linked to the oligonucleotide, wherein the functional moiety comprises any one of triamine, retinoic acid, DHA, DCA, α-tocopherol succinate, or LA.

[0010] In certain embodiments, two DHA functional moieties are linked to the oligonucleotide.

[0011] In certain embodiments, the oligonucleotide comprises an antisense oligonucleotide or siRNA.

[0012] In certain embodiments, the siRNA comprises a sense strand and an antisense strand. In certain embodiments, the antisense strand has a length of about 15 nucleotides to 25 nucleotides. In certain embodiments, the sense strand has a length of about 15 nucleotides to 25 nucleotides. In certain embodiments, the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length.

[0013] In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs to 20 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

[0014] In certain embodiments, the siRNA comprises at least one blunt end.

[0015] 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 single-stranded nucleotide overhang of 2 nucleotides or a single-stranded nucleotide overhang of 5 nucleotides.

[0016] In certain embodiments, the siRNA comprises naturally occurring nucleotides.

[0017] In certain embodiments, the siRNA comprises at least one modified nucleotide. In certain embodiments, modified nucleotides include 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 non-natural bases, or mixtures thereof.

[0018] In certain embodiments, the siRNA comprises at least one modified internucleotide bond. In certain embodiments, modified internucleotide bonds include phosphorothioate internucleotide bonds. In certain embodiments, the siRNA comprises 4 - 16 phosphorothioate internucleotide bonds. In certain embodiments, the siRNA comprises 8 - 13 phosphorothioate internucleotide bonds.

[0019] In certain embodiments, the siRNA comprises at least 80% chemically modified nucleotides. In certain embodiments, the siRNA is fully chemically modified.

[0020] In certain embodiments, the siRNA comprises at least 70% 2'-O-methyl nucleotide modification. In certain embodiments, the antisense strand comprises at least about 70% 2'-O-methyl nucleotide modification. In certain embodiments, the antisense strand comprises about 70% to 90% 2'-O-methyl nucleotide modification. In certain embodiments, the sense strand comprises at least about 65% 2'-O-methyl nucleotide modification. In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modification.

[0021] In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

[0022] In certain embodiments, the antisense strand comprises a 5'-phosphate, 5'-alkylphosphonate, 5'-alkylenephosphonate, or 5'-alkenylphosphonate. In certain embodiments, the antisense strand comprises a 5'-vinylphosphonate.

[0023] In certain embodiments, the functional moiety is attached to the 5'-end and / or 3'-end of the oligonucleotide.

[0024] In certain embodiments, the functional moiety is attached to the 5'-end and / or 3'-end of the sense strand or to the 5'-end and / or 3'-end of the antisense strand.

[0025] In certain embodiments, the functional moiety is attached to the 3'-end of the sense strand.

[0026] In certain embodiments, the functional moiety is attached to the antisense strand and / or the sense strand via a linker.

[0027] In certain embodiments, the linker comprises a divalent or trivalent linker.

[0028] In certain embodiments, the divalent or trivalent linker is selected from the group consisting of:

[0029]

[0030]

[0031] where n is 1, 2, 3, 4 or 5.

[0032] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, an aminophosphate, an amide, a carbamate or a combination thereof.

[0033] In certain embodiments, when the linker is a trivalent linker, the linker is further attached to a phosphodiester or a phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of:

[0034]

[0035] and

[0036]

[0037] where X is O, S or BH 3 .

[0038] In certain embodiments, the nucleotides at positions 1 and 2 at the 3'-end of the sense strand, and the nucleotides at positions 1 and 2 at the 5'-end of the antisense strand, are linked to adjacent ribonucleotides via phosphorothioate bonds.

[0039] In certain embodiments, two or more oligonucleotides in a branched oligonucleotide are interconnected by one or more moieties independently selected from linkers, spacers, and branch points. In certain embodiments, a linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate ester, a phosphonate, an aminophosphate, an ester, an amide, a triazole, or a combination thereof. In certain embodiments, a branch point comprises a polyvalent organic substance or a derivative thereof. In certain embodiments, a spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate ester, a phosphonate, an aminophosphate, an ester, an amide, a triazole, or a combination thereof.

[0040] In certain embodiments, a linker comprises structure L1:

[0041]

[0042] In certain embodiments, a linker comprises structure L2:

[0043]

[0044] In certain embodiments, a branched oligonucleotide consists of two oligonucleotides. In certain embodiments, a branched oligonucleotide consists of three oligonucleotides. In certain embodiments, a branched oligonucleotide consists of four oligonucleotides.

[0045] In certain embodiments, the oligonucleotides in a branched oligonucleotide are siRNAs.

[0046] In certain embodiments, an oligonucleotide conjugate or a branched oligonucleotide is administered by intravitreal injection.

[0047] In certain embodiments, an oligonucleotide conjugate or a branched oligonucleotide is delivered to ocular cells after administration to a subject.

[0048] In certain embodiments, ocular cells are selected from the group consisting of Müller glia cells, rod photoreceptor cells, cone photoreceptor cells, ganglion cells, amacrine cells, bipolar cells, and horizontal cells.

[0049] In certain embodiments, ocular cells are selected from the group consisting of ocular cells expressing glutamine synthetase (GS), ocular cells expressing rhodopsin, ocular cells expressing cone arrestin (CA), ocular cells expressing Vglut2, ocular cells expressing VGAT, ocular cells expressing protein kinase Cα (PKCa), and ocular cells expressing Lim1.

[0050] In certain embodiments, the eye cells are Müller glial cells, and: i) the oligonucleotide conjugate comprises DHA, DCA, α-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments, DHA, α-tocopherol succinate, and LA are phosphatidylcholine (PC)-esterified DHA (PC-DHA), α-tocopherol succinate (PC-TS), and LA (PC-LA).

[0051] In certain embodiments, the eye cells are rod photoreceptor cells, and: i) the oligonucleotide conjugate comprises DCA; or ii) the branched oligonucleotide consists of three or four oligonucleotides.

[0052] In certain embodiments, the eye cells are cone photoreceptor cells, and: i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments, retinoic acid and α-tocopherol succinate are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA) and α-tocopherol succinate (PC-TS). In certain embodiments, the oligonucleotide conjugate comprises two DHA functional moieties. In certain embodiments, the eye cells are ganglion cells, and the oligonucleotide conjugate comprises α-tocopherol succinate. In certain embodiments, α-tocopherol succinate is phosphatidylcholine (PC)-esterified α-tocopherol succinate (PC-TS). In certain embodiments, lithocholic acid (LA) is phosphatidylcholine (PC)-esterified lithocholic acid (PC-LA). In certain embodiments, natural lithocholic acid (LA) is phosphatidylcholine (PC)-esterified natural lithocholic acid (PC-natural LA). In certain embodiments, isomeric lithocholic acid (LA) is phosphatidylcholine (PC)-esterified isomeric lithocholic acid (PC-isomeric LA).

[0053] In certain embodiments, the eye cells are amacrine cells, and: i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides.

[0054] In certain embodiments, retinoic acid, DHA, α-tocopherol succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), α-tocopherol succinate (PC-TS), and LA (PC-LA). In certain embodiments, the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

[0055] In certain embodiments, the eye cell is a bipolar cell, and: i) the oligonucleotide conjugate comprises triamine, retinoic acid, DHA, DCA, alpha-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments, retinoic acid, DHA, alpha-tocopherol succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), alpha-tocopherol succinate (PC-TS), and LA (PC-LA). In certain embodiments, the oligonucleotide conjugate comprises two DHA moieties or two PC-DHA moieties.

[0056] In certain embodiments, the eye cell is a horizontal cell, and: i) the oligonucleotide conjugate comprises DCA; or ii) the branched oligonucleotide consists of two oligonucleotides.

[0057] In certain embodiments, the oligonucleotide conjugate comprises the following structure:

[0058]

[0059]

[0060]

[0061]

[0062] In certain embodiments, the branched oligonucleotide comprises the following structure:

[0063]

[0064] In certain embodiments, the expression of the target nucleic acid is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0065] In certain embodiments, the oligonucleotide conjugate has a selective affinity for retinal proteins.

[0066] In certain embodiments, the subject has an ocular disorder. In certain embodiments, administration of the oligonucleotide conjugate or the branched oligonucleotide results in treatment of the subject's ocular disorder.

[0067] 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.

[0068] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having 5' and 3' termini and complementary to a target nucleic acid; and ii) a di-docosahexaenoic acid (di-DHA) moiety linked to the oligonucleotide.

[0069] In certain embodiments, the di-DHA functional moiety is phosphatidylcholine (PC)-esterified di-DHA (PC-di-DHA).

[0070] In certain embodiments, the oligonucleotide conjugate has the following structure:

[0071]

[0072] 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 linked to 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.

[0073] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having 5'- and 3'-ends and complementary to a target nucleic acid; and ii) a triamine functional moiety linked to the oligonucleotide.

[0074] In certain embodiments, the triamine functional moiety is phosphatidylcholine (PC)-esterified triamine (PC-triamine).

[0075] In certain embodiments, the oligonucleotide conjugate has the following structure:

[0076]

[0077] 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 linked to the 5'-end and / or 3'-end of the antisense strand.

[0078] In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1Shows retinal cross-sections with 12 different siRNA distributions 3 days after injection of 0.3 nanomoles of siRNA (left inset: retinoic acid (RA), docosahexaenoic acid (DHA), phosphocholine (PC); alpha-tocopherol succinate (TS); docosanoic acid (DCA)). On the right: one example per group, showing the entire retinal cross-section with distribution across the whole retina. All siRNAs were labeled with Cy3 and shown in red. Glutamine synthetase (GS) expression was shown in green. Nuclear DAPI was shown in blue. On the right: one example per group, showing the entire retinal cross-section with distribution across the whole retina.

[0080] Figure 2 Shows the enrichment of siRNAs in different retinal cell types arranged by cell type. Bars show the relative protein levels of cell type-specific markers calibrated against the total retinal extract of uninjected mouse retinas.

[0081] Figure 3 Shows the enrichment of siRNAs in different retinal cell types arranged by modification. Bars show the relative protein levels of cell type-specific markers calibrated against the total retinal extract of uninjected mouse retinas. For each modification (i.e., monomer, dimer, trimer, etc.), each bar from left to right represents rhodopsin (rod cells), cone arrestin (CA) (cone cells), glutamine synthetase (GS) (Muller cells), Vglut2 (ganglion cells), VGAT (amacrine cells), protein kinase C alpha (PKCa) (bipolar cells), and Lim1 (horizontal cells).

[0082] Figure 4 Shows examples of siRNA distribution in retinal cross-sections 3 days after injection of 0.3 nanomoles of siRNA. The siRNAs were labeled with Cy3 and shown in red. All siRNAs targeted the Huntington gene. Non-targeting control (NTC), trimer, and tetramer with PC-TS modification were shown. Cone segments were highlighted in green, labeled by PNA (peanut agglutinin lectin), Muller glial cells were shown in cyan, labeled by glutamine synthetase (GS), and cell nuclei were shown in blue, labeled by DAPI.

