Oligonucleotides targeting S6K1

Abstract

Provided herein are oligonucleotides (e.g., siRNAs) that target S6K1, as well as methods for treating diseases associated with S6K1 expression.

Description

[Technical Field]

[0001] Related Applications This invention claims the benefit of U.S. Provisional Patent Application No. 63 / 412,092, filed September 30, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Ribosomal protein S6 kinase B1 (RPS6KB1), also known as S6K1, is a serine / threonine kinase that phosphorylates the S6 ribosomal protein to stimulate protein synthesis. S6K1 has been implicated in several pathologies, including cancer, obesity, diabetes and insulin resistance, and macular degeneration (e.g., age-related macular degeneration (AMD)).

[0003] Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in industrialized countries. The disease typically begins with the formation of drusen, lipoprotein-rich deposits that form between Bruch's membrane (BrM) and the retinal pigment epithelium (RPE) or between the RPE and photoreceptor (PR) outer segments. Twenty percent of individuals with drusen progress to an advanced stage of the disease, characterized by geographic atrophy (GA) or neovascular lesions of the RPE and underlying PR. The only available treatments to date involve neovascular lesions (also known as "wet AMD"), which involve the use of antiangiogenic antibodies to inhibit the action of vascular endothelial growth factor (VEGF). There are no treatments available to prevent progression from the early to advanced stages of the disease. There are also no treatments available for advanced GA (often referred to as "dry" AMD).

[0004] Oligonucleotides, such as small interfering RNA (siRNA) molecules, have been used to regulate gene expression levels across different organs. However, their application in the eye has been hindered by the low permeability of siRNA molecules to various cell types, the stability of siRNA, and the long-lasting knockdown effect. Herein, we describe oligonucleotides that are effective in silencing S6K1 expression. Summary of the Invention

[0005] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1 to 6 (i.e., the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6).

[0006] In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 7 to 12. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 8. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 10. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 12.

[0007] In certain embodiments, the siRNA comprises complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0008] In certain embodiments, the siRNA comprises no more than three mismatches with the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0009] In certain embodiments, the siRNA comprises perfect complementarity to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0010] In certain embodiments, the length of the antisense strand is about 15 to 25 nucleotides. In certain embodiments, the length of the sense strand is about 15 to 25 nucleotides. In certain embodiments, the length of the antisense strand is 20 nucleotides. In certain embodiments, the length of the antisense strand is 21 nucleotides. In certain embodiments, the length of the antisense strand is 22 nucleotides. In certain embodiments, the length of the sense strand is 15 nucleotides. In certain embodiments, the length of the sense strand is 16 nucleotides. In certain embodiments, the length of the sense strand is 18 nucleotides. In certain embodiments, the length of the sense strand is 20 nucleotides.

[0011] In certain embodiments, the siRNA comprises a double-stranded region of 15 to 20 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 16 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 18 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 20 base pairs.

[0012] In certain embodiments, the siRNA comprises at least one blunt end.

[0013] 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. In certain embodiments, the siRNA comprises a single-stranded nucleotide overhang of 5 nucleotides. In certain embodiments, the siRNA comprises naturally occurring nucleotides.

[0014] In certain embodiments, siRNA comprises at least one modified nucleotide.In certain embodiments, modified nucleotide comprises 2'-O-methyl modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, non-natural base containing nucleotide, or mixture thereof.

[0015] In certain embodiments, the siRNA comprises at least one modified internucleotide linkage. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the siRNA comprises 4 to 16 phosphorothioate internucleotide linkages. In certain embodiments, the siRNA comprises 8 to 13 phosphorothioate internucleotide linkages. In certain embodiments, the antisense strand comprises 2 to 10 phosphorothioate internucleotide linkages.

[0016] In certain embodiments, the siRNA comprises at least 80% chemically modified nucleotides, hi certain embodiments, the siRNA is fully chemically modified.

[0017] In certain embodiments, the siRNA comprises at least 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises at least 70% 2'-O-methyl nucleotide modifications.

[0018] In certain embodiments, the antisense strand contains about 70%-90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand contains at least 65% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand contains 100% 2'-O-methyl nucleotide modifications.

[0019] In certain embodiments, the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand. In certain embodiments, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.

[0020] In certain embodiments, the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate.

[0021] In certain embodiments, the antisense strand comprises a 5' vinyl phosphonate. In certain embodiments, the functional moiety is linked to the 5' and / or 3' end of the antisense strand. In certain embodiments, the functional moiety is linked to the 5' and / or 3' end of the sense strand. In certain embodiments, the functional moiety is linked to the 3' end of the sense strand.

[0022] In certain embodiments, the functional moiety comprises a hydrophobic moiety.

[0023] In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0024] In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LA).

[0025] In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

[0026] In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof.

[0027] In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0028] In certain embodiments, the functional moiety comprises any one of a triple amine, retinoic acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), alpha-tocopherol succinate, or lithocholic acid.

[0029] In certain embodiments, the lithocholic acid is a naturally occurring lithocholic acid.

[0030] In certain embodiments, the lithocholic acid is an isomeric lithocholic acid.

[0031] In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

[0032] In certain embodiments, the linker comprises a bivalent or trivalent linker.

[0033] In certain embodiments, the bivalent or trivalent linker is selected from the group consisting of: [ka] wherein n is 1, 2, 3, 4, or 5.

[0034] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0035] In certain embodiments, when the linker is a trivalent linker, the linker further links phosphodiesters or phosphodiester derivatives.

[0036] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] wherein X is O, S, or BH3.

[0037] In certain embodiments, the nucleotides at positions 1 and 2 from the 3' end of the sense strand and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate bonds.

[0038] In one aspect, the present disclosure provides a pharmaceutical composition for inhibiting the expression of the S6K1 gene in an organism, the pharmaceutical composition comprising the above-described siRNA and a pharmaceutically acceptable carrier.

[0039] In certain embodiments, the siRNA inhibits expression of the S6K1 gene by at least 20%. In certain embodiments, the siRNA inhibits expression of the S6K1 gene by at least 50%.

[0040] In one aspect, the present disclosure provides a method for inhibiting expression of the S6K1 gene in a cell, the method comprising: (a) introducing the above-described siRNA into a cell; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the S6K1 gene, thereby inhibiting expression of the S6K1 gene in the cell.

[0041] In one aspect, the present disclosure provides a method of treating or managing an ocular disease, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of the above-described siRNA.

[0042] In certain embodiments, the siRNA is administered to the eye of the patient.

[0043] In some embodiments, the siRNA is administered by intravitreal injection.

[0044] In certain embodiments, the siRNA inhibits expression of the S6K1 gene by at least 20%. In certain embodiments, the dsRNA inhibits expression of the S6K1 gene by at least 50%.

[0045] In one aspect, the disclosure provides a vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding a dsRNA substantially complementary to an S6K1 nucleic acid sequence of SEQ ID NOs: 1-6.

[0046] In certain embodiments, the dsRNA inhibits expression of the S6K1 gene by at least 20%. In certain embodiments, the dsRNA inhibits expression of the S6K1 gene by at least 50%.

[0047] In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NOs: 1-6.

[0048] In one aspect, the present disclosure provides a cell comprising the above-described vector.

[0049] In one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising the above-described vector and an AAV capsid.

[0050] In one aspect, the present disclosure provides branched RNA compounds comprising two or more of the above-described siRNAs covalently linked to each other.

[0051] In certain embodiments, the siRNAs are covalently linked to each other via a linker, spacer, or branch point. [Brief explanation of the drawings]

[0052] [Figure 1]Figure 1 shows retinal cross sections with the distribution of 12 different siRNAs 3 days after injection of 0.3 nmoles of siRNA (left panel: retinoic acid (RA), docosahexaenoic acid (DHA), phosphocholine (PC), α-tocopherol succinate (TS), docosanoic acid (DCA)). Right: One example per group shows a whole retinal cross section with its distribution throughout the retina. All siRNAs are Cy3 labeled and shown in red. Glutamine synthetase (GS) expression is shown in green. Nuclear DAPI is shown in blue. Right: One example per group shows a whole retinal cross section with its distribution throughout the retina.

[0053] [Figure 2] Figure 1 shows the enrichment of siRNAs in different retinal cell types ordered by cell type, with bars indicating relative protein levels of cell type-specific markers calibrated to whole retinal extracts of uninjected mouse retinas.

[0054] [Figure 3] Figure 1 shows the enrichment of siRNAs in different retinal cell types ordered by modification. Bars indicate relative protein levels of cell-type-specific markers calibrated to whole retinal extracts of uninjected mouse retinas. For each modification (i.e., monomer, dimer, trimer, etc.), each bar represents, from left to right, rhodopsin (rods), cone arrestin (CA) (cones), glutamine synthetase (GS) (Müller cells), Vglut2 (ganglion cells), VGAT (amacrine cells), protein kinase C alpha (PKCa) (bipolar cells), and Lim1 (horizontal cells).

[0055] [Figure 4]Figure 1 shows an example of siRNA distribution in a retinal cross section 3 days after injection of 0.3 nmoles of siRNA. The siRNAs are labeled with Cy3 and shown in red. All siRNAs target the Huntington gene. A non-targeting control (NTC) with PC-TS modification, trimers, and tetramers is shown. Cone segments are highlighted in green and labeled with PNA (peanut agglutinin lectin), Müller glial cells are shown in cyan and labeled with glutamine synthetase (GS), and nuclei are shown in blue and labeled with DAPI.

[0056] [Figure 5] An example of siRNA distribution in a retinal cross section 3 days after injection of 0.3 nmoles of siRNA is shown without GS staining in cyan. Additionally, for trimers and tetramers, only a high magnification of the outer nuclear layer (ONL) is shown to highlight the distribution of siRNA in the photoreceptor layer. Half of the panels show only siRNA to better visualize the signal.

[0057] [Figure 6] Figure 1 shows antibody staining of HTT protein in retinal cross sections 2 weeks after injection of Htt-siRNA. The first column shows staining in control mice injected with NTC-siRNA. The second column shows HTT protein expression after knockdown with PC-RA-Htt siRNA. Shown are examples from two different mice, each injected with approximately 0.3 nmoles of Htt-siRNA.

[0058] [Figure 7] Figure 1 shows quantification by Western blotting of total HTT protein 2 weeks after injection with Htt-siRNA. In the same experimental setting as in Figure 6 (different mice of the same injection batch), total HTT protein remaining from whole retina extraction is quantified.

[0059] [Figure 8]FIG. 10 Quantification by bDNA assay to quantify total Htt mRNA levels 2 weeks after Htt-siRNA injection using 0.1 nmoles per injection of the indicated siRNA modifications.

[0060] [Figure 9] Quantification by bDNA assay to quantify total Htt mRNA levels 3 days after Htt-siRNA injection using 0.3 nmoles per injection of the indicated siRNA modifications. Each dot represents one retina.