[0083] Figure 5 Shows examples of siRNA distribution in retinal cross-sections 3 days after injection of 0.3 nanomoles of siRNA (without GS staining in cyan). In addition, for the trimer and tetramer, only high magnification of the outer nuclear layer (ONL) is shown to highlight the distribution of siRNAs in the photoreceptor cell layer. Half of the inset shows only the siRNAs for better visualization of the signal.

[0084] Figure 6 Shows antibody staining against HTT protein on retinal cross-sections two weeks after injection of Htt-siRNA. The first column shows the staining of control mice injected with NTC-siRNA. The second column, the expression of HTT protein after knockdown with PC-RA-Htt siRNA. Examples of two different mice are shown, each injected with approximately 0.3 nanomoles of Htt-siRNA.

[0085] Figure 7 Shows quantification of total HTT protein by Western blot two weeks after injection of Htt-siRNA. Quantifies the remaining total HTT protein in total retinal extracts with Figure 6 the same experimental setup (different mice from the same injection batch).

[0086] Figure 8 Shows quantification of total Htt mRNA levels by bDNA assay two weeks after injection of Htt-siRNA (0.1 nanomoles of the siRNA modification per injection).

[0087] Figure 9 Shows quantification of total Htt mRNA levels by bDNA assay three days after injection of Htt-siRNA (0.3 nanomoles of the siRNA modification per injection). Each point represents 1 retina.

[0088] Figure 10 Shows quantification of total Htt mRNA levels by bDNA assay 100 days after injection of Htt-siRNA (0.3 nanomoles of the siRNA modification per injection). Each point represents 1 retina.

[0089] Figure 11 Shows representative fundus images over time of eyes injected with Cy3-labeled siRNA with the specified modifications. At any given time point, the fluorescence signal exposures of all 4 siRNAs are the same, but vary over time. This figure is supplementary to Figure 10 and shows Figure 10 fundus images of the mice used. All mice were injected intravitreally with 0.3 nanomoles of siRNA.

[0090] Figure 12Shows a dose escalation study of HTT-knockdown in the retina. Mice were injected with the amounts shown in the figure (1 - 60 micrograms [note: not nanomoles] of Cy3-labeled tetramer and Htt-siRNA) in a total volume of 2 microliters. Five mice were injected according to the siRNA amount. Tissues were collected two weeks after injection to quantify the remaining HTT protein in the retina by Western blot. Injecting 15 - 30 micrograms roughly corresponds to a knockdown of approximately 0.3 nanomoles observed in previous experiments.

[0091] Figure 13 Shows Figure 12 Fundus images of the dose escalation study shown in. Images were taken two weeks after injection, just before euthanasia. Conventional bright-field fundus images and Cy3 images for each concentration are shown.

[0092] Figure 14 Shows from Figure 12 and 13 Cross-sections of the eye retina from the dose escalation study shown. The images show Cy3 distributed throughout the retinal cross-section, indicating uniform absorption of siRNA throughout the study.

[0093] Figure 15 Shows from Figure 14 Cross-sections of the eye retina from the dose escalation study shown, stained with Iba1 (green) to identify Iba1-positive cells that migrate to the outer nuclear layer (ONL) where photoreceptor cells are located. Half of each inset (dashed line) shows only the Iba1 signal to better visualize the signal. The nucleus is shown in blue with DAPI.

[0094] Figure 16 Shows from Figure 14 Cross-sections of the eye retina from the dose escalation study shown, stained with GFAP (red) to identify reactive gliosis in Müller glial cells. siRNA is not shown as these are sections from the same eyes as those shown in Figure 15 The nucleus is shown in blue with DAPI, and the cone photoreceptor segments containing peanut agglutinin lectin (PNA) are marked in green.

[0095] Figure 17 Shows photoreceptor and retinal function measured by electroretinogram under scotopic (0.01 cd.s / m2 - 1 cd.s / m2) and photopic conditions (3 and 10 flashes). The a-wave and b-wave were recorded at several injected amounts.

[0096] Figure 18Shows the fluorescence intensity of the tetramer-Htt-Cy3 after intravitreal delivery in porcine eyes. The amount of siRNA delivered (100 - 1500 micrograms of tetramer) is shown at the top of each inset. The top row shows the Cy3 fluorescence of the unfixed tissue after opening the eyes. The bottom of the figure is a high magnification of a region of the top inset.

[0097] Figure 19 Shows the knockdown of huntingtin in suids as measured by western blot analysis of eyes as Figure 18 shown. The knockdown was compared to the huntingtin protein levels in the NTC injected with 250 ug of tetramer-siRNA-Cy3. The top panel shows the knockdown presented as bar graphs seen in the four major retinal quadrants (DT: dorsal temporal; DN: dorsal nasal; VT: ventral temporal; VN: ventral nasal). The middle inset shows the knockdown on a flat mount cartoon, where the corresponding values of regional knockdown are shown in the bar graph. Bottom inset: The average knockdown of huntingtin in the whole retina was calculated by taking the mean of the knockdown seen in each quadrant of each retina. The data shown represent one biological sample for each amount of siRNA delivered. The error bars in the first inset were generated from technical replicates. The error bars in the last inset were generated by taking the mean of 4 data points in each quadrant of each retina.

[0098] Figure 20 Shows antibody staining of huntingtin on eye sections injected with different amounts as Figure 19 shown. Figure 19 The middle inset of

[0099] Figure 21 Shows antibody staining of GFAP (glial fibrillary acidic protein) and Iba1 (ionized calcium-binding adapter molecule 1) on retinal sections of eyes injected with different amounts as Figure 18 and 19 shown, as in the mice of Figure 15 and 16 shown, to determine dose-dependent toxicity. Both GFAP and Iba1 are shown in green as designated on the left side of each row. Red staining shows the distribution of siRNA on the retinal sections. Nuclei are labeled with nuclear DAPI. Only half of each inset shows the target signal (siRNA, GFAP, or Iba1) to better visualize the signal.

[0100] Figure 22 Shows the in vitro initial knockdown efficiency of siRNA duplexes formed by the sense and antisense strands shown in Tables 3 and 4.

[0101] Figure 23 Shows Figure 22Dose-response curves of duplexes 2, 3, 9, and 10 in

[0102] Figure 24 Show RNA-Scope in situ hybridization performed on cross-sections of mouse retinas to detect siRNA-tetramers against S6K1. The top row shows cross-sections of 3 mice injected with NTC against S6K1 in tetrameric configuration. The middle row shows cross-sections of 3 mice injected with siRNA against S6K1 in tetrameric configuration at 3 μg / eye. The last row shows cross-sections of 3 mice injected with siRNA against S6K1 in tetrameric configuration at 6 μg / eye. The siRNA was delivered intravitreally, and the animals were euthanized two weeks after injection.

[0103] Figure 25A – Figure 25B Show knockdown of S6K1 after intravitreal injection of 6 μg of siRNA in tetrameric configuration in mice. Figure 25A Show S6K1 protein levels detected by Western blot two weeks after injection. Figure 25B Show data similar to the first graph two months after injection. Each point in the graph represents a biological sample (retina) from one animal.

[0104] Figure 26 Show knockdown of S6K1 protein in non-human primates (NHPs). Western blot data using retinal protein extracts from the superotemporal (ST) region (aka: dorsal temporal) of one NHP intravitreally injected with 225 μg S6K1-tetramer (75 μL), and from 6 naïve NHP retinas from the same region. The first set of bar graphs shows a comparison between the uninjected contralateral eye and one eye injected with S6K1 siRNA to allow direct comparison of the two eyes in the animal. The second bar graph shows a comparison between 6 naïve NHPs and the NHP injected with S6K1 siRNA. NHP eyes were collected one month after injection. Phosphorylation of ribosomal protein S6, a canonical target of S6K1, is also shown. Similar to the S6K1 knockdown data, the comparison within the animal is shown on the left, and the comparison with several NHPs is shown on the right.

[0105] Figure 27 Show knockdown of S6K1 protein on cross-sections of non-human primate (NHP) retinas after siRNA treatment. Data were generated from one injected eye and the uninjected contralateral eye. As Figure 19As shown for the pig, the cross-section was obtained from the central region. Left side: The entire cross-section covering the fovea. Right side: High magnification of the temporal and nasal regions and the fovea. The top row shows the uninjected eye, and the bottom row shows the eye intravitreally injected with 225 μg of S6K1-tetramer (75 μL).

[0106] Figure 28 Reduction of phosphorylated S6 protein (pS6) in the retinal cross-section of non-human primates (NHP) after siRNA treatment is shown. Data are the same as Figure 27 shown, except that the staining probe was used for the expression of pS6 (red signal). In each panel, the green and blue signals have been removed from half of the panels (dashed lines) to better visualize the knockdown of pS6. Blue shows nuclear DAPI, and green shows cone segments labeled with peanut agglutinin lectin (PNA).

[0107] Figure 29 Expression of inflammatory markers in NHP after siRNA treatment with S6K1 siRNA (75 μL, 225 μg of siRNA in tetrameric configuration) is shown. Data are the same as Figure 27 and Figure 28 shown, except that the staining probes were used for the expression of Iba1 (red signal, first set) and GFAP (red signal, second set). The untreated contralateral eye is in the first row of each set, and the treated contralateral eye is in the second row. In each panel, the green and blue signals have been removed from half of the panels (dashed lines) to better visualize the Iba1 and GFAP signals. Blue shows nuclear DAPI, and green shows cone segments labeled with peanut agglutinin lectin (PNA). DETAILED DESCRIPTION

[0108] 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 exhibit cell-specific delivery to eye cells after administration.

[0109] The oligonucleotide conjugates and branched oligonucleotides described herein facilitate simple, effective, and non-toxic delivery of oligonucleotides (e.g., siRNA) and promote efficient silencing of therapeutic targets in multiple eye cell types in vivo.

[0110] Unless otherwise specified, the nomenclature used herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, is that which is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are carried out according to conventional methods that are well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. Unless otherwise provided with an exact definition, the nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, and the laboratory procedures and techniques in these chemical fields, are those that are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient treatment.

[0111] Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Additionally, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms such as "includes" and "included" is not limiting.

[0112] To facilitate a better understanding of the present disclosure, certain terms are first defined.

[0113] As used herein in the context of oligonucleotide sequences, "A" represents a nucleoside containing the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside containing the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside containing the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside containing the base cytosine (e.g., cytidine or a chemically modified derivative thereof).

[0114] 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. Other exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, nucleothymidine, 2'-O-methylguanosine, and N2,N2-dimethylguanosine (also referred to as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups linked to the sugar moiety by an ester bond. Exemplary nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked together by phosphodiester or phosphorothioate bonds between the 5' and 3' carbon atoms.

[0115] The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30 or more ribonucleotides). The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or DNA transcription, respectively). RNA can be post-transcriptionally modified. 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" refers to single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. During protein synthesis, this information is translated when ribosomes bind to the mRNA.