[0061] [Figure 10] Quantification by bDNA assay to quantify total Htt mRNA levels 100 days after Htt-siRNA injection using 0.3 nmoles per injection of the indicated siRNA modifications. Each dot represents one retina.

[0062] [Figure 11] Representative fundus images over time of eyes injected with Cy3-labeled siRNA with the indicated modifications are shown. The fluorescent signal exposure is the same for all four siRNAs at any given time point, but not over time. This figure supplements Figure 10 and shows fundus images of the mice used in Figure 10. All mice were injected intravitreally with 0.3 nmoles of siRNA.

[0063] [Figure 12] Figure 1 shows a dose-escalation study of HTT knockdown in the retina. Mice were injected with the indicated amounts (1–60 micrograms [note: not nanomoles] of Cy3-labeled tetramer with Htt-siRNA) in a total volume of 2 microliters. Five mice were injected per siRNA amount. Tissues were harvested two weeks post-injection, and residual HTT protein in the retina was quantified by Western blotting. Injections of 15–30 micrograms roughly correspond to the same knockdown seen in previous experiments with approximately 0.3 nanomoles.

[0064] [Figure 13] 13A-13C are fundus images from the dose escalation study shown in Figure 12. Images were taken two weeks post-injection prior to euthanasia. Normal brightfield fundus images and Cy3 images are shown for each concentration.

[0065] [Figure 14] Figure 14 shows retinal cross sections of eyes from the dose escalation study shown in Figures 12 and 13. The images show Cy3 distribution throughout the retinal section, indicating that the siRNA was taken up uniformly throughout the eye.

[0066] [Figure 15] Retinal cross sections of eyes from the dose-escalation study shown in Figure 14 were stained with Iba1 (green) to identify Iba1-positive cells migrating to the outer nuclear layer (ONL) where photoreceptors reside. Half of each panel (dotted line) shows only Iba1 signal to better visualize the signal. Blue indicates nuclear DAPI.

[0067] [Figure 16] Figure 15 shows retinal cross sections of eyes from the dose-escalation study shown in Figure 14 stained with GFAP (red) to identify reactive gliosis in Müller glial cells. siRNA is not shown as these are sections from the same eyes shown in Figure 15. Blue indicates nuclear DAPI, and green marks cone photoreceptor segments with peanut agglutinin lectin (PNA).

[0068] [Figure 17] Figure 1 shows measurements of photoreceptor and retinal function by electroretinography under scotopic (0.01 cd.s / m² to 1 cd.s / m²) and photopic conditions (3 and 10 flashes). A- and b-waves are recorded at several injected doses.

[0069] [Figure 18]This figure shows the fluorescence intensity of tetramer-Htt-Cy3 after intravitreal delivery into pig eyes. The top of each panel indicates the amount of siRNA delivered (100-1500 micrograms of tetramer). The top row shows Cy3 fluorescence in unfixed tissue immediately after opening the eye. The bottom of the figure is a higher magnification of the area in the top panel.

[0070] [Figure 19] Figure 18 shows huntingtin protein knockdown in pigs, measured by Western blot analysis from the eye shown in Figure 18. Knockdown was compared to huntingtin protein levels in NTCs injected with 250 μg of tetramer-siRNA-Cy3. The top panel shows the knockdown observed in the four major retinal quadrants (DT: dorsal-temporal, DN: dorsal-nasal, VT: temporal-nasal, VN: ventral-nasal) in a bar graph. The middle panel shows the knockdown in a flat-mount view, with corresponding values ​​for regional knockdown shown in a bar graph. Bottom panel: Average huntingtin protein knockdown across the entire retina, calculated by averaging the knockdown observed in each quadrant per retina. Data shown represent one biological sample per amount of siRNA delivered. Error bars in the first panel are generated by technical replicates. Error bars in the last panel are generated by averaging four data points in each quadrant per retina.

[0071] [Figure 20] 19 shows antibody staining of huntingtin protein in sections of eyes injected with different doses as shown in Figure 19. Areas of the sections are shown in the middle panel of Figure 19.

[0072] [Figure 21]Figure 1 shows antibody staining of GFAP (glial fibrillary acidic protein) and Iba1 (ionized calcium-binding adaptor protein 1) (as shown for mice in Figures 15 and 16) expression in retinal sections from eyes injected with different doses as shown in Figures 18 and 19 to determine dose-dependent toxicity. Both GFAP and Iba1 are shown in green, as indicated on the left side of each row. Red staining indicates the distribution of siRNA across the retinal section. Nuclei are marked with nuclear DAPI. Half of each panel shows only the signal of interest (siRNA, GFAP, or Iba1) to better visualize the signal.

[0073] [Figure 22] FIG. 1 shows the initial in vitro knockdown efficiency of siRNA duplexes formed from the sense and antisense strands shown in Tables 3 and 4.

[0074] [Figure 23] FIG. 23 shows dose-response curves for duplexes 2, 3, 9, and 10 of FIG. 22.

[0075] [Figure 24] Figure 1 shows RNA-Scope in situ hybridization in mouse retinal cross sections to detect siRNA tetramers against S6K1. The top row shows sections from three mice injected with NTC against S6K1 in a tetrameric configuration. The middle row shows sections from three mice injected with 3 μg / eye of siRNA against S6K1 in a tetrameric configuration. The last row shows sections from three mice injected with 6 μg / eye of siRNA against S6K1 in a tetrameric configuration. siRNA was delivered intravitreally, and animals were euthanized two weeks after injection.

[0076] [Figure 25A](A-B) Knockdown of S6K1 in mice after intravitreal injection of 6 μg of tetramer-configured siRNA. (A) S6K1 protein levels detected by Western blot 2 weeks after injection. (B) Data similar to the first graph 2 months after injection. Each dot in the graph represents one biological sample (retina) from one animal. [Figure 25B] Same as above.

[0077] [Figure 26] This figure shows S6K1 protein knockdown in non-human primates (NHPs). Western blot data are shown for retinal protein extracts from the superior temporal (ST) region (also known as the dorsal-temporal region) of one NHP intravitreally injected with 225 μg of S6K1-tetramer (in 75 μL) and six naive NHP retinas from the same region. The first set of bar graphs shows a comparison of the uninjected contralateral eye with an S6K1 siRNA-injected eye to allow for direct intraanimal comparison between eyes. The second bar graph shows a comparison of six naive NHPs with NHPs injected with S6K1 siRNA. NHP eyes were harvested one month post-injection. Phosphorylation of the ribosomal protein S6, a canonical target of S6K1, is also shown. As with the S6K1 knockdown data, intraanimal comparisons are shown on the left, and comparisons across multiple NHPs are shown on the right.

[0078] [Figure 27] Figure 1 shows knockdown of S6K1 protein in non-human primate (NHP) retinal cross sections after siRNA treatment. Data were generated from one injected eye and the contralateral uninjected eye. Sections were taken from the central region as shown for the pig in Figure 19. Left: Full cross section including the fovea. Right: Magnified view of the temporal and nasal regions and the fovea. The top row shows an uninjected eye, and the bottom row shows an eye intravitreally injected with 225 μg of S6K1 tetramer (in 75 μL).

[0079] [Figure 28]This figure shows the reduction of phosphorylated S6 protein (pS6) in retinal cross sections from non-human primates (NHPs) after siRNA treatment. The data are the same as those shown in Figure 27, except for the staining probe for pS6 expression (red signal). In each panel, the green and blue signals have been removed from half of the panel (dotted line) to better visualize pS6 knockdown. Blue indicates nuclear DAPI, and green indicates cone segments marked with peanut agglutinin lectin (PNA).

[0080] [Figure 29] Figure 2 shows the expression of inflammatory markers in NHPs after siRNA treatment with S6K1 siRNA (75 microliters, 225 μg of tetramer-configured siRNA). The data are the same as those shown in Figures 27 and 28, except for staining probes for Iba1 (red signal, first set) and GFAP (red signal, second set) expression. Untreated contralateral eyes are in the first row of each set, and treated eyes are in the second row. In each panel, the green and blue signals have been removed from half of the panel (dotted lines) to better visualize the Iba1 and GFAP signals. Blue indicates nuclear DAPI, and green indicates cone segments marked with peanut agglutinin lectin (PNA). DETAILED DESCRIPTION OF THE INVENTION

[0081] The present disclosure relates to oligonucleotide conjugates and branched oligonucleotides capable of efficient gene knockdown in the eye. Several different functional moieties and branched oligonucleotides have demonstrated ocular cell-specific delivery upon administration.

[0082] The oligonucleotide conjugates and branched oligonucleotides described herein facilitate simple, efficient, and non-toxic delivery of oligonucleotides (e.g., siRNA) and potent silencing of therapeutic targets in various ocular cell types in vivo.

[0083] Unless otherwise specified, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein is that which is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The terminology used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0084] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the case of potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise indicated. The use of the term "including" and other forms such as "include" and "included" is not limiting.

[0085] In order that this disclosure may be more readily understood, certain terms are first defined.

[0086] 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 adenine (e.g., cytidine or a chemically modified derivative thereof).

[0087] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also referred to as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety by ester linkages. Exemplary nucleotides include nucleoside monophosphate, diphosphate, and triphosphate. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked together by phosphodiester or phosphorothioate linkages between the 5' and 3' carbon atoms.

[0088] The terms "RNA" or "RNA molecule" or "ribonucleic acid molecule" refer to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refer to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be modified post-transcriptionally. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

[0089] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNA") refers to an RNA (or RNA analog) comprising approximately 10-50 nucleotides (or nucleotide analogs) and capable of inducing or mediating RNA interference. An siRNA is a duplex formed by a sense strand and an antisense strand, which are sufficiently complementary to each other to form the duplex. In certain embodiments, an siRNA comprises approximately 15-30 nucleotides or nucleotide analogs, or approximately 16-25 nucleotides (or nucleotide analogs), or approximately 18-23 nucleotides (or nucleotide analogs), or approximately 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA comprising approximately 21 nucleotides (or nucleotide analogs), e.g., 19, 20, 21, or 22 nucleotides. The term "long" siRNA refers to an siRNA containing approximately 24 to 25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. A short siRNA may optionally contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, a long siRNA may optionally contain more than 26 nucleotides, provided that the long siRNA retains its ability to mediate RNAi without further processing, e.g., enzymatic processing, into a short siRNA.

[0090] The terms "nucleotide analog" or "altered nucleotide" or "modified nucleotide" or "chemically modified nucleotide" refer to non-standard nucleotides, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide but retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position (e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5- Nucleotide analogs include those at positions 6 (e.g., 6-(2-amino)propyluridine), 8 (e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc.) of adenosine and / or guanosine. Nucleotide analogs also include deazanucleotides (e.g., 7-deaza-adenosine), O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or others known in the art) nucleotides, and other heterocyclic-modified nucleotide analogs (such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310).

[0091] Nucleotide analogs may also contain modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438. In certain embodiments, the nucleotide analog contains a 2'-O-methyl modification. In certain embodiments, the nucleotide analog contains a 2'-fluoro modification.

[0092] The phosphate group of a nucleotide may also be modified, for example, by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioate) or by other substitutions that enable the nucleotide to perform its intended function (e.g., those described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21; Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45; Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25; Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85; and U.S. Pat. No. 5,684,143). Certain of the above modifications (e.g., phosphate group modifications) reduce the hydrolysis rate of polynucleotides containing the analogs, for example, in vivo or in vitro.

[0093] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs. The term "oligonucleotide" includes, but is not limited to, antisense oligonucleotides (ASOs), siRNAs, and microRNAs.

[0094] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to a corresponding unmodified or unmodified RNA, but retains the same or similar properties or functions as the corresponding unmodified or unmodified RNA. As described above, oligonucleotides may be linked with linkages that reduce the rate of hydrolysis of the RNA analog compared to RNA molecules with phosphodiester linkages. For example, the nucleotides of the analog may contain methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and / or phosphorothioate linkages. Some RNA analogs include sugar- and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include the addition of non-nucleotide material, such as to the end(s) or internal (one or more nucleotides of the RNA) of the RNA. An RNA analog need only be sufficiently similar to natural RNA to have the ability to mediate RNA interference.

[0095] As used herein, the term "RNA interference" ("RNAi") refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells and removes foreign RNA (such as viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA, and these fragments guide the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by human hands, for example, to silence the expression of target genes.

[0096] An RNAi agent (e.g., an RNA silencing agent) having a strand that is "sufficiently complementary to a target mRNA sequence to induce target-specific RNA interference (RNAi)" means that the strand has sufficient sequence to cause destruction of the target mRNA by the RNAi machinery or process.

[0097] As used herein, "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material or culture medium when produced by recombinant techniques, or that is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0098] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression), which result in the inhibition or "silencing" of expression of corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0099] The term "in vitro" has its art-recognized meaning and includes, for example, purified reagents or extracts, e.g., cellular extracts. The term "in vivo" also has its art-recognized meaning and includes, for example, living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0100] As used herein, "target" refers to a specific nucleic acid sequence (e.g., a gene, mRNA, miRNA, etc.) to which an oligonucleotide conjugate or branched oligonucleotide of the present disclosure binds and / or otherwise affects its expression. In certain embodiments, the target is expressed in the eye. In certain embodiments, the target is expressed in specific ocular cells. In other embodiments, the target is associated with a specific disease or disorder in a subject.

[0101] As used herein, the term "target gene" refers to a gene whose expression is substantially inhibited or "silenced." This silencing can be achieved by RNA silencing, e.g., cleavage of the target gene's mRNA or translational repression of the target gene. The term "non-target gene" refers to a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., 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 paralog) of the target gene.

[0102] As used herein, the term "RNA silencing agent" refers to an RNA that can inhibit or "silence" the expression of a target gene. In certain embodiments, an RNA silencing agent can prevent 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 containing paired strands, as well as precursor RNAs that can generate such small non-coding RNAs. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, short hairpin RNAs (shRNAs), and dual-function oligonucleotides, as well as their precursors. In one embodiment, an RNA silencing agent can induce RNA interference. In another embodiment, an RNA silencing agent can mediate translational repression.

[0103] As used herein, the term "rare nucleotide" refers to a rare naturally occurring nucleotide, such as a rare naturally occurring deoxyribonucleotide or ribonucleotide (e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine). Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0104] The term "engineered" indicates that the precursor or molecule is not found in nature, such as an engineered RNA precursor or engineered nucleic acid molecule, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by machinery within the cell. Thus, an RNA precursor produced within a cell from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0105] As used herein, the term "microRNA" ("miRNA"), also known in the art as "small temporal RNA" ("stRNA"), refers to small (e.g., 10-50 nucleotide) RNAs that are genetically encoded (e.g., by viral, mammalian, or plant genomes) and can induce or mediate RNA silencing. "miRNA disorder" refers to a disease or disorder characterized by aberrant expression or activity of miRNA.

[0106] As used herein, the term "dual-function oligonucleotide" refers to an RNA silencing agent having the formula TL-μ, where T is an mRNA targeting moiety, L is a linking moiety, and μ is an miRNA recruitment moiety. As used herein, the terms "mRNA targeting moiety," "targeting moiety," "mRNA targeting portion," or "targeting portion" refer to a domain, portion, or region of a dual-function oligonucleotide that is of sufficient size and sufficient complementarity to a portion or region of an mRNA that is selected or targeted for silencing (i.e., the portion has sufficient sequence to capture the target mRNA).

[0107] As used herein, the term "linking moiety" or "linking portion" refers to a domain, portion, or region of an RNA silencing agent that covalently binds or links an mRNA.

[0108] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA, refers to the strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides, of the mRNA of a gene targeted for silencing. The antisense strand, or first strand, has a sequence sufficiently complementary to the desired target mRNA sequence to induce target-specific silencing, e.g., sufficient complementarity to cause destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or sufficient complementarity to cause translational repression of the desired target mRNA.

[0109] The term "sense strand" or "second strand" of an RNA silencing agent, such as an siRNA or RNA silencing agent, refers to the strand complementary to the antisense strand or first strand. The antisense strand and the sense strand may also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and the second strand or first strand, respectively, is complementary to the first strand or second strand. The miRNA duplex intermediate or siRNA-like duplex includes an miRNA strand that is sufficiently complementary to a section of approximately 10-50 nucleotides of the mRNA of the gene targeted for silencing, and an miRNA* strand that is sufficiently complementary to form a duplex with the miRNA strand.

[0110] As used herein, the term "guide strand" refers to the strand of an RNA silencing agent, e.g., the antisense strand of an siRNA duplex or siRNA sequence, that enters the RISC complex and guides cleavage of a target mRNA.

[0111] As used herein, the term "asymmetry" refers to the unequal binding strength or base pairing strength between the ends of an RNA silencing agent (for example, between the terminal nucleotide on the first strand or stem portion and the terminal nucleotide on the opposite second strand or stem portion), such as the asymmetry of the double-stranded region of an RNA silencing agent (for example, the stem of an shRNA), so that the 5'-end of one strand of the duplex is more frequently in a transient unpaired state (for example, single-stranded state) than the 5'-end of the complementary strand.This structural difference determines that one strand of the duplex is preferentially incorporated into the RISC complex.The strand whose 5'-end is less tightly paired with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0112] As used herein, the term "binding strength" or "base pair strength" refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., an siRNA duplex), and is primarily due to H-bonding, van der Waals interactions, etc. between the nucleotides (or nucleotide analogs).

[0113] As used herein, "5' end" refers to the 5'-terminal nucleotide, e.g., 1 to about 5 nucleotides at the 5' end of the antisense strand, as in the 5' end of the antisense strand. As used herein, "3' end" refers to the region, e.g., 1 to about 5 nucleotides, complementary to the 5'-terminal nucleotide of the complementary antisense strand, as in the 3' end of the sense strand.

[0114] As used herein, the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analog that can form a base pair with a second nucleotide or nucleotide analog, resulting in a base pair with a lower binding strength than conventional base pairs (i.e., Watson-Crick base pairs). In certain embodiments, the destabilizing nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide can form a wobble base pair with the second nucleotide. In still other embodiments, the destabilizing nucleotide can form an ambiguous base pair with the second nucleotide.

[0115] As used herein, the term "base pair" refers to the interaction between a pair of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), primarily due to H-bonding, van der Waals interactions, etc. between the nucleotides (or nucleotide analogs). As used herein, the term "binding strength" or "base pair strength" refers to the strength of the base pair.

[0116] As used herein, the term "mismatch base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair, e.g., a base pair that is not the usual complementary G:C, A:T, or A:U base pair. As used herein, the term "ambiguous base pair" (also known as a promiscuous base pair) refers to a base pair formed by a universal nucleotide.

[0117] As used herein, the term "universal nucleotide" (also known as "neutral nucleotide") includes nucleotides (e.g., certain destabilizing nucleotides) that have bases ("universal bases" or "neutral bases") that do not significantly discriminate against bases on complementary polynucleotides when base-pairing. Universal nucleotides are primarily hydrophobic molecules that can efficiently assemble into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0118] As used herein, the term "sufficient complementarity" or "sufficient degree of complementarity" means that the RNA silencing agent has sufficient sequence (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruitment portion) to bind to the desired target and induce RNA silencing of the target mRNA, respectively.

[0119] As used herein, the term " translational repression " refers to the selective inhibition of mRNA translation.Natural translational repression proceeds through miRNA cleaved from shRNA precursor.Both RNAi and translational repression are mediated by RISC.Both RNAi and translational repression can occur naturally or can be initiated by human hands, for example, to silence the expression of target genes.

[0120] Various methodologies of the present disclosure include comparing a value, level, characteristic, property, or the like to a "suitable control" (interchangeably referred to herein as "appropriate control"). A "suitable control" or "suitable control" is any control or standard useful for comparison purposes and familiar to those of skill in the art. In one embodiment, a "suitable control" or "suitable control" is a value, level, characteristic, property, or the like determined prior to performing an RNAi methodology as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular property or property, genotype, phenotype, or the like can be determined prior to introducing an RNA silencing agent of the present disclosure into a cell or organism. In another embodiment, a "suitable control" or "suitable control" is a value, level, characteristic, property, or the like determined, for example, in a cell or organism exhibiting a normal property (e.g., a control or normal cell or organism). In yet another embodiment, a "suitable control" or "suitable control" is a predefined value, level, characteristic, property, or the like.

[0121] In one aspect, instead of the RNA silencing agent being an interfering ribonucleic acid, such as the above-mentioned siRNA or shRNA, the RNAi agent can code an interfering ribonucleic acid, such as the above-mentioned shRNA.In other words, the RNAi agent can be a transcription template for an interfering ribonucleic acid.Therefore, the RNAi agent of the present disclosure can also include small hairpin RNA (shRNA) and the expression construct that is engineered to express shRNA.It is believed that the transcription of shRNA starts at polymerase III (pol III) promoter and terminates at the 2nd position of the 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into stem-loop structures with 3' UU overhangs, after which the ends of these shRNAs are processed, converting them into siRNA-like molecules of approximately 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, supra; Miyagishi et al., 2002; Paddison et al., 2002, supra; Paul et al., 2002, supra; Sui et al., 2002, supra; Yu et al., 2002, supra). Further information regarding the design and use of shRNAs can be found on the Internet at the following addresses: kkatandin.cshl.org:9331 / RNAi / docs / BseRI-BamHI_Strategy.pdf and katandin.cshl.org:9331 / RNAi / docs / Web_version_of_PCR_strategy1.pdf).