[0116] As used herein, the term "small interfering RNA" ("siRNA") (also known in the art as "short interfering RNA") refers to an RNA (or RNA analog) comprising from about 10 to 50 nucleotides (or nucleotide analogs) that is capable of directing or mediating RNA interference. An siRNA is a duplex formed by a sense strand and an antisense strand having sufficient complementarity between the two strands to form the duplex. In certain embodiments, the siRNA comprises from about 15 to 30 nucleotides or nucleotide analogs, or from about 16 to 25 nucleotides (or nucleotide analogs), or from about 18 to 23 nucleotides (or nucleotide analogs), or from about 19 to 22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA comprising from about 21 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides). The term "long" siRNA refers to an siRNA comprising from about 24 to 25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. In some cases, a short siRNA may comprise fewer than 19 nucleotides, such as 16, 17, or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Similarly, in some cases, a long siRNA may comprise more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi without further processing (e.g., enzymatic processing) into a short siRNA.

[0117] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" or "chemically modified nucleotide" refers to a non-standard nucleotide, including a non-naturally occurring ribonucleotide or deoxyribonucleotide. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide, but still 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-propynyluridine, 5-allyluridine, etc.; 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, etc. Nucleotide analogs also include deazapurines, e.g., 7-deazaadenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, as known in the art) nucleotides; and other heterocyclic modified nucleotide analogs, e.g., those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0118] The nucleotide analogs can also include modifications to the nucleotide sugar moiety. For example, the 2'-OH group can be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH 2 , NHR, NR 2 or COOR, where R is a substituted or unsubstituted C 1 -C 6 alkyl, alkenyl, alkynyl, aryl, etc. Other modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438. In certain embodiments, the nucleotide analogs include a 2'-O-methyl modification. In certain embodiments, the nucleotide analogs include a 2'-fluoro modification.

[0119] The phosphate group of the nucleotide can also be modified, for example, by replacing one or more of the oxygens of the phosphate group with sulfur (such as phosphorothioate), or by making other substitutions that allow the nucleotide to perform its intended function, such as those described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. April 2000, 10(2):117-21, Rusckowski et al., Antisense Nucleic Acid Drug Dev. October 2000, 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. October 2001, 11(5):317-25, Vorobjev et al., Antisense Nucleic Acid Drug Dev. April 2001, 11(2):77-85 and U.S. Patent No. 5,684,143. Some of the above-mentioned modifications (such as phosphate group modifications) reduce the hydrolysis rate of, for example, polynucleotides containing such analogs in vivo or in vitro.

[0120] The term "oligonucleotide" refers to short polymers of nucleotides and / or nucleotide analogs. The term "oligonucleotide" includes, but is not limited to, antisense oligonucleotides (ASO), siRNA, and microRNA.

[0121] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one altered or modified nucleotide compared to the corresponding unaltered or unmodified RNA, but retains the same or similar properties or functions as the corresponding unaltered or unmodified RNA. As discussed above, oligonucleotides can be linked by bonds that result in a lower hydrolysis rate for the RNA analog compared to an RNA molecule with phosphodiester bonds. For example, the nucleotides of the analog can contain methylene glycol, ethylene glycol, oxymethylthio, oxyethylthio, oxycarbonyloxy, diamidophosphate, amidophosphate, and / or phosphorothioate bonds. Some RNA analogs include ribonucleotides and / or deoxyribonucleotides with sugar and / or backbone modifications. Such alterations or modifications can further include the addition of non-nucleotide materials, such as addition to the ends or within the RNA (at one or more nucleotides of the RNA). The RNA analog only needs to be similar enough to natural RNA such that it has the ability to mediate RNA interference.

[0122] As used herein, the term "RNA interference" ("RNAi") refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove foreign RNA (such as viral RNA). Natural RNAi proceeds via fragments cleaved from free dsRNA that direct the degradation machinery to other similar RNA sequences. Alternatively, RNAi can be initiated artificially, for example to silence the expression of a target gene.

[0123] An RNAi agent having a strand that is "sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)", such as an RNA silencing agent, means that the strand has a sequence sufficient to trigger the destruction of the target mRNA via the RNAi mechanism or process.

[0124] As used herein, the term "isolated RNA" (such as "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material, or medium, when produced by recombinant techniques, or substantially free of chemical precursors or other compounds when chemically synthesized.

[0125] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (such as RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational inhibition), resulting in the suppression or "silencing" of the expression of the corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0126] The term "in vitro" has its generally recognized meaning in the art, e.g., relating to purified reagents or extracts, such as cell extracts. The term "in vivo" also has its generally recognized meaning in the art, e.g., relating to living cells, such as immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0127] As used herein, a "target" refers to a specific nucleic acid sequence (e.g., a gene, mRNA, miRNA, etc.) to which the oligonucleotide conjugates or branched oligonucleotides of the present disclosure bind and / or otherwise affect its expression. In certain embodiments, the target is expressed in the eye. In certain embodiments, the target is expressed in specific eye cells. In other embodiments, the target is associated with a particular disease or disorder of a subject.

[0128] As used herein, the term "target gene" is a gene whose expression will be substantially inhibited or "silenced". Such silencing can be achieved by RNA silencing, e.g., by cleavage of the mRNA of the target gene or translational inhibition of the target gene. The term "non-target gene" is a gene whose expression will not be 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 gene and the non-target gene can differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target gene and the non-target gene can share less than 100% sequence identity. In another embodiment, the non-target gene can be a homolog (e.g., an ortholog or a paralog) of the target gene.

[0129] As used herein, the term "RNA silencing agent" refers to an RNA that is capable of inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing the 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 comprising paired strands, and precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include siRNA, miRNA, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and bifunctional oligonucleotides, as well as their precursors. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational inhibition.

[0130] As used herein, the term "rare nucleotide" refers to a naturally occurring nucleotide that occurs infrequently, including a naturally occurring deoxyribonucleotide or ribonucleotide that occurs infrequently, such as 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, 2'-O-methylguanosine, and 2,2',N,N'-dimethylguanosine.

[0131] As used herein, the term "engineered" in an engineered RNA precursor or an engineered nucleic acid molecule means that the precursor or molecule does not exist in nature because all or part of the nucleic acid sequence of the precursor or molecule is produced or selected by a human. Once the sequence is produced or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by intracellular mechanisms. Thus, an RNA precursor produced intracellularly from a transgene comprising an engineered nucleic acid molecule is an engineered RNA precursor.

[0132] As used herein, the term "microRNA" ("miRNA"), also known in the art as "small temporal RNA" ("stRNA"), refers to a small (10-50 nucleotides) RNA that is genetically encoded (e.g., by a viral, mammalian, or plant genome) and capable of directing or mediating RNA silencing. "miRNA disorder" shall mean a disease or disorder characterized by abnormal expression or activity of a miRNA.

[0133] As used herein, the term "bifunctional oligonucleotide" refers to an RNA silencer having the formula T-L-μ, where T is an mRNA targeting moiety, L is a linking moiety, and μ is a miRNA recruitment moiety. As used herein, the terms "mRNA targeting moiety", "targeting moiety", "mRNA targeting portion", or "targeting portion" refer to a domain, portion, or region of a bifunctional oligonucleotide that has sufficient size and sufficient complementarity to a portion or region of an mRNA selected or targeted for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA).

[0134] As used herein, the term "linking moiety" or "linking portion" refers to a domain, portion, or region of an RNA silencer that covalently attaches or links to an mRNA.

[0135] As used herein, the "antisense strand" of a term RNA silencing agent (e.g., siRNA) refers to a strand that is substantially complementary to a portion of about 10-50 nucleotides (e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides) of the mRNA of the 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, such as complementarity sufficient to trigger the RNAi mechanism or process (RNAi interference) to disrupt the desired target mRNA or complementarity sufficient to trigger translational inhibition of the desired target mRNA.

[0136] The term "sense strand" or "second strand" of an RNA silencing agent such as siRNA or an RNA silencing agent refers to a strand that is complementary to the antisense strand or first strand. The antisense and sense strands may also be referred to as the first or second strand, the first or second strand has complementarity with the target sequence, and the corresponding second or first strand has complementarity with the first or second strand. The miRNA duplex intermediate or siRNA-like duplex includes a miRNA strand having sufficient complementarity to a portion of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA* strand having sufficient complementarity to form a duplex with the miRNA.

[0137] As used herein, the term "guide strand" refers to the strand of an RNA silencing agent, such as the antisense strand of an siRNA duplex or siRNA sequence, that enters the RISC complex and directs cleavage of the target mRNA.

[0138] As used herein, the term "asymmetric", such as in the asymmetry of an RNA silencing agent duplex region (e.g., the stem of an shRNA), refers to an inequality in the bond strength or base pairing strength between the ends of an RNA silencing agent (e.g., between the terminal nucleotides on the first strand or stem portion and the terminal nucleotides on the opposing second strand or stem portion), such that the 5' end of one strand of the duplex is more frequently in a transiently unpaired state, such as a single-stranded state, than the 5' end of the complementary strand. This structural difference determines which strand of the duplex is preferentially incorporated into the RISC complex. The strand with the less tightly paired 5' end will be preferentially incorporated into the RISC and mediate RNAi.

[0139] As used herein, the terms "bond strength" or "base pair strength" refer to the strength of the interaction between paired nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), which is primarily due to hydrogen bonding, van der Waals interactions, etc. between the nucleotides (or nucleotide analogs).

[0140] As used herein, the "5'-end" in the 5'-end of an antisense strand refers to the 5'-terminal nucleotide in the 5'-end of the antisense strand, such as between 1 and about 5 nucleotides. As used herein, the "3'-end" in the 3'-end of an antisense strand refers to the region complementary to the nucleotide at the 5'-end of the complementary antisense strand, such as a region between 1 and about 5 nucleotides.

[0141] As used herein, the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analogue that is capable of forming a base pair with a second nucleotide or nucleotide analogue such that the bond strength of the base pair is lower than that of a conventional base pair (i.e., a Watson-Crick base pair). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatched base pair with a second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with a second nucleotide. In still other embodiments, the destabilizing nucleotide is capable of forming a fuzzy base pair with a second nucleotide.

[0142] As used herein, the term "base pair" refers to the interaction between paired nucleotides (or nucleotide analogues) on opposite strands of an oligonucleotide duplex (such as a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), which is mainly due to hydrogen bonding, van der Waals interactions, etc. between the nucleotides (or nucleotide analogues). As used herein, the term "bond strength" or "base pair strength" refers to the strength of a base pair.

[0143] As used herein, the term "mismatched base pair" refers to a base pair composed of non-complementary or non-Watson-Crick base pairs, for example, an abnormal complementary G:C, A:T or A:U base pair. As used herein, the term "fuzzy base pair" (also referred to as a non-discriminating base pair) refers to a base pair formed by universal nucleotides.