[0122] Expression constructs of the present invention include any construct suitable for use in an appropriate expression system, including, but not limited to, retroviral vectors, linear expression cassettes, plasmids, and viral or virus-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems (e.g., U6 snRNA promoter or H1 RNA polymerase III promoter), or other promoters known in the art. The construct may contain one or both strands of the siRNA. Expression constructs expressing both strands may also include a loop structure connecting both strands, or each strand may be transcribed separately from a separate promoter within the same construct. Each strand may also be transcribed from a separate expression construct (Tuschl, T., 2002, supra). (Tuschl, T., 2002, supra).

[0123] Synthetic siRNA can be delivered to cells by methods known in the art, such as cationic liposome transfection and electroporation. To achieve long-term suppression of a target gene (e.g., the S6K1 gene) and to facilitate delivery under certain circumstances, one or more siRNAs can be expressed in cells from recombinant DNA constructs. Methods for expressing siRNA duplexes in cells from recombinant DNA constructs to enable long-term suppression of a target gene in cells are known in the art and include mammalian Pol III promoter systems (e.g., H1 or U6 / snRNA promoter systems (Tuschl, T., 2002, supra)) that can express functional double-stranded siRNAs (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). Transcription termination by RNA PolIII occurs at a series of four consecutive T residues within the DNA template, providing a mechanism for terminating siRNA transcripts at specific sequences. The siRNA is complementary to the target gene sequence in the 5'-3' and 3'-5' directions, and the two strands of the siRNA can be expressed in the same or separate constructs. Hairpin siRNAs expressed in cells driven by the H1 or U6 snRNA promoter can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). Constructs containing siRNA sequences under the control of the T7 promoter also produce functional siRNAs when cotransfected into cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, supra).A single construct may contain multiple siRNA-encoding sequences, such as multiple regions of the S6K1-encoding gene targeting the same gene or multiple genes, and may be driven, for example, by separate PolIII promoter sites.

[0124] Animal cells express a series of approximately 22-nucleotide noncoding RNAs called microRNAs (miRNAs), which can regulate gene expression at the post-transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from approximately 70-nucleotide precursor RNA stem-loops, presumably by the RNase III enzyme Dicer or its homologs. By replacing the stem sequence of a miRNA precursor with a sequence complementary to the target mRNA, vector constructs expressing the engineered precursors can be used to produce siRNAs and initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., supra, 2002). When expressed by a DNA vector containing a polymerase III promoter, microRNA-designed hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms can also be useful for inhibiting the translation of mutant proteins in the absence of siRNA-mediated gene silencing. Such applications may be useful, for example, in situations where the designed siRNA caused off-target silencing of a wild-type protein.

[0125] Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through the expression of siRNA, for example, by generating recombinant adenoviruses carrying siRNA under the transcriptional control of an RNA Pol II promoter (Xia et al., 2002, supra). Infection of HeLa cells with these recombinant adenoviruses can reduce the expression of endogenous target genes. Injection of recombinant adenoviral vectors into transgenic mice expressing the target gene of the siRNA results in in vivo reduction of target gene expression (ibid.). The compound of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38, having Id. In animal models, whole-embryo electroporation can efficiently deliver synthetic siRNA to postimplantation mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be achieved by a "high-pressure" delivery technique, i.e., rapid (within 5 seconds) injection of a large volume of siRNA-containing solution into the animal via the tail vein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis et al., 2002). Nanoparticles and liposomes can also be used to deliver siRNA to animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs to cells, such as neuronal cells (e.g., brain cells) (U.S. Patent Application Nos. 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766).

[0126] The nucleic acid compositions of the present disclosure include both unmodified siRNAs and modified siRNAs (e.g., crosslinked siRNA derivatives or derivatives having a non-nucleotide moiety linked to their 3' or 5' ends, for example). Modifying siRNA derivatives in this manner can improve the cellular uptake or cell targeting activity of the resulting siRNA derivative compared to the corresponding siRNA, and is also useful for tracking the siRNA derivative within cells or improving the stability of the siRNA derivative compared to the corresponding siRNA.

[0127] As described herein, engineered RNA precursors introduced into cells or whole organisms lead to the production of desired siRNA molecules.Then, these siRNA molecules associate with the endogenous protein components of the RNAi pathway to bind to and target specific mRNA sequences for cleavage and destruction.In this way, the mRNA targeted by the siRNA produced from engineered RNA precursors is depleted from cells or organisms, thereby reducing the concentration of the protein coded by that mRNA in cells or organisms.RNA precursors are typically nucleic acid molecules that individually code one strand of dsRNA or code the entire nucleotide sequence of an RNA hairpin loop structure.

[0128] The nucleic acid compositions of the present invention may be unconjugated or may be conjugated to another moiety, such as a nanoparticle, to enhance the properties of the composition, e.g., pharmacokinetic parameters such as absorption, efficacy, bioavailability, and / or half-life. Attachment can be achieved using methods known in the art, such as those described in Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles), Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describing nucleic acids bound to nanoparticles), Schwab et al., Ann. Oncol. 5 Suppl. 4: 55-8 (1994) (describing nucleic acids linked to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles), and Godard et al., Eur. J. Biochem. 232(2): 404-10 (1995) (describing nucleic acids linked to nanoparticles).

[0129] The nucleic acid molecules of the present disclosure can also be labeled using any method known in the art.For example, the nucleic acid composition can be labeled with a fluorophore, such as Cy3, fluorescein, or rhodamine.Labeling can be carried out using a kit, such as SILENCER™ siRNA labeling kit (Ambion).In addition, siRNA can be, for example, 3 H, 32 It can be radiolabeled using P or another suitable isotope.

[0130] Furthermore, because RNAi is believed to proceed through at least one single-stranded RNA intermediate, those skilled in the art will understand that ss-siRNAs (e.g., the antisense strand of ds-siRNAs) can also be designed (e.g., by chemical synthesis), generated (e.g., enzymatically generated), or expressed (e.g., from a vector or plasmid) as described herein and utilized in accordance with the claimed methodologies. Furthermore, in invertebrates, RNAi can be effectively induced by long dsRNAs (e.g., dsRNAs of about 100-1000 nucleotides in length, e.g., about 200-500, e.g., about 250, 300, 350, 400, or 450 nucleotides in length) that function as effectors of RNAi (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4;98(25):14428-33. Epub 2001 Nov. 27.).

[0131] Oligonucleotides targeting S6K1 Ribosomal protein S6 kinase B1 (RPS6KB1), also known as S6K1, is a serine / threonine kinase that phosphorylates the S6 ribosomal protein to stimulate protein synthesis. The S6K1 gene is provided in NCBI reference sequence NG_029513.1. Described herein are oligonucleotides (e.g., siRNAs, antisense oligonucleotides, and short hairpin RNAs (shRNAs)) that target S6K1 mRNA and effectively silence S6K1 expression.

[0132] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to an S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0133] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO:1.

[0134] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO:2.

[0135] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO:3.

[0136] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO:4.

[0137] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO:5.

[0138] In one aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO:6.

[0139] In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 7 to 12. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 8. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 10. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 11. In certain embodiments, the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NO: 12.

[0140] In certain embodiments, the siRNA comprises complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0141] In certain embodiments, the siRNA comprises no more than three mismatches with the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0142] In certain embodiments, the siRNA comprises perfect complementarity to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

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

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

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

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

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

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

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

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

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

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

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

[0154] Anti-S6K1 short hairpin RNA (shRNA) molecules In a particular characteristic embodiment, the present disclosure provides shRNA that can mediate the RNA silencing of S6K1 target sequence with enhanced selectivity.In contrast to siRNA, shRNA mimics the natural precursor of microRNA (miRNA) and enters the top of gene silencing pathway.Therefore, shRNA is believed to mediate gene silencing more efficiently by being delivered through the entire natural gene silencing pathway.

[0155] miRNAs are approximately 22-nucleotide non-coding RNAs that can regulate gene expression at the post-transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from approximately 70-nucleotide precursor RNA stem-loops, called pre-miRNAs, presumably by the RNase III enzyme Dicer or its homologs. Naturally occurring miRNA precursors (pre-miRNAs) generally have a single strand that forms a double-stranded stem containing two complementary portions and a loop connecting the two portions of the stem. In a typical pre-miRNA, the stem contains one or more bulges, e.g., extra nucleotides that form a single-nucleotide "loop" in one portion of the stem, and / or one or more unpaired nucleotides that form a gap in the mutual hybridization of the two portions of the stem. The short hairpin RNAs or engineered RNA precursors of the present application are artificial constructs based on these naturally occurring pre-miRNAs but engineered to deliver a desired RNA silencing agent (e.g., the siRNA of the present disclosure). By replacing the stem sequence of the pre-miRNA with a sequence complementary to the target mRNA, an shRNA is formed, which is processed throughout the cellular gene silencing pathway, thereby efficiently mediating RNAi.

[0156] Essential elements of an shRNA molecule include a first and a second portion that are sufficiently complementary to anneal or hybridize to form a double-stranded or double-stranded stem portion. The two portions do not need to be completely or perfectly complementary. The first and second "stem" portions are connected by a portion whose sequence is insufficiently complementary to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as the "loop" portion within the shRNA molecule. shRNA molecules are processed to produce siRNA. shRNAs can also contain one or more bulges, i.e., extra nucleotides that form small nucleotide "loops" in portions of the stem, e.g., one-, two-, or three-nucleotide loops. The stem portions can be the same length, or some can include overhangs, e.g., one to five nucleotides. Overhanging nucleotides can include, for example, uracils (U), e.g., all Us. Such Us are specifically encoded by thymidines (T) in the DNA encoding the shRNA, which signal the termination of transcription.

[0157] In shRNAs (or engineered precursor RNAs) of the present disclosure, one portion of the double-stranded stem is a nucleic acid sequence complementary (or antisense) to the APP target sequence. In certain embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to the target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of the target RNA via RNA interference (RNAi). Thus, the engineered RNA precursor comprises a two-part double-stranded stem and a loop connecting the two stem portions. The antisense portion can be located at the 5' or 3' end of the stem. The stem portion of the shRNA is about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portion should be 21 nucleotides or greater. When used in mammalian cells, the length of the stem portion should be less than about 30 nucleotides to avoid eliciting non-specific responses such as the interferon pathway. In non-mammalian cells, the stem may be longer than 30 nucleotides. In fact, the stem may contain a much larger section (up to the entire mRNA) that is complementary to the target mRNA. In fact, the stem portion may contain a much larger section (up to the entire mRNA) that is complementary to the target mRNA.

[0158] The two portions of the double-stranded stem must be sufficiently complementary to hybridize and form a double-stranded stem. Thus, the two portions can be, but do not need to be, completely or perfectly complementary. Furthermore, the two stem portions can be the same length, or one portion can contain an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotide can, for example, contain uracil (U), e.g., all U. The loop in the shRNA or engineered RNA precursor can differ from the natural pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Thus, the loop in the shRNA or engineered RNA precursor can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length, for example, 15 or 20 or more nucleotides.

[0159] The loop in an shRNA or engineered RNA precursor can differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Thus, the loop portion in an shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides, e.g., 15 or 20 nucleotides or more. In certain embodiments, the loop consists of or includes a "tetraloop" sequence. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA (where N is any nucleotide and R is a purine nucleotide), GGGG, and UUUU.

[0160] In certain embodiments, the shRNA of the present application comprises the sequence of the desired siRNA molecule described above. In other embodiments, the sequence of the antisense portion of the shRNA can be designed essentially as described above, or generally by selecting an 18, 19, 20, 21, or longer sequence from within the target RNA (e.g., APP mRNA), for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the translation start point. Generally, the sequence can be selected from any part of the target RNA (e.g., mRNA), such as the 5' untranslated region (UTR), coding sequence, or 3' UTR. This sequence can optionally immediately follow a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected as UU. This approximately 21-nucleotide sequence is used to form one portion of the double-stranded stem in the shRNA. This sequence can replace the stem portion of the wild-type pre-miRNA sequence, for example, enzymatically, or can be included in a synthetically synthesized complete sequence. For example, DNA oligonucleotides encoding the entire stem-loop engineered RNA precursor, or only the portion to be inserted into the double-stranded stem of the precursor, can be synthesized and restriction enzymes used to assemble the engineered RNA precursor construct, e.g., from the wild-type pre-miRNA.

[0161] The engineered RNA precursor contains, in its double-stranded stem, approximately 21–22 nucleotides of the desired siRNA or siRNA-like duplex to be produced in vivo. Thus, the stem portion of the engineered RNA precursor contains at least 18 or 19 nucleotide pairs corresponding to the sequence of an exon of the gene whose expression is to be reduced or inhibited. The two 3′ nucleotides flanking this region of the stem are selected to maximize siRNA production from the engineered RNA precursor and maximize the effectiveness of the resulting siRNA in targeting the corresponding mRNA for translational repression or disruption by RNAi in vivo and in vitro.

[0162] In certain embodiments, the shRNA of the present disclosure comprises an miRNA sequence, optionally a terminally modified miRNA sequence, to enhance entry into RISC. The miRNA sequence can be similar or identical to the sequence of any naturally occurring miRNA (see, for example, The miRNA Registry, Griffiths-Jones S, Nuc. Acids Res., 2004). To date, more than 1,000 naturally occurring miRNAs have been identified, and they are thought to collectively account for approximately 1% of all predicted genes in the genome. Many natural miRNAs are clustered together within the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms that predict the ability of candidate miRNA genes to form stem-loop structures in pre-mRNAs (e.g., MiRScan, MiRSeeker) (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003). Online registries provide searchable databases of all publicly available miRNA sequences (The miRNA Registry, Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004).Exemplary naturally occurring miRNAs include lin-4, let-7, miR-10, miR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other naturally occurring miRNAs from humans and certain model organisms, including Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thalania, Mus musculus, and Rattus norvegicus, as described in International PCT Publication No. WO 03 / 029459.

[0163] Naturally occurring miRNAs are expressed by endogenous genes in vivo and processed by Dicer or other RNAses from hairpin or stem-loop precursors (pre-miRNA or pri-miRNA) (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). Although miRNAs can exist transiently in vivo as double strands, only one strand is incorporated into the RISC complex to induce gene silencing. Certain miRNAs, such as plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs, thereby inducing cleavage of the target mRNA. Other miRNAs have less-than-perfect complementarity to their target mRNAs, thereby inducing translational repression of the target mRNA. The degree of complementarity between a miRNA and its target mRNA is thought to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA predicts a cleavage mechanism (Yekta et al., Science, 2004), while less-than-perfect complementarity predicts a translational repression mechanism. In certain embodiments, the miRNA sequence is a naturally occurring miRNA sequence, the abnormal expression or activity of which is correlated with a miRNA disorder.

[0164] Modified anti-S6K1 RNA silencing agents In certain aspects of the present disclosure, the RNA silencing agents of the present application (or any portion thereof) may be modified, as described above, to further improve the activity of the agent. For example, the RNA silencing agents described in Section II above may be modified with any of the modifications described below. The modifications may act, in part, to further improve target discrimination, improve the stability of the agent (e.g., to prevent degradation), facilitate cellular uptake, improve targeting efficiency, improve the effectiveness of binding (e.g., to the target), improve patient tolerance to the agent, and / or reduce toxicity.

[0165] 1) Modifications to improve target discrimination In certain embodiments, the RNA silencing agents of the present application may be substituted with destabilizing nucleotides to improve single-nucleotide target discrimination (see U.S. Application No. 11 / 698,689, filed January 25, 2007, and U.S. Provisional Application No. 60 / 762,225, filed January 25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to abolish the specificity of the RNA silencing agent for non-target mRNAs (e.g., wild-type mRNAs) without significantly affecting the specificity of the RNA silencing agent for target mRNAs (e.g., gain-of-function mutant mRNAs).

[0166] In certain embodiments, the RNA silencing agent of the present application is modified by introducing at least one universal nucleotide into its antisense strand. A universal nucleotide contains a base moiety that can indiscriminately base pair with any of the four conventional nucleotide bases (e.g., A, G, C, U). Universal nucleotides are contemplated because they have a relatively small effect on the stability of an RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base moiety or an inosine analog base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In certain embodiments, the universal nucleotide is an inosine residue or a naturally occurring analogue thereof.

[0167] In certain embodiments, the RNA silencing agent of the present disclosure is modified by introducing at least one destabilizing nucleotide within 5 nucleotides of the specificity-determining nucleotide (i.e., the nucleotide that recognizes the disease-associated polymorphism). For example, the destabilizing nucleotide may be introduced within 5, 4, 3, 2, or 1 nucleotide(s) of the specificity-determining nucleotide. In an exemplary embodiment, the destabilizing nucleotide is introduced three nucleotides from the specificity-determining nucleotide (i.e., such that there are two stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide). In an RNA silencing agent having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide may be introduced into the strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced into the same strand or strand portion that contains the specificity-determining nucleotide.

[0168] 2) Modifications to increase efficacy and specificity In certain embodiments, the RNA silencing agent of the present disclosure can be modified according to asymmetric design rules to easily improve the efficacy and specificity of mediating RNAi (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such modifications facilitate the antisense strand of siRNA (for example, siRNA designed using the method of the present invention, or siRNA produced from shRNA) to enter RISC in favor of the sense strand. This allows the antisense strand to preferentially induce cleavage or translational suppression of target mRNA, thereby increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of the RNA silencing agent is improved by decreasing the base pairing strength between the 5' end of the antisense strand (AS5') and the 3' end of the sense strand (S3') of the RNA silencing agent relative to the binding strength or base pairing strength between the 3' end of the antisense strand (AS3') and the 5' end of the sense strand (S'5) of the RNA silencing agent.

[0169] In one embodiment, the asymmetry of the RNA silencing agent of the present disclosure can be improved so that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than there are G:C base pairs between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present disclosure can be improved so that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In certain embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the asymmetry of the RNA silencing agent of the present disclosure can be improved so that there is at least one wobble base pair, for example, G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present disclosure can be improved by the presence of at least one base pair containing a rare nucleotide, such as inosine (I). In certain embodiments, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the asymmetry of the RNA silencing agent of the present disclosure can be improved by the presence of at least one base pair containing a modified nucleotide. In certain embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0170] 3) RNA silencing agents with improved stability The RNA silencing agent of the present application can be modified to improve stability in serum or cell culture growth medium.To improve stability, the 3'-residue can be stabilized against degradation, and can be selected to be composed of purine nucleotides, such as adenosine or guanosine nucleotides.Alternatively, the substitution of pyrimidine nucleotides with modified analogs, for example, the substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNA interference.

[0171] In one aspect, the present application features an RNA silencing agent comprising a first and a second strand, wherein the second strand and / or the first strand is modified by substituting an internal nucleotide with a modified nucleotide to enhance in vivo stability compared to the corresponding unmodified RNA silencing agent. As defined herein, an "internal" nucleotide is one that is present at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. The internal nucleotide may be within a single-stranded molecule or within a strand of a double-stranded or duplex molecule. In one embodiment, the sense strand and / or antisense strand is modified by substituting at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand is modified by substituting at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more internal nucleotides. In another embodiment, the sense and / or antisense strands are modified by substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of their internal nucleotides, hi yet another embodiment, the sense and / or antisense strands are modified by substitution of all of their internal nucleotides.

[0172] In one aspect, the present application features an RNA silencing agent that is at least 80% chemically modified.In certain embodiments, the RNA silencing agent can be completely chemically modified, that is, 100% of nucleotides are chemically modified.In another aspect, the present application features an RNA silencing agent that comprises at least 80% chemically modified 2'-OH ribose group.In certain embodiments, the RNA silencing agent comprises about 80%, 85%, 90%, 95% or 100% chemically modified 2'-OH ribose group.

[0173] In certain embodiments, RNA silencing agent can comprise at least one modified nucleotide analogue.Nucleotide analogue can be located at the position where target-specific silencing activity, such as RNAi-mediated activity or translational repression activity, is not substantially affected, for example, at the 5'-end and / or 3'-end of siRNA molecule.In addition, by incorporating modified nucleotide analogue, end can be stabilized.

[0174] Exemplary nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., modifications to the phosphate sugar backbone). For example, the phosphodiester linkage of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate ester group linked to adjacent ribonucleotides is replaced with a modified group, such as a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH-group is replaced with a group selected from H, OR, R, halo, SH, SR, NH, NHR, NR, or ON, where R is C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0175] In certain embodiments, the modifications are 2'-fluoro, 2'-amino, and / or 2'-thio modifications. Modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and / or 5-amino-allyl-uridine. In certain embodiments, 2'-fluoro ribonucleotides are all uridines and cytidines. Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluorouridine. 2'-deoxy-nucleotides and 2'-ome nucleotides can also be used in the modified RNA silencing agent moiety of the present disclosure. Additional modified residues include deoxyabasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside, and ribavirin. In certain embodiments, the 2' moiety is a methyl group, so that the linking moiety is a 2'-O-methyl oligonucleotide.

[0176] In certain embodiments, the RNA silencing agent of the present application comprises a locked nucleic acid (LNA). LNAs contain sugar-modified nucleotides that are resistant to nuclease activity (highly stable) and have single-nucleotide discrimination against mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1):439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have 2'-O,4'-C-ethylene-bridged nucleic acids, which may have modifications such as 2'-deoxy-2'-fluorouridine. Furthermore, LNAs enhance the specificity of oligonucleotides by constraining the sugar moiety to a 3'-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by 10°C per base.