[0144] As used herein, the term "universal nucleotide" (also referred to as "neutral nucleotide") includes those nucleotides (such as certain destabilizing nucleotides) that have a base ("universal base" or "neutral base") that does not significantly discriminate between bases on a complementary polynucleotide when forming a base pair. Universal nucleotides are mainly hydrophobic molecules, and due to stacking interactions, they can effectively assemble into an antiparallel double-stranded nucleic acid (such as double-stranded DNA or RNA). The base portion of a universal nucleotide usually contains a nitrogen-containing aromatic heterocyclic moiety.

[0145] As used herein, the term "sufficient complementarity" or "sufficient degree of complementarity" means that an RNA silencing agent has a sequence (such as in an antisense strand, an mRNA targeting portion or an miRNA recruitment portion) sufficient to bind to a desired target RNA accordingly and trigger RNA silencing of the target mRNA.

[0146] As used herein, the term "translational repression" refers to the selective inhibition of mRNA translation. Natural translational repression occurs via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated artificially, such as to silence the expression of a target gene.

[0147] The various methods of the present disclosure include steps involving comparing a value, level, feature, characteristic, property, etc. to a "suitable control" that can be interchangeably referred to herein as an "appropriate control". A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art for comparison purposes. In one embodiment, the "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing the RNAi method as described herein. For example, the transcription rate, mRNA level, translation rate, protein level, biological activity, cell feature or property, genotype, phenotype, etc. can be determined prior to introducing the RNA silencing agent of the present disclosure into a cell or organism. In another embodiment, the "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism (such as a control or normal cell or organism) that exhibits, for example, normal characteristics. In yet another embodiment, the "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

[0148] Oligonucleotide conjugate

[0149] The oligonucleotide conjugates described herein comprise an oligonucleotide linked to a functional moiety. The functional moiety provides enhanced ocular delivery of the oligonucleotide, including ocular cell-specific delivery.

[0150] In one aspect, the present disclosure provides a method for delivering an oligonucleotide conjugate to the eye of a subject, the method comprising administering the oligonucleotide conjugate to the subject, wherein the oligonucleotide conjugate comprises: i) an oligonucleotide having 5' and 3' termini and complementary to a target nucleic acid (such as a target gene or target mRNA); and ii) a functional moiety linked to the oligonucleotide, wherein the functional moiety comprises any one of triamine, retinoic acid (RA), docosahexaenoic acid (DHA), docosanoic acid (DCA), alpha-tocopherol succinate (TS), or lithocholic acid (LA).

[0151] The structure of each of the functional moieties described above is shown in the figures below. The functional moieties can have different isomeric configurations than those presented in the present disclosure.

[0152]

[0153] (TS)

[0154]

[0155] In certain embodiments, two DHA functional moieties are linked to an oligonucleotide.

[0156] In certain embodiments, the oligonucleotide comprises an antisense oligonucleotide or siRNA.

[0157] In certain embodiments, the siRNA comprises a sense strand and an antisense strand. In certain embodiments, the antisense strand has a length of about 15 to 25 nucleotides (e.g., a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). In certain embodiments, the antisense strand has a length of 20 nucleotides, a length of 21 nucleotides, or a length of 22 nucleotides. In certain embodiments, the sense strand has a length of about 15 to 25 nucleotides (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). In certain embodiments, the sense strand has a length of 15 nucleotides, a length of 16 nucleotides, a length of 18 nucleotides, or a length of 20 nucleotides.

[0158] In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs 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 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

[0159] In certain embodiments, the siRNA comprises at least one blunt end. In certain embodiments, the siRNA comprises two blunt ends.

[0160] 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 a single-stranded nucleotide overhang of about 2 to 5 nucleotides (e.g., an overhang of 2, 3, 4, or 5 nucleotides). In certain embodiments, the siRNA comprises a single-stranded nucleotide overhang of 2 nucleotides or a single-stranded nucleotide overhang of 5 nucleotides.

[0161] In certain embodiments, the siRNA comprises naturally occurring nucleotides (i.e., unmodified ribonucleotides).

[0162] In certain embodiments, the siRNA comprises at least one modified nucleotide. In certain embodiments, the modified nucleotides include 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 comprising non-natural bases, or mixtures thereof.

[0163] In certain embodiments, the siRNA comprises at least one modified internucleotide bond. In certain embodiments, the modified internucleotide bonds include phosphorothioate internucleotide bonds. In certain embodiments, the siRNA comprises 4-16 phosphorothioate internucleotide bonds. In certain embodiments, the siRNA comprises 8-13 phosphorothioate internucleotide bonds.

[0164] 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 fully chemically modified.

[0165] 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% of 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% of 2'-O-methyl nucleotide modifications). In certain embodiments, the antisense strand comprises about 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% of 2'-O-methyl nucleotide modifications). In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0166] In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

[0167] In certain embodiments, the antisense strand comprises a 5'-phosphate, 5'-alkylphosphonate, 5'-alkylenephosphonate or 5'-alkenylphosphonate. In certain embodiments, the antisense strand comprises a 5'-vinylphosphonate.

[0168] In certain embodiments, the functional moiety is attached to the 5'-end and / or the 3'-end of the oligonucleotide. In certain embodiments, the functional moiety is attached to the 5'-end and / or the 3'-end of the sense strand or attached to the 5'-end and / or the 3'-end of the antisense strand. In certain embodiments, the functional moiety is attached to the 3'-end of the sense strand.

[0169] In certain embodiments, the functional moiety is attached to the antisense strand and / or the sense strand via a linker.

[0170] In certain embodiments, the linker comprises a divalent or trivalent linker.

[0171] In certain embodiments, the divalent or trivalent linker is selected from the group consisting of:

[0172]

[0173] where n is 1, 2, 3, 4, or 5.

[0174] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, an aminophosphate, an amide, a carbamate, or a combination thereof.

[0175] In certain embodiments, when the linker is a trivalent linker, the linker is further linked to a phosphodiester or a phosphodiester derivative.

[0176] In certain embodiments, the phosphodiester or the phosphodiester derivative is selected from the group consisting of:

[0177]

[0178]

[0179] and

[0180]

[0181] where X is O, S, or BH 3 。

[0182] The partial Zc1 described above is phosphatidylcholine (PC). Any of the functional moieties described herein may include derivatives esterified with phosphatidylcholine (PC), namely phosphatidylcholine (PC)-esterified triamine (PC-triamine), phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), phosphatidylcholine (PC)-esterified docosahexaenoic acid (PC-DHA), phosphatidylcholine (PC)-esterified docosanoic acid (PC-DCA), phosphatidylcholine (PC)-esterified alpha-tocopherol succinate (PC-TS), phosphatidylcholine (PC)-esterified lithocholic acid (PC-TS).

[0183] In certain embodiments, the nucleotides at positions 1 and 2 at the 3'-end of the sense strand, and the nucleotides at positions 1 and 2 at the 5'-end of the antisense strand, are linked to adjacent ribonucleotides via phosphorothioate bonds.

[0184] In certain embodiments, the oligonucleotide conjugate comprises the following structure:

[0185]

[0186]

[0187]

[0188] For any of the above structures, the term "oligonucleotide" corresponds to any of the oligonucleotides described herein, such as an ASO or siRNA. In certain embodiments, the term "oligonucleotide" in the above structure corresponds to the sense strand of an siRNA. In certain embodiments, the oxygen adjacent to the term "oligonucleotide" in the structure is attached to the 3' end of the sense strand of the siRNA.

[0189] Di-DHA oligonucleotide conjugate

[0190] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having 5' and 3' ends and complementary to a target nucleic acid; and ii) a di-docosahexaenoic acid (di-DHA) functional moiety attached to the oligonucleotide.

[0191] In certain embodiments, the di-DHA functional moiety is phosphatidylcholine (PC)-esterified di-DHA (PC-di-DHA).

[0192] In certain embodiments, the oligonucleotide conjugate has the following structure:

[0193]

[0194]

[0195] In certain embodiments, the oligonucleotide corresponds to an antisense oligonucleotide or siRNA.

[0196] In certain embodiments, the siRNA comprises a sense strand and an antisense strand.

[0197] In certain embodiments, the functional moiety (i.e., di-DHA or PC-di-DHA) is attached to the 5' end and / or 3' end of the sense strand or attached to the 5' end and / or 3' end of the antisense strand. In certain embodiments, the functional moiety is attached to the 3' end of the sense strand.

[0198] Triamine oligonucleotide conjugate

[0199] In one aspect, the present disclosure provides an oligonucleotide conjugate comprising: i) an oligonucleotide having 5' and 3' ends and complementary to a target nucleic acid; and ii) a triamine functional moiety attached to the oligonucleotide.

[0200] In certain embodiments, the triamine functional moiety is phosphatidylcholine (PC)-esterified triamine (PC-triamine).

[0201] In certain embodiments, the oligonucleotide conjugate comprises the following structure:

[0202]

[0203]

[0204] In certain embodiments, the oligonucleotide corresponds to an antisense oligonucleotide or siRNA.

[0205] In certain embodiments, the siRNA comprises a sense strand and an antisense strand.

[0206] In certain embodiments, the functional moiety (i.e., triamine or PC-triamine) is attached to the 5'-end and / or 3'-end of the sense strand or attached to the 5'-end and / or 3'-end of the antisense strand. In certain embodiments, the functional moiety is attached to the 3'-end of the sense strand.

[0207] Branched oligonucleotide

[0208] The branched oligonucleotides described herein comprise two or more oligonucleotides linked together. The different branched oligonucleotides described herein (e.g., branched oligonucleotides having two, three, or four oligonucleotides) enhance the ocular delivery of oligonucleotides, including ocular cell-specific delivery.

[0209] In one aspect, the present disclosure provides a method for delivering a branched oligonucleotide to the eye of a subject, the method comprising administering the branched oligonucleotide to the subject, wherein the branched oligonucleotide comprises two or more oligonucleotides, each oligonucleotide comprising 5'- and 3'-ends and being complementary to a target nucleic acid.

[0210] In certain embodiments, one or more of the oligonucleotides in the branched oligonucleotide further comprises a functional moiety attached to the oligonucleotide, wherein the functional moiety comprises any one of triamine, retinoic acid, DHA, DCA, α-tocopherol succinate, or LA. The functional moieties as described in the oligonucleotide conjugate portion above can be applied to the oligonucleotides of the branched oligonucleotide. Similarly, the oligonucleotides described above in the oligonucleotide conjugate portion can serve as the oligonucleotides of the branched oligonucleotide, including type (ASO or siRNA), strand length, and chemical modification.

[0211] In certain embodiments, two or more of the oligonucleotides in the branched oligonucleotide are interconnected by one or more moieties independently selected from linkers, spacers, and branch points.

[0212] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate ester, a phosphonate ester, an aminophosphate ester, an ester, an amide, a triazole, or a combination thereof.

[0213] In certain embodiments, the branch point comprises a multivalent organic substance or a derivative thereof.