[0177] In another exemplary embodiment, the RNA silencing agent of the present application comprises a peptide nucleic acid (PNA), which contains modified nucleotides in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety that can form a polyamide backbone, making the molecule more resistant to nuclease digestion and providing improved binding specificity (Nielsen, et al., Science, (2001), 254:1497-1500).

[0178] Also contemplated are nucleobase-modified ribonucleotides, i.e., ribonucleotides, which contain at least one non-naturally occurring nucleobase instead of naturally occurring nucleobases.The base can be modified to block the activity of adenosine deaminase.Examples of modified nucleobases include, but are not limited to, uridine and / or cytidine modified at 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, adenosine and / or guanosine modified at 8-position, such as 8-bromoguanosine, deazanucleotides, such as 7-deaza-adenosine, O- and N-alkylated nucleotides, such as N6-methyladenosine, and are preferred.It should be noted that the above modifications can also be combined.

[0179] In other embodiments, crosslinking can be used to change the pharmacokinetics of the RNA silencing agent, for example, to extend its half-life in the body. Thus, the present application includes RNA silencing agents having two complementary nucleic acid strands, where the two strands are crosslinked. The present application also includes RNA silencing agents that are bound (e.g., at the 3' end) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like, or that are not bound. By modifying siRNA derivatives in this way, the cellular uptake or cell targeting activity of the resulting siRNA derivative can be improved compared to the corresponding siRNA, and this can also be useful for tracking the siRNA derivative in cells, or the stability of the siRNA derivative can be improved compared to the corresponding siRNA.

[0180] Other exemplary modifications include: (a) 2' modifications, such as providing a 2'OMe moiety on a U in the sense or antisense strand, particularly in the sense strand, or providing a 2'OMe moiety in a 3' overhang, for example, at the 3' end (3' end means the 3' atom or the 3'-most portion of the molecule, for example, the 3'-most P or 2' position, as indicated by the context); (b) backbone modifications, such as by substituting O with S in the phosphate backbone, for example, providing a phosphorothioate modification to U or A or both, particularly in the antisense strand; for example, by substituting O with S; (c) substitution of U with a C5 amino linker; (d) substitution of A with G (sequence changes may, in certain embodiments, be placed in the sense strand but not the antisense strand); and (d) modifications at the 2', 6', 7', or 8' positions. Exemplary embodiments are those in which one or more of these modifications are present on the sense strand but not on the antisense strand, or in which the antisense strand has few such modifications. Still other exemplary modifications include the use of a methylated P in the 3' overhang, e.g., at the 3' end, a combination of 2' modifications, e.g., providing a 2'OMe moiety and modifying the backbone, e.g., substituting O with S, e.g., providing a phosphorothioate modification, or the use of a methylated P in the 3' overhang, e.g., at the 3' end, a 3' alkyl modification, a modification in the 3' overhang, e.g., at the 3' end, with an abasic pyrrolidone, a modification with naproxen, ibuprofen, or other moieties that inhibit degradation at the 3' end.

[0181] Highly modified RNA silencing agents In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0182] In certain embodiments, the RNA silencing agent is 2'-O-methyl rich, i.e., contains greater than 50% 2'-O-methyl content. In certain embodiments, the RNA silencing agent contains at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2'-O-methyl nucleotide content. In certain embodiments, the RNA silencing agent contains at least about 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent contains about 70% to about 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand. In certain embodiments, the antisense strand contains at least about 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand contains about 70% to about 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises about 70% to about 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0183] 2'-O-methyl-rich RNA silencing agents and specific chemical modification patterns are further described in US20200087663 and US20210115442, each of which is incorporated herein by reference.

[0184] Internucleotide bond modifications In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA silencing agent comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4 to 16 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8 to 13 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5' end and a 3' end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5'-end of the sense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3'-end of the sense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5'-end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 through 1-8 from the 3'-end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3'-end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, nucleotides 1-2 through 1-7 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages.

[0185] In one aspect, the disclosure provides a modified oligonucleotide, the oligonucleotide having a 5' end, a 3' end complementary to a target, the oligonucleotide comprising a sense and an antisense strand and at least one modified intersubunit linkage of formula (I): [ka] During the ceremony, B is a base pairing moiety, W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is halo, hydroxy, and C 1-6 alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH, Z is selected from the group consisting of O and CH2; R is a protecting group, [ka] is an optional double bond.

[0186] In an embodiment of Formula (I), when W is CH: [ka] is a double bond.

[0187] In an embodiment of Formula (I), when W is selected from the group consisting of O, OCH2, OCH, CH2: [ka] is a single bond.

[0188] In an embodiment of Formula (I), Y is O - Then either Z or W is not O.

[0189] In an embodiment of Formula (I), Z is CH2 and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II). [ka]

[0190] In one embodiment of Formula (I), Z is CH and W is O. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III). [ka]

[0191] In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV). [ka]

[0192] In one embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V. [ka]

[0193] In an embodiment of Formula (I), Z is O and W is OCH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI. [ka]

[0194] In one embodiment of Formula (I), Z is CH and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII. [ka]

[0195] In an embodiment of Formula (I), the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0196] In one embodiment, the modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end complementary to the target, and is complementary to the target, the siRNA comprising a sense strand and an antisense strand, and at least one modified inter-subunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII).

[0197] In one embodiment, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, is complementary to a target, and comprises a sense and an antisense strand, and the siRNA comprises at least one modified intersubunit linkage of Formula VIII: [ka] During the ceremony, D is selected from the group consisting of O, OCH2, OCH2, CH2, and CH; C is O - , OH, OR 1 , N.H. - , NH2, S - and SH, A is selected from the group consisting of O and CH2; R 1 is a protecting group, [ka] is any double bond, Between the subunits, two optionally modified nucleosides are bridged.

[0198] In one embodiment, C is O - If so, then either A or D is not O.

[0199] In one embodiment, D is CH. In another embodiment, the modified intersubunit linkage of formula VIII is a modified intersubunit linkage of formula (IX). [ka]

[0200] In one embodiment, D is O. In another embodiment, the modified intersubunit linkage of formula VIII is a modified intersubunit linkage of formula (X). [ka]

[0201] In one embodiment, D is CH. In another embodiment, the modified intersubunit linkage of formula (VIII) is a modified intersubunit linkage of formula (XI). [ka]

[0202] In one embodiment, D is CH. In another embodiment, the modified intersubunit linkage of formula VIII is a modified intersubunit linkage of formula (XII). [ka]

[0203] In another embodiment, the modified intersubunit linkage of formula (VII) is a modified intersubunit linkage of formula (XIV). [ka]

[0204] In one embodiment, D is OCH. In another embodiment, the modified intersubunit linkage of formula (VII) is a modified intersubunit linkage of formula (XIII). [ka]

[0205] In another embodiment, the modified intersubunit linkage of formula (VII) is a modified intersubunit linkage of formula (XXa). [ka]

[0206] In one embodiment of the modified siRNA conjugate, each optionally modified nucleoside at each occurrence is independently selected from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0207] In certain exemplary embodiments of Formula (I), W is O. In other embodiments, W is CH. In yet other embodiments, W is CH.

[0208] In certain exemplary embodiments of Formula (I), X is OH. In other embodiments, X is OCH. In yet other embodiments, X is halo.

[0209] In certain embodiments of Formula (I), the modified siRNA does not contain a 2'-fluoro substituent.

[0210] In an embodiment of Formula (I), Y is O - In another embodiment, Y is OH. In yet another embodiment, Y is OR. In yet another embodiment, Y is NH - In one embodiment, Y is NH. In another embodiment, Y is S - In yet another embodiment, Y is SH.

[0211] In one embodiment of Formula (I), Z is O. In another embodiment, Z is CH.

[0212] In one embodiment, the modified intersubunit bond is inserted at positions 1-2 of the antisense strand. In another embodiment, the modified intersubunit bond is inserted at positions 6-7 of the antisense strand. In yet another embodiment, the modified intersubunit bond is inserted at positions 10-11 of the antisense strand. In yet another embodiment, the modified intersubunit bond is inserted at positions 19-20 of the antisense strand. In one embodiment, the modified intersubunit bond is inserted at positions 5-6 and 18-19 of the antisense strand.

[0213] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), C is O - In another embodiment, C is OH. In yet another embodiment, C is OR 1 In yet another embodiment, C is NH - In one embodiment, C is NH. In another embodiment, C is S - In yet another embodiment, C is SH.

[0214] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), A is O. In another embodiment, A is CH. In yet another embodiment, C is OR. 1 In yet another embodiment, C is NH - In one embodiment, C is NH. In another embodiment, C is S - In yet another embodiment, C is SH.

[0215] In certain embodiments of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is adenosine.In another embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is guanosine.In another embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is cytidine.In another embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is uridine.

[0216] In one embodiment of the modified siRNA linkage, the linkage is inserted at positions 1-2 of the antisense strand. In another embodiment, the linkage is inserted at positions 6-7 of the antisense strand. In yet another embodiment, the linkage is inserted at positions 10-11 of the antisense strand. In yet another embodiment, the linkage is inserted at positions 19-20 of the antisense strand. In one embodiment, the linkages are inserted at positions 5-6 and 18-19 of the antisense strand.

[0217] In certain embodiments of Formula (I), base pairing moiety B is adenine. In certain embodiments of Formula (I), base pairing moiety B is guanine. In certain embodiments of Formula (I), base pairing moiety B is cytosine. In certain embodiments of Formula (I), base pairing moiety B is uracil.

[0218] In one embodiment of Formula (I), W is O. In one embodiment of Formula (I), W is CH. In one embodiment of Formula (I), W is CH.

[0219] In an embodiment of Formula (I), X is OH. In an embodiment of Formula (I), X is OCH. In an embodiment of Formula (I), X is halo.

[0220] In an exemplary embodiment of Formula (I), the modified oligonucleotide does not include a 2'-fluoro substituent.

[0221] In an embodiment of Formula (I), Y is O -In an embodiment of Formula (I), Y is OH. In an embodiment of Formula (I), Y is OR. In an embodiment of Formula (I), Y is NH - In an embodiment of Formula (I), Y is NH. In an embodiment of Formula (I), Y is S - In an embodiment of Formula (I), Y is SH.

[0222] In an embodiment of Formula (I), Z is O. In one embodiment of Formula (I), Z is CH2.

[0223] In an embodiment of Formula (I), the bond is inserted at positions 1-2 of the antisense strand. In another embodiment of Formula (I), the bond is inserted at positions 6-7 of the antisense strand. In yet another embodiment of Formula (I), the bond is inserted at positions 10-11 of the antisense strand. In yet another embodiment of Formula (I), the bond is inserted at positions 19-20 of the antisense strand. In an embodiment of Formula (I), the bond is inserted at positions 5-6 and 18-19 of the antisense strand.