[0214] In another embodiment, the branch point is an amino acid derivative. In another embodiment, the branch point is selected from the following formulas:

[0215]

[0216] A multivalent organic substance is a moiety containing carbon and three or more valences (i.e., attachment points to moieties such as S, L, or N as defined above). Non-limiting examples of multivalent organic substances include triols (such as glycerol, phloroglucinol, etc.), tetrols (such as ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tricarboxylic acids (such as citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimellitic acid, etc.), tetracarboxylic acids (such as ethylenediaminetetraacetic acid, pyromellitic acid, etc.), tertiary amines (such as triallylamine, triethanolamine, etc.), triamines (such as diethylenetriamine, etc.), tetraamines, and substances containing a combination of hydroxyl, thiol, amino, and / or carboxyl moieties (such as amino acids, such as lysine, serine, cysteine, etc.).

[0217] In certain embodiments, the spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate ester, a phosphonate ester, an aminophosphate ester, an ester, an amide, a triazole, or a combination thereof.

[0218] In certain embodiments, the linker comprises structure L1:

[0219]

[0220] In certain embodiments, the linker comprises structure L2:

[0221]

[0222] 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 siRNA.

[0223] In certain embodiments, the branched oligonucleotide comprises the following structure:

[0224]

[0225]

[0226] For any of the above structures, the term "oligonucleotide" corresponds to any of the oligonucleotides described herein, such as an ASO or siRNA. In certain embodiments, the term "oligonucleotide" in the above structure corresponds to the sense strand of an siRNA. In certain embodiments, the oxygen adjacent to the term "oligonucleotide" in the structure is linked to the 3' end of the sense strand of the siRNA.

[0227] Branched oligonucleotides, including methods of synthesis and use, are described in more detail in WO2017 / 132669, which is incorporated herein by reference. More details regarding synthesis are provided in the Materials and Methods section of the Examples.

[0228] Method for delivering to eye cells

[0229] The oligonucleotide conjugates and branched oligonucleotides described herein are capable of achieving ocular cell-specific delivery and effectively silencing target genes. Any given oligonucleotide conjugate and branched oligonucleotide can be effectively delivered to more than one type of ocular cell.

[0230] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is administered by intravitreal injection.

[0231] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is delivered to ocular cells after administration to a subject.

[0232] In certain embodiments, the ocular cells are selected from the group consisting of Müller glia cells, rod photoreceptor cells, cone photoreceptor cells, ganglion cells, amacrine cells, bipolar cells, and horizontal cells.

[0233] In certain embodiments, the ocular cells are selected from the group consisting of ocular cells that express glutamine synthetase (GS), ocular cells that express rhodopsin, ocular cells that express cone arrestin (CA), ocular cells that express Vglut2, ocular cells that express VGAT, ocular cells that express protein kinase Cα (PKCa), and ocular cells that express Lim1.

[0234] In certain embodiments, the oligonucleotide conjugate has a selective affinity for retinal proteins.

[0235] In certain embodiments, the eye cell is a Müller glial cell and: i) the oligonucleotide conjugate comprises DHA, DCA, alpha-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments of Müller glial cell delivery, DHA, alpha-tocopherol succinate, and LA are phosphatidylcholine (PC)-esterified DHA (PC-DHA), alpha-tocopherol succinate (PC-TS), and LA (PC-LA).

[0236] In certain embodiments, the eye cell is a rod photoreceptor cell and: i) the oligonucleotide conjugate comprises DCA; or ii) the branched oligonucleotide consists of three or four oligonucleotides.

[0237] In certain embodiments, the eye cell is a cone photoreceptor cell and: i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, alpha-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments of cone photoreceptor cell delivery, retinoic acid and alpha-tocopherol succinate are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA) and alpha-tocopherol succinate (PC-TS). In certain embodiments of cone photoreceptor cell delivery, the oligonucleotide conjugate comprises two DHA functional moieties.

[0238] In certain embodiments, the eye cell is a ganglion cell and the oligonucleotide conjugate comprises alpha-tocopherol succinate. In certain embodiments of ganglion cell delivery, alpha-tocopherol succinate is phosphatidylcholine (PC)-esterified alpha-tocopherol succinate (PC-TS).

[0239] In certain embodiments, the eye cell is an amacrine cell and: i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, alpha-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments of amacrine cell delivery, retinoic acid, DHA, alpha-tocopherol succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), alpha-tocopherol succinate (PC-TS), and LA (PC-LA). In certain embodiments of amacrine cell delivery, the oligonucleotide conjugate comprises two DHA functional moieties or two PC-DHA functional moieties.

[0240] In certain embodiments, the eye cell is a bipolar cell, and: i) the oligonucleotide conjugate comprises triamine, retinoic acid, DHA, DCA, α-tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides. In certain embodiments of bipolar cell delivery, retinoic acid, DHA, α-tocopherol succinate, and LA are phosphatidylcholine (PC)-esterified retinoic acid (PC-RA), DHA (PC-DHA), α-tocopherol succinate (PC-TS), and LA (PC-LA). In certain embodiments of bipolar cell delivery, the oligonucleotide conjugate comprises two DHA moieties or two PC-DHA moieties.

[0241] In certain embodiments, the eye cell is a horizontal cell, and: i) the oligonucleotide conjugate comprises DCA; or ii) the branched oligonucleotide consists of two oligonucleotides.

[0242] Gene silencing method / Therapeutic method

[0243] The oligonucleotide conjugates and branched oligonucleotides described herein are capable of silencing target genes (i.e., target nucleic acids) in the eye and within specific eye cells.

[0244] In certain embodiments, the expression of the target nucleic acid is reduced by at least 20%, at least 30%, at least 40%, or at least 50% in the eye cell.

[0245] In certain embodiments, in eye 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, the expression of the target nucleic acid is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0246] In certain embodiments, in eye cells selected from the group consisting of eye cells expressing glutamine synthetase (GS), eye cells expressing rhodopsin, eye cells expressing cone arrestin (CA), eye cells expressing Vglut2, eye cells expressing VGAT, eye cells expressing protein kinase Cα (PKCa), and eye cells expressing Lim1, the expression of the target nucleic acid is reduced by at least 20%, at least 30%, at least 40%, or at least 50%.

[0247] 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 a branched oligonucleotide described herein, thereby treating the ocular disorder.

[0248] In certain embodiments, administering the oligonucleotide conjugate or the branched oligonucleotide results in treatment of the ocular disorder in the subject.

[0249] In certain embodiments, administration of an oligonucleotide conjugate or branched oligonucleotide results in a decrease in the expression of a gene from a target nucleic acid associated with an ocular disorder in a subject.

[0250] In certain embodiments, the oligonucleotide conjugate or branched oligonucleotide is administered by intravitreal injection.

[0251] 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.

[0252] Examples

[0253] Materials and methods

[0254] Synthesis of lipid-functionalized solid support

[0255] Non-phosphocholine (PC) lipid moieties (except α-tocopherol succinate) are directly attached via a peptide bond to controlled pore glass (CPG) functionalized with a C7 linker as previously described (Nika n 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. Bio conjug. Chem. 2017;28:758–1766). Briefly, Fmoc-L-serine tert-butyl (TCIA merica) was phosphitylated using 2′-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (ChemGenes). The resulting phosphoramidite was coupled to choline tosylate (Alfa Aesar) using 5-(ethylthio)-1H-tetrazole (ETT) as an activator. The phosphite 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 attached via a peptide bond to amino C7 CPG to form phosphocholine-functionalized CPG. The Fmoc group was removed, and the selected lipid moiety was attached via a peptide bond to the CPG. All lipid-functionalized solid supports with a loading of 55 μmol / g were obtained.

[0256] Synthesis of α-tocopherol succinate-conjugated oligonucleotide

[0257] Attach α-tocopherol succinate to the amino group at the 3'-end of the purified oligonucleotide synthesized on amino C7 CPG or phosphocholine-functionalized amino C7 CPG. Combine N-hydroxysuccinimide α-tocopherol succinate and the purified oligonucleotide in a 0.1 M sodium bicarbonate, 20% (v / v) dimethylformamide solution and incubate overnight at room temperature. Add one-tenth volume of 3 M sodium acetate (pH 5.2) to obtain a final concentration of 0.3 M sodium acetate. Add three volumes of 95% (v / v) ethanol and vortex the mixture, then place at -80 °C for 1 hour. Centrifuge the solution at 5200 × g for 30 minutes to precipitate. Dissolve the precipitate containing the lipid-conjugated siRNA sense strand in water and purify and desalt as described below.

[0258] Oligonucleotide synthesis

[0259] Oligonucleotides were synthesized by phosphoramidite solid-phase synthesis on a Dr Oligo 48 (Biolytic, Fremont, CA) or MerMade12 (Biosearch Technologies, Novato, CA) using phosphoramidites modified with 2'-F or 2'-O-Me with standard protecting groups. The 5'-(E)-vinyltetraphosphonate (pivaloyloxymethyl) 2'-O-methyl-uridine 3'-CE phosphoramidite (VP) for in vivo unconjugated oligonucleotides was purchased from Hongene Biotech (Hongene Biotech, USA), and the Quasar 570CE phosphoramidite (Cy3) was purchased from GenePharma (GenePharma, Shanghai, China). The bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite (5'P) for in vitro unconjugated oligonucleotides and all other phosphoramidites used were purchased from ChemGenes (ChemGenes, Wilmington, MA). The phosphoramidites were prepared at 0.1 M in anhydrous acetonitrile (ACN), except that the 2'-O-methyl-uridine phosphoramidite was dissolved in anhydrous ACN containing 15% dimethylformamide. 5-(Benzylthio)-1H-tetrazole (BTT) was used as an activator at 0.25 M, and all phosphoramidites were used with 10 eq for a coupling time of 4 minutes. Deprotection of the trityl group was carried out 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-dimethylpyridine in ACN). The reagents for capping and deprotection were purchased from AIC (AIC, Framingham, MA). The oxidation of phosphite to phosphate or phosphorothioate was carried out using a 0.05 M solution of iodine in pyridine-H 2 O (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) for 4 minutes. Unconjugated oligonucleotides were synthesized on a long-chain alkylamine (LCAA) controlled pore glass (CPG) (ChemGenes) functionalized with Unylinker. In Cholesterol-conjugated oligonucleotides were synthesized on an LCA A-CPG support, where the cholesterol moiety was attached to tetra-ethylene glycol via a succinate linker (ChemGenes, Wilmington, MA). Lipid-conjugated oligonucleotides were synthesized on the modified solid support (synthesis as described above). Divalent oligonucleotides (dimers) were synthesized on the modified solid support, and the synthesis has been previously described (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)).