[0224] Modified intersubunit linkages are further described in U.S. Patent Publication Nos. 2020 / 0385740A1 and 2022 / 0010309, each of which is incorporated herein by reference.

[0225] 4) Binding functional part In other embodiments, the RNA silencing agent may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moiety enhances cellular uptake by target cells (e.g., neuronal cells). Thus, the present disclosure includes RNA silencing agents that are conjugated (e.g., at the 5' and / or 3' ends) with or without other moieties (e.g., non-nucleic acid moieties such as peptides), organic compounds (e.g., dyes), etc. Attachment can be achieved using methods known in the art, such as those described in Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles), Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describing nucleic acids bound to nanoparticles), Schwab et al., Ann. Oncol. 5 Suppl. 4: 55-8 (1994) (describing nucleic acids linked to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles), and Godard et al., Eur. J. Biochem. 232(2): 404-10 (1995) (describing nucleic acids linked to nanoparticles).

[0226] In certain embodiments, the functional moiety is a hydrophobic moiety. In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LA). In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0227] In certain embodiments, the disclosed RNA silencing agent is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, and cationic dyes (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0228] In certain embodiments, the functional moiety may include one or more ligands tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with the target nucleic acid, targeting to specific tissues or cell types, or cell permeability, e.g., by endocytosis-dependent or -independent mechanisms. The ligand and associated modifications can also enhance sequence specificity and, consequently, reduce off-site targeting. The tethering factor ligand can include one or more modified bases or sugars that can function as intercalators. These can also be located within internal regions, such as within the bulge, of the RNA silencing agent / target duplex. The intercalator can be aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. Polycyclic intercalators can have stacking capabilities and include systems with two, three, or four fused rings. The universal bases described herein can be included in the ligand. In one embodiment, the ligand can include a cleavage group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleavage group can be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. Metal ion chelating groups can include, for example, Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote selective cleavage of target RNA at the bulge site by free metal ions such as Lu(III). In some embodiments, a peptide ligand can be tethered to the RNA silencing agent to promote cleavage of target RNA, for example, at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be attached to a peptide (eg, by an amino acid derivative) to promote target RNA cleavage.The tethered ligand can be an aminoglycoside ligand, which can provide the RNA silencing agent with improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine linkages of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of an acridine analog can enhance sequence specificity. For example, neomycin B has a higher affinity for RNA compared to DNA, but lower sequence specificity. The acridine analog neo-5-acridine has a higher affinity for the HIV Rev-response element (RRE). In some embodiments, a guanidine analog (guanidinoglycoside) of the aminoglycoside ligand is tethered to the RNA silencing agent. In guanidinoglycosides, the amine group on the amino acid is replaced with a guanidine group. The attachment of a guanidine analogue can increase the cell permeability of RNA silencing agents. The anchoring ligand can be a polyarginine peptide, peptoid, or peptidomimetic, which can increase the cellular uptake of oligonucleotide agents.

[0229] Exemplary ligands are coupled to the ligand-binding carrier directly or indirectly via an intervening tether. In certain embodiments, the coupling is via a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, the ligand changes the distribution, targeting or life span of the RNA silencing agent that it is incorporated into. In certain embodiments, the ligand provides, for example, a higher affinity to a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cell or organ compartment, a tissue, an organ or a region of the body, compared to a species that does not have such a ligand.

[0230] Exemplary ligands can improve transport, hybridization, and specificity properties, and may also improve nuclease resistance of the resulting natural or modified RNA silencing agent, or polymer molecule comprising any combination of the monomers described herein and / or natural or modified ribonucleotides. Ligands generally can include therapeutic modifiers, e.g., to increase uptake, diagnostic compounds or reporter groups, e.g., to monitor distribution, crosslinkers, nuclease-resistance-conferring moieties, and natural or unusual nucleobases. Common examples include lipophilic substances, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein-binding agents, integrin-targeting molecules, polycations, peptides, polyamines, and peptidomimetics. Ligands can include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), amino acids, or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0231] The ligand can also include a targeting group that binds to a specific cell type, such as a kidney cell, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody. The targeting group can also be thyroid-stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine (GalNAc) or a derivative thereof, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or an RGD peptide or RGD peptidomimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidium aminoguanidium aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and its thio analogs), cholic acid, cholanic acid, lithocholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters thereof (e.g., mono-, bis-, or tris-fatty acid esters, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C18 , C 19 or C 20 fatty acids) and ethers, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 Alkyl, e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., ante Napedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocycles of Eu 3+ complex), dinitrophenyl, HRP, or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

[0232] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for co-ligands, or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-kB.

[0233] The ligand can be a substance, such as a drug, that can increase the uptake of an RNA silencing agent into cells, for example, by disrupting the cytoskeleton of the cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of an RNA silencing agent into cells, for example, by activating an inflammatory response. Exemplary ligands with such effects include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or gamma interferon. In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule can bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) improve the conjugate's resistance to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to regulate binding to serum proteins, such as HSA. The lipid-based ligand can be used to regulate, e.g., control, the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds strongly to HSA is less likely to target the kidney and therefore less likely to be eliminated from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney. In certain embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate is distributed to non-renal tissues.However, it is contemplated that the affinity is not so strong that it cannot reverse the HSA-ligand binding. In another embodiment, the lipid-based ligand binds weakly or not at all to HSA, so that the conjugate is distributed to the kidney. Other moieties that target kidney cells can also be used instead of or in addition to the lipid-based ligand.

[0234] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These may be particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B (e.g., folic acid, B12, riboflavin, biotin, pyridoxal) or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0235] In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennopedia. If the agent is a peptide, it may be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent may be an alpha-helical agent, which may have a lipophilic phase and a lipophobic phase.

[0236] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, such as by improving cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. The peptide moiety can be an L-peptide or a D-peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). The peptide or peptidomimetic can be encoded by a random DNA sequence, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In an exemplary embodiment, the peptide or peptidomimetic tethered to the RNA silencing agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can undergo structural modifications, for example, to improve stability or induce conformational properties. Any of the structural modifications described below can be used.

[0237] In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the RNA silencing agent of the present disclosure. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the antisense strand of the RNA silencing agent of the present disclosure. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand of the RNA silencing agent of the present disclosure. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand of the RNA silencing agent of the present disclosure.

[0238] In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3' end of the sense strand by a linker. In certain embodiments, the linker comprises a bivalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the bivalent or trivalent linker is selected from the following: [ka] wherein n is 1, 2, 3, 4 or 5.

[0239] In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative, in certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] wherein X is O, S, or BH3.

[0240] Various functional moieties of the present disclosure and means for attaching them to RNA silencing agents are described in further detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0241] Anti-S6K1 oligonucleotide conjugates for ocular delivery The S6K1-targeting oligonucleotides of the present disclosure may be linked to a functional moiety for ocular delivery, which functional moiety enhances ocular delivery of the oligonucleotide, including ocular cell-specific delivery.

[0242] In certain embodiments, the functional moiety comprises any one of a triple amine, retinoic acid (RA), docosahexaenoic acid (DHA), docosanoic acid (DCA), alpha-tocopherol succinate, or lithocholic acid (LA).

[0243] Each of the above functional moieties is structurally represented below: The functional moieties may have different isomeric configurations than those presented in this disclosure. [ka] [ka]

[0244] In certain embodiments, two DHA functional moieties are linked to an oligonucleotide.

[0245] In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the oligonucleotide. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand or the 5'-end and / or 3'-end of the antisense strand. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand.

[0246] In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

[0247] In certain embodiments, the linker comprises a bivalent or trivalent linker.

[0248] In certain embodiments, the bivalent or trivalent linker is selected from the group consisting of: [ka] wherein n is 1, 2, 3, 4, or 5.

[0249] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0250] In certain embodiments, when the linker is a trivalent linker, the linker further links phosphodiesters or phosphodiester derivatives.

[0251] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] wherein X is O, S, or BH3.

[0252] The moiety Zc1 is phosphatidylcholine (PC). Any one of the functional moieties described herein can include a phosphatidylcholine (PC) esterified derivative, i.e., phosphatidylcholine (PC) esterified triple amine (PC-triple amine), phosphatidylcholine (PC) esterified retinoic acid (PC-RA), phosphatidylcholine (PC) esterified docosahexaenoic acid (PC-DHA), phosphatidylcholine (PC) esterified docosanoic acid (PC-DCA), phosphatidylcholine (PC) esterified α-tocopherol succinate (PC-TS), or phosphatidylcholine (PC) esterified lithocholic acid (PC-LA).

[0253] In certain embodiments, the oligonucleotide conjugate targeting S6K1 comprises the following structure: [ka] [ka] [ka]

[0254] For any of the structures listed above, the term "oligonucleotide" refers to an oligonucleotide comprising a sequence substantially complementary to an S6K1 nucleic acid sequence. In certain embodiments, the oligonucleotide is an siRNA comprising an antisense strand and a sense strand. In certain embodiments, the antisense strand comprises a sequence substantially complementary to an S6K1 nucleic acid sequence of any one of SEQ ID NOS: 1-6.

[0255] Branched Oligonucleotides The S6K1 oligonucleotides described herein may be comprised in a branched oligonucleotide structure. Branched oligonucleotides comprise two or more oligonucleotides linked together. Different branched oligonucleotides described herein (e.g., branched oligonucleotides having two, three, or four oligonucleotides) have enhanced ocular delivery of oligonucleotides, including ocular cell-specific delivery.

[0256] In certain embodiments, the two or more oligonucleotides in the branched oligonucleotide are linked to each other by one or more moieties independently selected from a linker, a spacer, and a branch point.

[0257] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

[0258] In certain embodiments, the branch points comprise a polyvalent organic species or derivatives thereof.

[0259] In another embodiment, the branch points are amino acid derivatives. In another embodiment, the branch points are selected from the following formulas: [ka]

[0260] Polyvalent organic species are moieties that contain carbon and three or more valencies (i.e., points of attachment to moieties such as S, L, or N, as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol, etc.), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, etc.), tetracarboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, etc.), tertiary amines (e.g., tripropargylamine, triethanolamine, etc.), triamines (e.g., diethylenetriamine, etc.), tetramines, and species that contain combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, etc.).

[0261] In certain embodiments, the spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

[0262] In certain embodiments, the linker comprises the structure L1. [ka]

[0263] In certain embodiments, the linker comprises the structure L2. [ka]

[0264] In certain embodiments, the branched oligonucleotide consists of two oligonucleotides. In certain embodiments, the branched oligonucleotide consists of three oligonucleotides. In certain embodiments, the branched oligonucleotide consists of four oligonucleotides. In certain embodiments, the oligonucleotide is an siRNA.

[0265] In certain embodiments, the branched oligonucleotide comprises the following structure: [ka]

[0266] In any of the structures listed above, the term "oligonucleotide" corresponds to any of the oligonucleotides described herein, such as ASO or siRNA. In certain embodiments, the term "oligonucleotide" in the structures listed above corresponds to the sense strand of siRNA. In certain embodiments, the oxygen directly adjacent to the term "oligonucleotide" in the structure is linked to the 3' end of the sense strand of siRNA.