[0260] Branched oligonucleotide synthesis

[0261] Synthesis of branched oligonucleotides was performed by phosphoramidite solid phase on an AKTA Oligopilot 10 (Cytiva, Marlborough, MA) using the parameters described above or as otherwise specified herein. Commercial trebler and doublet phosphoramidites purchased from Glen Research (Glen Research, Sterling, VA) were used to prepare trimer and tetramer branched oligonucleotides, respectively. The trimer linker was produced in two steps as follows: First, 10 eq of DMT-tetraethoxy-ethylene glycol CED phosphoramidite (ChemGenes) was coupled to thymine 3'-LCAA-CPG (ChemGenes) for 8 minutes, and then 10 eq was coupled to the trebler phosphoramidite for 8 minutes. Subsequently, 30 eq was used to grow the trivalent oligonucleotide on this linker. The tetramer linker was produced in three steps as follows: First, 10 eq of DMT-tetraethoxy-ethylene glycol CED phosphoramidite was coupled to thymine 3'-LCAA-CPG for 8 minutes; second, 10 eq of the doublet phosphoramidite was coupled for 8 minutes; and third, subsequently 20 eq of the doublet phosphoramidite was coupled for 8 minutes. Subsequently, 40 eq was used to grow the tetravalent oligonucleotide. On thymine 3'-LCAA-CPG (ChemGenes), 10 eq of DMT-tetraethoxy-ethylene glycol CED phosphoramidite (ChemGenes) was coupled for 8 minutes, and then 10 eq was coupled to the trebler phosphoramidite for 8 minutes. Subsequently, 30 eq was used to grow the trivalent oligonucleotide on this linker. The tetramer linker was produced in three steps as follows: First, On thymine 3'-LCAA-CPG, 10 eq of DMT-tetraethoxy-ethylene glycol CED phosphoramidite was coupled for 8 minutes; second, 10 eq of the doublet phosphoramidite was coupled for 8 minutes; and third, subsequently 20 eq of the doublet phosphoramidite was coupled for 8 minutes. Subsequently, 40 eq was used to grow the tetravalent oligonucleotide.

[0262] Deprotection and purification of oligonucleotides for screening sequences

[0263] Before deprotection, the synthesis column containing the oligonucleotide was treated with 10% diethylamine (DEA) in ACN to deprotect the cyanoethyl groups. In the synthesis column, both the un-conjugated and cholesterol-conjugated oligonucleotides on the solid support were deprotected with methylamine gas (Airgas) for one hour at room temperature. The deprotected oligonucleotides released from the solid support were precipitated on the support by passing (i) a mixture solution of 0.1 M sodium acetate in 85% ethanol and then (ii) an 85% ethanol solution through the synthesis column. The excess ethanol on the solid support was dried by an air stream, and the oligonucleotides were flushed out by passing water through the column. This procedure provides pure oligonucleotides for in vitro experiments.

[0264] Deprotection and purification of oligonucleotides for in vivo experiments

[0265] Before deprotection, the synthesis column containing the oligonucleotide was treated with 10% diethylamine (DEA) in ACN to deprotect the cyanoethyl groups. The Cy3-labeled and lipid-conjugated oligonucleotides were cleaved and deprotected in 28 - 30% ammonium hydroxide, 40% aqueous methylamine (1:1, v / v) (AM A) for 2 hours at room temperature. The un-conjugated, divalent, trivalent, and tetravalent oligonucleotides, both Cy3-labeled and unlabeled, were cleaved and deprotected by AMA treatment at 45 °C for 2 hours. The oligonucleotides containing VP were not pretreated with DEA after synthesis but were cleaved and deprotected as described previously (O'Shea J, Theile CS, Das R et al., An efficient deprotection method for 5′-[O,O-bis(pivaloyloxymethyl)]-(E)-vinyl phosphonate containing oligonucleotides. Tetrahedron 74, 6182 - 6186 (2018)). Briefly, the CPG containing VP-oligonucleotides was treated with a solution of 3% DEA in 28 - 30% ammonium hydroxide at 35 °C for 20 hours.

[0266] All solutions containing cleaved oligonucleotides were filtered to remove CPG and dried under vacuum. The resulting precipitate was resuspended in water containing 5% 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 25x150 mm column packed with Source 15Q anion exchange resin (Cytiva, Marlborough, MA); running conditions: eluent A, 10 mM Tris-HCl buffer (pH 9) / aqueous solution containing 7.5% ACN; eluent B, 1 M sodium perchlorate / 10 mM Tris-HC buffer (pH 9) / aqueous solution containing 7.5% ACN; linear gradient, B from 12% to 35% in 40 minutes at 50 °C. Lipid-conjugated and Cy3-labeled oligonucleotides were purified using a 21.2x150 mm PRP-C18 column (Hamilton Co, Reno, NV); running conditions: eluent A, 50 mM sodium acetate (pH 6) / aqueous solution containing 5% ACN; eluent B, 100% acetonitrile; linear gradient, B from 15% to 60% in 40 minutes at 60 °C. The flow rate was 40 mL / min in both methods, and the peaks of unlabeled oligonucleotides were monitored at 260 nm and the peaks of labeled oligonucleotides were monitored at 550 nm. The Cy3-labeled oligonucleotides used a separate column to avoid cross-contamination. The fractions were analyzed by liquid chromatography mass spectrometry (LC-MS), the pure fractions were combined and dried under vacuum. The oligonucleotides were resuspended in 5% ACN and desalted by size exclusion using an isocratic method on a 25×250 mm custom column packed with Sephadex G-25 medium (Cytiva, Marlborough, MA) using HPLC grade water (Honeywell Chemicals, Charlotte, NC), and finally the oligonucleotides were lyophilized.

[0267] LC-MS analysis of oligonucleotides

[0268] The identity of the oligonucleotides was verified by performing LC-MS analysis using an Agilent 6530 Accurate-Mass Q-TOF under 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 for 5 min (unconjugated, divalent, trivalent, and tetravalent oligonucleotides); linear gradient 50–100% B for 5 min (lipid-conjugated and Cy3-labeled oligonucleotides); temperature, 60 °C; flow rate, 0.85 ml / min. LC peaks were monitored at 260 nm, and labeled oligonucleotides were monitored at 550 nm. MS parameters: source, electrospray ionization; ion polarity, negative mode; range, 100–3,200 m / z; scan rate, 2 spectra / sec; capillary voltage, 4,000; fragmentation voltage, 200 V; gas temperature, 325 °C.

[0269] In vivo experiments

[0270] As described above, intravitreal injections were performed in adult mice (Venkatesh A, Ma S, Langellotto F et al. Retinal gene delivery by rAAV and DNA electroporation. Curr Protoc Microbiol: 2013; Chapter 14: Unit 14D 14.). Using a FemtoJet from Eppendorf, injections were performed with a glass needle (Clunbury Scientific LLC; catalog number B100-58-50) at a constant pressure and an injection time of 300 psi and 1.5 s, respectively, to deliver approximately 2 μL of liquid into the vitreous. All concentrations were adjusted to obtain the required amount of siRNA using a 2 μL injection volume. Intravitreal injections in adult pigs were performed by injecting 100 μL of siRNA into the vitreous approximately 2-3 mm from the temporal side using an insulin injection needle. Anesthesia and euthanasia of pigs were performed by the animal medicine department according to standard procedures. Before injecting siRNA, the cornea was treated with proparacaine and ophthalmic Betadine. After injection, the eyes were rinsed with saline eye wash. The excised pig eyes and mouse eyes were processed as described below (Venkatesh A, Ma S, Langellotto F et al. Retinal gene delivery by rAAV and DNA electroporation. Curr Protoc Microbiol: 2013; Chapter 14: Unit 14D 14).

[0271] Example 1: siRNA Delivery for Treating Ocular Diseases.

[0272] Preliminary screening was performed on different siRNAs (all targeting the Htt gene with the same sequence) to study their distribution and knockdown efficiency in the eye.

[0273] It was difficult to determine cell distribution solely from the Cy3 tag. Therefore, a lower dose of the siRNA compound (0.1 nanomole) ( Figure 1 where the siRNA labeled with Cy3 is shown in red) was injected. Three days later, the tissue was dissociated, and Cy3-positive cells were sorted by FACS, and cell type-specific antibodies were used to determine which cell types could be enriched by which siRNA modification.

[0274] Figure 1All siRNAs were labeled with Cy3 and appear red, while glutamine synthetase (GS), which is specific to Müller glial cells, appears green, and nuclear DAPI appears blue. All siRNAs are visible across the entire retinal cross-section, but the cellular distribution varies slightly. Figure 1 The right side shows an example of each group, showing the entire retinal cross-section and its distribution across the retina: one half of the section shows nuclear DAPI, GS, and siRNA, and the other half shows only siRNA.

[0275] The overall goals were A) to determine the cellular distribution and B) to identify modifications that allow optimal entry into cone photoreceptors, rod photoreceptors, and Müller glial cells, as these three cell types are the most important cell types targeted in many retinal diseases. Figure 2 and 3 shows the enrichment results of siRNAs in different retinal cell types, arranged by cell type ( Figure 2 ) and by modification ( Figure 3 ). The results indicate that the monomeric configuration is suitable for cone photoreceptors, the tetramer for rod photoreceptors, and the dimer is most suitable for Müller glial cells. Other good Müller glial cell compounds are PC-TS, PC-DHA, and DCA.

[0276] Repeated injections and high magnification of a subset of these compounds showed that PC-TS accumulates well in Müller glial cells, while trimers and tetramers accumulate well in rod photoreceptors ( Figure 4 and 5 ).

[0277] To determine whether the tetramer could knockdown HTT protein in photoreceptor cells better than other configurations, antibody staining for HTT protein was performed two weeks after intravitreal injection of 0.3 nanomoles of siRNA ( Figure 6 shown). Figure 6 The first column in shows antibody staining in control mice injected with NTC-siRNA. HTT protein is expressed throughout the retina, particularly enriched in the inner segments (IS) of photoreceptor cells, the outer plexiform layer (OPL) (where photoreceptor cells make synaptic connections with bipolar cells and horizontal cells); moderately enriched in the inner nuclear layer (INL) (where bipolar cells, amacrine cells, horizontal cells, and Müller glial cells are located); and highly enriched in the inner plexiform layer (IPL) (where synaptic connections of amacrine cells, bipolar cells, and ganglion cells are located). The second column, HTT protein expression after knockdown with PC-RA-Htt siRNA. As Figure 2 and 3As shown, the siRNA tended to preferentially accumulate in bipolar cells and amacrine cells. Therefore, the use of this siRNA could more effectively reduce the expression in the OPL and IPL. The third column shows the HTT protein expression in two different mice, each mouse was injected with approximately 0.3 nanomoles of Htt-siRNA after knockdown with the tetramer-HttsiRNA. This configuration tended to be able to accumulate effectively in rod photoreceptor cells and bipolar cells. Consistent with this, the expression in the photoreceptor IS was reduced much more than with the PC-RA-Htt siRNA.

[0278] Two weeks after injection of Htt-siRNA, the same experimental setup ( Figure 6 used) was also used, with different mice from the same injection batch, and the remaining total HTT protein in the total retinal extract was quantified by Western blot. Note that when quantifying the total HTT protein, the knockdown at 2 weeks after injection was at approximately 50% relative expression. These two configurations targeted different cell populations in the retina with different efficiencies. Nevertheless, since HTT is ubiquitously expressed in the retina, the overall knockdown was similar.

[0279] Example 2: Evaluation of Htt mRNA knockdown by bDNA assay.

[0280] Two weeks after injection of Htt-siRNA (0.1 nanomole was used for each injection of the siRNA modification), the total Htt mRNA level was quantified by bDNA assay. As Figure 8 shown, when quantifying the Htt mRNA level, 0.1 nanomole resulted in approximately 20%-30% knockdown at 2 weeks after injection.

[0281] Three days after injection of Htt-siRNA (0.3 nanomole was used for each injection of the siRNA modification), the bDNA assay was also used to quantify the total Htt mRNA level. Note that when quantifying the Htt mRNA level, 0.3 nanomole resulted in approximately 30%-60% knockdown at 3 days after injection. In addition, when compared with the total protein measurement at 2 weeks after injection, PC-RA showed a similar percentage of knockdown ( Figure 7 : 60% knockdown), which indicates that for the Htt gene in the retina, the two quantification methods are similar, and there is a direct correlation between the mRNA and protein levels of this gene (each point represents 1 retina).

[0282] Example 3: Long-term evaluation of Htt mRNA knockdown by bDNA assay

[0283] Figure 10Shows the quantification results of total Htt mRNA levels quantified by bDNA assay 100 days after injection of Htt-siRNA (0.3 nanomoles of the siRNA modification were used per injection). Note that compared to Figure 9 the knockdown effect only changed by about 10% (from 60% to 50% knockdown) over a time window of approximately 100 days, indicating that the knockdown was very stable ( Figure 10 each point in represents 1 retina). Figure 11 Shows representative fundus images of eyes injected with Cy3-labeled siRNA with the modifications shown over time. The fluorescence signal exposure for all 4 siRNAs was the same at any given time point, but varied over time. Figure 11 Supplemented Figure 10 to show fundus images of the mice used in Figure 10 . All mice were injected intravitreally with 0.3 nanomoles of siRNA.

[0284] Example 4: Dose Escalation Study of HTT Knockdown in Mice with Tetramer Configuration

[0285] Figure 12 Shows the results of a dose escalation study of HTT knockdown in the retina. Mice were injected with the amounts shown in the figure (1 - 60 Cy3-labeled tetramers and Htt-siRNA) in a total volume of 2 microliters. Five mice were injected according to the siRNA amount. Tissues were collected two weeks after injection to quantify the remaining HTT protein in the retina by Western blot. Injecting 15 - 30 micrograms was roughly equivalent to the approximately 0.3 nanomole knockdown observed in previous experiments.

[0286] Figure 14 Shows cross-sections of the eye retina from a dose escalation study of mouse HTT knockdown with the tetramer configuration, the results of which are as shown in Figure 12 and 13 . The images show Cy3 distributed throughout the retinal cross-section, indicating uniform absorption of the siRNA throughout the eye.

[0287] To determine toxicity, antibody staining was performed on cross-sections of the eye retina shown in Figure 14 to identify Iba1-positive cells and changes in GFAP. Figure 15 Shows these retinal cross-sections of eyes stained with Iba1 (green) to identify Iba1-positive cells that migrated to the outer nuclear layer (ONL) where photoreceptor cells are located. Iba1-positive cells in the ONL were visible at 60 micrograms per injection and occasionally at 30 micrograms per injection, indicating an inflammatory response at 60 micrograms and a mild response at 30 micrograms. Half of each inset ( Figure 15The dashed lines (on [the figure]) only show the Iba1 signal to better visualize the signal. The nucleus DAPI is shown in blue. Figure 16 shows a retinal cross-section of the eye from Figure 14 the dose escalation study shown, stained with GFAP (red) to identify reactive gliosis in Müller glial cells. Although a slight increase in GFAP expression was visible at the level of the ganglion cell layer (GCL) where astrocytes are located, this expression did not extend upwards into the Müller glial cells. The expression of GFAP in astrocytes was normal. The increased expression was 60 micrograms, consistent with the results seen with Iba1. However, the absence of reactive gliosis indicates that siRNA does not induce severe retinal degenerative events. The siRNA is not shown because these are sections from the same eye as Figure 15 shown in [the figure]. In Figure 16 blue indicates the nuclear DAPI, and cone photoreceptor segments containing peanut agglutinin lectin (PNA) are marked in green. Figure 17 Presents the photoreceptor and retinal function measured by electroretinogram under scotopic (0.01 cd.s / m2 - 1 cd.s / m2) and photopic conditions (3 and 10 flashes). The a-wave and b-wave recordings respectively show normal photoreceptor and inner retina function for all injection amounts. As shown in the above two charts, there were no statistically significant differences between the recorded results (n = 5 mice / siRNA amount). There were also no significant differences in the implicit times of the a-wave and b-wave between the different groups of injected mice (the following two charts).

[0288] Example 5: Large animal model: siRNA in the eyes of suids (all data shown below were generated by the tetramer-Htt-siRNA-Cy3 and its NTC in suids)

[0289] For the translational purpose of siRNA technology, the applicability of this technology in a large animal model was tested to determine distribution, knockdown efficiency, and toxicity. For this purpose, a pig model was selected because the eye size of pigs is similar to that of humans (35 kg pigs were used). The only difference is the absence of a fovea. As a preliminary test run, 3 pigs were injected with 5 different amounts of siRNA with the same chemical configuration, keeping the injection volume constant at 100 microliters. The following data represents an overview of the injection of siRNA against HTT in the form of a tetramer 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 after intravitreal delivery of the tetramer-Htt-Cy3 in the porcine eye. Figure 18 The delivery amount of siRNA (100 - 1500 micrograms of the tetramer) is shown at the top of each small graph in [the figure]. The fluorescence intensity was evenly distributed throughout the eye.Figure 18 The top row shows the Cy3 fluorescence of unfixed tissue after opening the eyes, while the bottom inset is a high magnification of a region in the top inset.

[0290] Knockdown of huntingtin in swine was measured from the porcine eyes shown by Figure 18 western blot analysis. The knockdown was compared to the huntingtin protein levels in the NTC injected with 250 ug of tetramer-siRNA-Cy3. In Figure 19 , the top panel shows the knockdown plotted as bar graphs seen in the four major retinal quadrants (DT: dorsal temporal; DN: dorsal nasal; VT: ventral temporal; VN: ventral nasal), where the error bars are generated from technical replicates. The knockdown efficiency in each quadrant depends on the needle positioning and insertion angle. The needle is typically inserted from the temporal side and pointed towards the center of the eye. Figure 19 The middle inset of Figure 19 shows the knockdown on a flat-locked sketch, where the corresponding values of regional knockdown are shown in the bar graph. Figure 19 The bottom inset of

[0291] Figure 20 shows the average knockdown of huntingtin across the entire retina, which is derived by taking the mean of the knockdown in each quadrant of each retina, where the error bars are derived by taking the mean of 4 data points in each quadrant of each retina. Figure 19 The data shown in the bottom inset of Figure 19 represents one biological sample for each amount of siRNA delivered.

[0292] Figure 21 shows antibody staining of huntingtin on eye sections injected with different amounts as shown in Figure 18 and 19 . Antibody staining of GFAP (glial fibrillary acidic protein) and Iba1 (ionized calcium-binding adapter molecule 1) (as shown in the mice of Figure 15 and 16 ) was performed on eye retinal sections injected with different amounts as shown in Figure 21 to determine dose-dependent toxicity. Both GFAP and Iba1 are shown in green in Figure 21The red staining in shows the siRNA distribution on the retinal section, while the nuclei are labeled with nuclear DAPI. The expressions of GFAP and Iba1 increase significantly in a dose-dependent manner. The expression of IBA1 and GFAP siRNA rarely enters the outer nuclear layer (ONL) where photoreceptor cells are located at up to 500 μg. At 1000 μg and 1500 μg, the expressions of GFAP and Iba1 in the ONL increase significantly. In addition, many siRNAs seem to be absorbed by Iba1-positive cells, which reflects that macrophages may absorb excessive extracellular substances. Figure 21 Half of each small figure in only shows the target signals (siRNA, GFAP or Iba1) for better visualization of the signals.

[0293] Overview of porcine data: After a single intravitreal injection of the tetramer-Htt-siRNA into the large eyes of porcine animals, it is evenly distributed throughout the retina. This is particularly important because the porcine eye is similar in size to the human eye. Except for the lack of a fovea, the porcine eye is the animal model closest to the human eye. For distribution studies, it is more relevant than most laboratory NHPs due to its similar size. Figure 19 The dose response in and Figure 21 The toxicity in shows that for this specific compound, doses in the range of 100 - 500 μg can be used for further research. This should result in approximately 50% knockdown of HTT, similar to that seen with the tetramer in mice. The toxicity can be reduced by removing the Cy3 molecules still attached in the current study.

[0294] Example 6: Development of siRNA against S6K1 (RPS6KB1: Ribosomal Protein S6 Kinase B1)

[0295] A preliminary bioinformatics screening was conducted to identify potential siRNA sequences against S6K1. The list of sequences identified in the preliminary bioinformatics screening is shown in Tables 1 and 2 below.

[0296] Table 1 - Target sites of the 45-nucleotide gene region of S6K1

[0297]

[0298]

[0299] For the 45-nucleotide gene region described above, the sequence corresponds to the DNA gene sequence, but the mRNA encoded by the S6K1 gene will have the same sequence except that the T nucleotide is replaced by the U nucleotide. Thus, for example, an siRNA with an antisense strand targeting SEQ ID NO:1 will target the mRNA sequence corresponding to the gene region of SEQ ID NO:1.

[0300] Table 2 - S6K1 20 - nucleotide target sites

[0301]

[0302]

[0303] Table 3 - S6K1 sense strand

[0304]

[0305]

[0306] Table 4 - S6K1 sense strand

[0307]

[0308]

[0309]

[0310] For the sense and antisense sequences of 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 bond, "P" corresponds to a 5'-phosphate, and "TegChol" corresponds to a cholesterol moiety linked by three or four ethylene glycols.

[0311] Figure 22 The in vitro initial knockdown efficiency of the duplexes formed by the sense and antisense strands shown in Tables 3 and 4 is shown. The candidates with the best knockdown results are duplexes 2, 3, 7, 9, 10, and 19. Figure 23 is shown Figure 22 The dose - response curves of the 4 sequences highlighted in red in

[0312] The main purpose of the siRNA against S6K1 is to knockdown S6K1 in photoreceptor cells for the treatment of AMD. Based on the data generated by different HTT - siRNA conjugates, an siRNA in a tetrameric configuration without any Cy3 tags was initially developed to reduce toxicity. Then, based on the tetrameric configuration of duplex 2 (Rps6k1b_459), in vivo data from mice and NHP were generated.

[0313] Figure 24 RNA - Scope in situ hybridization performed on mouse retinal cross - sections to detect the siRNA - tetramer against S6K1 is shown. Figure 24The top row shows cross-sections from 3 mice injected intravitreally with NTC against S6K1 in a tetrameric configuration; the middle row shows cross-sections from 3 mice injected with siRNA against S6K1 in a tetrameric configuration at 3 μg / eye; and the bottom row shows cross-sections from 3 mice injected with siRNA against S6K1 in a tetrameric configuration at 6 μg / eye. The siRNA was delivered intravitreally, and the animals were euthanized 2 weeks after injection.

[0314] Figure 25A and Figure 25B shows knockdown of S6K1 after intravitreal injection of 6 μg of siRNA in a tetrameric configuration in mice. Both graphs in Figure 25 used rod TSC1− / − mice, which have been shown to develop pathologies similar to age-related macular degeneration. rod TSC1+ / + mice were used as Cre-negative littermate controls that do not develop pathologies. Figure 25A shows S6K1 protein levels detected by Western blot 2 weeks after injection. There was a slight trend towards decreased S6K1 protein compared to uninjected littermate or NTC mice. Figure 25B shows data similar to the first graph at 2 months after injection. Strong knockdown (40 - 45%) was seen with 6 μg siRNA. Each point in the graphs of Figure 25 represents a biological sample (retina) from one animal.

[0315] Figure 26 shows knockdown of S6K1 protein in non-human primates (NHPs). Western blot data using retinal protein extracts from the superotemporal (ST) region (also known as: dorsotemporal) of one NHP intravitreally injected with 225 μg of S6K1-tetramer (75 μL), and from 6 naïve NHPs from the same region. The first set of bar graphs shows a comparison between the uninjected contralateral eye and one eye injected with S6K1 siRNA to allow for a direct comparison of the two eyes within the animal. The second bar graph shows a comparison between 6 naïve NHPs and the NHP injected with S6K1 siRNA. In both cases, the knockdown efficiency of S6K1 was around 50%. NHP eyes were collected 1 month after injection. Also shown is a decrease in phosphorylation of ribosomal protein S6, a canonical target of S6K1. Similar to the S6K1 knockdown data, the comparison within the animal is shown on the left, and the comparison with several NHPs is shown on the right.

[0316] Figure 27 shows knockdown of S6K1 protein in cross-sections of non-human primate (NHP) retinas after siRNA treatment. The data is from one injected eye (also see Figure 26) and generated from the uninjected contralateral eye. As Figure 19 shown for the pig, the cross-sections were obtained from the central region. Figure 27 The entire cross-section covering the fovea is shown on the left side. Figure 27 A high magnification of the temporal and nasal regions and the fovea is shown on the right side. Figure 27 The top row shows the uninjected eye, and the bottom row shows the eye intravitreally injected with 225 μg of S6K1-tetramer (75 μL). Consistent with Figure 26 the Western blot data generated using the superotemporal region of the same eye presented in

[0317] Figure 28 a decrease in phosphorylated S6 protein (pS6) is shown on the retinal cross-section of non-human primates (NHP) after siRNA treatment. Figure 28 The data in Figure 27 are the same as those shown in Figure 28 except that the staining probe was used for the expression of pS6 (red signal). A significant decrease in pS6 is visible throughout the retina, especially in the photoreceptor cells (including foveal cones) as well. In Figure 28 each panel of

[0318] Figure 29 the green and blue signals have been removed from half of the panels (dashed lines) to better visualize the pS6 knockdown. Figure 29 The blue in Figure 27 and 28 shows nuclear DAPI, while the green shows the cone segments labeled with peanut agglutinin lectin (PNA). Figure 29 The data presented in Figure 29In each of the insets, the green and blue signals have been removed from half of the insets (dashed lines) to better visualize the Iba1 and GFAP signals. In Figure 29 , blue indicates nuclear DAPI while green indicates cone segments labeled with peanut agglutinin lectin (PNA).

[0319] Overview of S6K1-siRNA data. After a single intravitreal delivery of 6 μg of the tetramer-S6K1-siRNA in the mouse eye, it was able to distribute evenly throughout the retina. The knockdown efficiency initially appeared slow but became very robust over time. Therapeutically, achieving approximately 50% knockdown of S6K1 protein in photoreceptor cells seems achievable. Duplex 2 selected from the preliminary screening worked extremely efficiently in vivo, indicating that the S6K1 target site of SEQ ID NO:1 is a useful target for knocking down S6K1. Based on the dose-response curve of HTT-tetramer in mice, a group of mice will be injected with 25 μg / eye in the near future, and markers of S6K1 knockdown and age-related macular degeneration will be analyzed to determine if disease progression is improved. Injections in NHP confirmed that knockdown is equally effective in larger eyes, widely distributed, can reach the therapeutic range, and there is no severe inflammatory response to the treatment. Overall, the data indicate that human gene knockdown is feasible for treating various retinal diseases.

Claims

1. A method for delivering an oligonucleotide conjugate to the eye of a subject, the method comprising administering the oligonucleotide conjugate to the subject, wherein the oligonucleotide conjugate comprises: i) an oligonucleotide having a 5' end and a 3' end and complementary to a target nucleic acid; and ii) a functional moiety linked to the oligonucleotide, wherein the functional moiety comprises any one of triamine, retinoic acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), α-tocopherol 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, wherein the branched oligonucleotide comprises two or more oligonucleotides, each oligonucleotide having a 5' and a 3' end and being complementary to a target nucleic acid.

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

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

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

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

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

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

9. The method according to any one of claims 6-8, wherein the antisense strand has a length of 20 nucleotides, 21 nucleotides, or 22 nucleotides.

10. The method according to any one of claims 6-8, wherein the sense strand has a length of 15 nucleotides, 16 nucleotides, 18 nucleotides, or 20 nucleotides.

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

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

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

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

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

17. The method according to any one of claims 5-16, wherein the siRNA comprises naturally occurring nucleotides.

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

19. The method according to claim 18, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, an aminophosphate, a nucleotide containing a non-natural base, or a mixture thereof.

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

21. The method according to claim 20, wherein the modified internucleotide bond comprises a phosphorothioate internucleotide bond.

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

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

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

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

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

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

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

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

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

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

32. The method according to any one of claims 6-30, wherein the antisense strand comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, or a 5'-alkenylphosphonate.

33. The method according to claim 32, wherein the antisense strand comprises a 5'-vinylphosphonate.

34. The method according to any one of claims 1-33, wherein the functional moiety is attached to the 5'-end and / or the 3'-end of the sense strand.

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

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

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

38. The method according to claim 37, wherein the linker comprises a divalent or trivalent linker.

39. The method according to claim 38, wherein the divalent or trivalent linker is selected from the group consisting of: where n is 1, 2, 3, 4 or 5.

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

41. The method according to claim 38 or 39, wherein when the linker is a trivalent linker, the linker is further attached to a phosphodiester or a phosphodiester derivative.

42. The method according to claim 41, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of: and where X is O, S or BH 3 .

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

44. The method according to any one of claims 2 - 43, wherein two or more oligonucleotides in the branched oligonucleotide are interconnected by one or more moieties independently selected from linkers, spacers, and branch points.

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

46. The method according to claim 44, wherein the branch point comprises a multivalent organic substance or a derivative thereof.

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

48. The method according to claim 44, wherein the linker comprises structure L1:

49. The method according to claim 44, wherein the conjugate comprises structure L2:

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

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

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

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

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

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

56. The method according to claim 55, wherein the ocular cells are selected from the group consisting of Müller glial cells, rod photoreceptor cells, cone photoreceptor cells, ganglion cells, amacrine cells, bipolar cells, and horizontal cells.

57. The method according to claim 55, wherein the ocular cells are selected from the group consisting of ocular cells expressing glutamine synthetase (GS), ocular cells expressing rhodopsin, ocular cells expressing cone arrestin (CA), ocular cells expressing Vglut2, ocular cells expressing VGAT, ocular cells expressing protein kinase Cα (PKCa), and ocular cells expressing Lim1.

58. The method according to any one of claims 55 - 57, wherein the ocular cell is a Müller glial cell, and: i) the oligonucleotide conjugate comprises DHA, DCA, α - tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides.

59. The method according to claim 58, wherein the DHA, α - tocopherol succinate, and LA are phosphatidylcholine (PC) - esterified DHA (PC - DHA), dα - tocopherol succinate (PC - TS), and LA (PC - LA).

60. The method according to any one of claims 55 - 57, wherein 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.

61. The method according to any one of claims 55 - 57, wherein the ocular cell is a cone photoreceptor cell, and: i) the oligonucleotide conjugate comprises retinoic acid, DHA, DCA, α - tocopherol succinate, or LA; or ii) the branched oligonucleotide consists of two, three, or four oligonucleotides.

62. The method according to claim 61, wherein the retinoic acid and α - tocopherol succinate are phosphatidylcholine (PC) - esterified retinoic acid (PC - RA) and α - tocopherol succinate (PC - TS).

63. The method according to claim 61, wherein the oligonucleotide conjugate comprises two DHA functional moieties.

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

65. The method according to claim 64, wherein the α - tocopherol succinate is phosphatidylcholine (PC) - esterified α - tocopherol succinate (PC - TS).

66. The method according to any one of claims 55 - 57, wherein the ocular cell is an amacrine cell, and: i) The oligonucleotide conjugate comprises retinoic acid, DHA, DCA, alpha-tocopherol succinate, or LA; or ii) The branched oligonucleotide consists of two, three, or four oligonucleotides.

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

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

69. The method according to any one of claims 55-57, wherein the eye cell is a bipolar cell, and: i) The oligonucleotide conjugate comprises triamine, retinoic acid, DHA, DCA, alpha-tocopherol succinate, or LA; or ii) The branched oligonucleotide consists of two, three, or four oligonucleotides.

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

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

72. The method according to any one of claims 55-57, wherein the eye cell is a horizontal cell, and: i) The oligonucleotide conjugate comprises DCA; or ii) The branched oligonucleotide consists of two oligonucleotides.

73. The method according to any one of claims 1-72, wherein the oligonucleotide conjugate comprises the following structure:

74. The method according to any one of claims 1-72, wherein the branched oligonucleotide comprises the following structure:

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

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

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

78. The method according to any one of claims 1-77, wherein administering the oligonucleotide conjugate or the branched oligonucleotide results in treatment of the eye disorder in the subject.

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

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

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

82. The oligonucleotide conjugate according to claim 80, which comprises the following structure:

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

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

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

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

87. An oligonucleotide conjugate, which comprises: i) an oligonucleotide having a 5'-end and a 3'-end and complementary to a target nucleic acid; and ii) a triamine functional moiety linked to the oligonucleotide.

88. The oligonucleotide conjugate according to claim 87, wherein the triamine functional moiety is phosphatidylcholine (PC)-esterified triamine (PC-triamine).

89. The oligonucleotide conjugate according to claim 87, which comprises the following structure:

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

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

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

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

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