[0267] Branched oligonucleotides, including their synthesis and methods of use, are described in further detail in WO2017 / 132669, which is incorporated herein by reference. Further details regarding synthesis are provided in the "Materials and Methods" section of the Examples.

[0268] Nucleic acids, vectors and methods for introducing host cells The RNA silencing agent of the present disclosure can be directly introduced into cells (e.g., ocular cells) (i.e., intracellularly), or can be introduced extracellularly into a cavity, interstitial space, or into the circulation of an organism, or can be introduced by immersing the cell or organism in a solution containing nucleic acid. Vascular or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are sites where nucleic acids can be introduced.

[0269] The RNA silencing agent of the present disclosure can be introduced using nucleic acid delivery methods known in the art, such as injecting a solution containing nucleic acid, bombarding with particles covered with nucleic acid, immersing cells or organisms in a solution of nucleic acid, or electroporating cell membranes in the presence of nucleic acid.Other methods known in the art for introducing nucleic acid into cells can also be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate.Nucleic acid can be introduced with other components that perform one or more of the following activities: enhance the uptake of nucleic acid by cells, or otherwise increase the inhibition of target genes.

[0270] Physical methods for introducing nucleic acids include injection of a solution containing RNA, bombardment with RNA-coated particles, immersion of cells or organisms in a solution of RNA, or electroporation of cell membranes in the presence of RNA. Viral constructs packaged in viral particles achieve both efficient introduction of the expression construct into cells and transcription of the RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids into cells, such as lipid-mediated carrier transport or chemical-mediated transport, such as calcium phosphate, may also be used. Thus, RNA may be introduced with components that perform one or more activities, such as enhancing RNA uptake by cells, inhibiting single-strand annealing, stabilizing single strands, or otherwise increasing target gene inhibition.

[0271] The cells bearing the target gene may be derived from germline or somatic cells, totipotent or pluripotent, dividing or non-dividing, parenchymal or epithelial, immortalized or transformed, etc. The cells may be stem cells or differentiated cells. Differentiated cell types include eye cells, adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelial cells, neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

[0272] Depending on the specific target gene and the dose of double-stranded RNA material delivered, this process may result in partial or complete loss of target gene function. A reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of the target cells is typical. Inhibition of gene expression refers to the absence (or observable reduction) of protein and / or mRNA product levels from the target gene. Specificity refers to the ability to inhibit the target gene without appreciably affecting other genes in the cell. Inhibition results can be confirmed by examining the external characteristics of the cell or organism (as demonstrated in the examples below) or by biochemical techniques, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0273] For RNA-mediated inhibition in cell lines or whole organisms, gene expression is conveniently assayed by using reporter or drug resistance genes whose protein products are easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and their derivatives. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Depending on the assay, quantification of gene expression can determine inhibition of greater than 10%, 33%, 50%, 90%, 95%, or 99% compared to cells not treated according to the present disclosure. Smaller doses of injected material and longer times after administration of the RNAi agent may result in inhibition in a smaller percentage of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of target cells). Quantification of intracellular gene expression may indicate similar amounts of inhibition at the level of target mRNA accumulation or target protein translation. As an example, the efficiency of inhibition may be determined by assessing the amount of intracellular gene product. mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or the translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

[0274] The RNA can be introduced in an amount that allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) of the agent may result in more effective inhibition, while lower doses may also be useful for certain applications.

[0275] In an exemplary embodiment, the efficacy of an RNAi agent (e.g., an siRNA targeting an S6K1 target sequence) of the present disclosure is tested for its ability to specifically degrade mutant mRNA (e.g., production of S6K1 mRNA and / or S6K1 protein) in cells (such as cells of the central nervous system). In certain embodiments, cells of the central nervous system include, but are not limited to, neurons (e.g., striatal or cortical neuron clonal lines and / or primary neurons), glial cells, and astrocytes. Other easily transfectable cells, such as HeLa cells or COS cells, are also suitable for cell-based validation assays. Cells are transfected with human wild-type or mutant cDNA (e.g., human wild-type or mutant S6K1 cDNA). Standard siRNA, modified siRNA, or a vector capable of producing siRNA from U-loop mRNA is co-transfected. Selective reduction in target mRNA (e.g., S6K1 mRNA) and / or target protein (e.g., S6K1 protein) is measured. The reduction of target mRNA or protein can be compared with the level of target mRNA or protein in the absence of RNAi agent or in the presence of RNAi agent that does not target S6K1 mRNA.Exogenously introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison.When using neural cells, which are known to be quite resistant to standard transfection techniques, it may be desirable to introduce RNAi agent (for example, siRNA) by passive uptake.

[0276] Recombinant adeno-associated viruses and vectors In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their related vectors can be used to deliver one or more siRNAs to cells, such as neural cells (e.g., brain cells). AAVs can infect many different cell types, with infection efficiencies varying based on the serotype, determined by the sequence of the capsid protein. Several native AAV serotypes have been identified, with serotypes 1 through 9 being the most commonly used recombinant AAVs. AAV-2 is the most well-studied and published serotype. The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ / 8. These serotypes were generated by DNA shuffling of multiple AAV serotypes to produce AAVs with hybrid capsids that offer improved transduction efficiency in vitro (AAV-DJ) and in vivo (AAV-DJ / 8) in various cells and tissues.

[0277] In certain embodiments, widespread central nervous system (CNS) delivery can be achieved by intravascular delivery of recombinant adeno-associated virus 7 (rAAV7), RAAV9, and rAAV10, or other suitable rAAV (Zhang et al. (2011) Mol. Ther. 19(8):1440-8. doi:10.1038 / mt.2011.98. Epub 2011 May 24). rAAVs and their associated vectors are well known in the art and are described in U.S. Patent Application Nos. 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766, each of which is incorporated by reference in its entirety for all purposes.

[0278] rAAV can be delivered to the subject in the composition according to any suitable method known in the art.rAAV can be suspended in a physiologically compatible carrier (i.e., composition) and can be administered to the subject, i.e., host animals such as humans, mice, rats, cats, dogs, sheep, rabbits, horses, cows, goats, pigs, guinea pigs, hamsters, chickens, turkeys, non-human primates (e.g., macaques).In certain embodiments, the animal is a non-human host animal.

[0279] Delivery of one or more rAAVs to a mammalian subject can be achieved, for example, by intramuscular injection or by administration into the mammalian subject's bloodstream. Administration into the bloodstream can be by injection into a vein, artery, or any other vascular conduit. In certain embodiments, one or more rAAVs are administered into the bloodstream by isolated limb perfusion, a technique well known in the surgical arts; this method essentially allows one skilled in the art to isolate a limb from the general circulation prior to administration of rAAV virions. A variation of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 can also be used by those skilled in the art to administer virions to the vasculature of an isolated limb, potentially enhancing transduction of muscle cells or tissues. Additionally, in some cases, it may be desirable to deliver virions to the subject's central nervous system (CNS). By "CNS" is meant all cells and tissues of the vertebrate brain and spinal cord. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and the like. Recombinant AAV can be delivered directly to the CNS or brain using neurosurgical techniques known in the art, such as stereotactic injection with a needle, catheter, or related device, for example, by injection into the ventricular region, as well as the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule (e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000).

[0280] The compositions of the present disclosure can include rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In certain embodiments, the compositions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs, each carrying one or more different transgenes.

[0281] The effective amount of rAAV is sufficient to target infection of animals and target desired tissues.In some embodiments, the effective amount of rAAV is sufficient to create a stable somatic transgenic animal model.The effective amount mainly depends on factors such as the species, age, weight, health status and target tissue of the subject, and therefore may vary depending on animals and tissues.For example, the effective amount of one or more rAAVs is generally about 10 9 ~10 16 The volume ranges from about 1 ml to about 100 ml of solution containing about 10 genome copies. 11 ~10 12 In certain embodiments, a dose of 10 rAAV genome copies is appropriate. 12 rAAV genome copies are effective in targeting heart, liver, and pancreatic tissues. In some cases, stable transgenic animals are produced by multiple administrations of rAAV.

[0282] In some embodiments, the rAAV composition is particularly effective when high rAAV concentrations are present (e.g., about 10 13 The composition is formulated to reduce aggregation of AAV particles (genome copies / mL or higher). Methods for reducing rAAV aggregation are well known in the art, including, for example, adding detergents, adjusting pH, adjusting salt concentration, etc. (e.g., Wright et al. (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference).

[0283] A "recombinant AAV (rAAV) vector" comprises, at a minimum, a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector that is packaged into capsid proteins and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence, which encodes a polypeptide, protein, functional RNA molecule (e.g., siRNA), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and / or expression of the transgene in cells of the target tissue.

[0284] The AAV sequence of the vector typically includes cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, for example, BJ Carter, "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). ITR sequences are usually about 145 base pairs in length. In certain embodiments, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modifications of these sequences are tolerated. It is within the skill of the art to be able to modify these ITR sequences (see, for example, the texts Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such a molecule used in the present disclosure is a "cis-acting" plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences, which can be obtained from any known AAV, including the mammalian AAV types further described herein.

[0285] Treatment method In one aspect, the disclosure provides both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) developing a disease associated with S6K1 expression. In one embodiment, the disease is cancer. In one embodiment, the disease is obesity. In one embodiment, the disease is diabetes. In one embodiment, the disease is an ocular disease. In one embodiment, the ocular disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, central axial cataract, normal tension glaucoma, macular edema, and glaucoma.

[0286] As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or a vector or transgene encoding same) to a patient with a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder, or to a tissue or cell line isolated from that patient, for the purpose of curing, curing, mitigating, alleviating, altering, treating, ameliorating, improving, or affecting the disease or disorder, symptom of the disease or disorder, or predisposition to a disease.

[0287] In one aspect, the present disclosure provides a method for preventing the above-mentioned disease or disorder in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding the same) to the subject.Subjects at risk of the disease can be identified, for example, by any one or a combination of diagnostic or prognostic assays described herein.The administration of the prophylactic agent can be carried out before the symptoms characteristic of the disease or disorder appear, thereby preventing or delaying the progression of the disease or disorder.

[0288] Another aspect of the present disclosure relates to a method for therapeutically treating a subject, i.e., a method for altering the onset of symptoms of a disease or disorder. In an exemplary embodiment, the modulatory method of the present disclosure comprises contacting a CNS cell that expresses S6K1 with a therapeutic agent (e.g., an RNAi agent or a vector or transgene encoding the same) that is specific to a target sequence in a gene (e.g., an S6K1 target sequence in Table 1), thereby achieving sequence-specific interference with the gene. These methods can be carried out in vitro (e.g., by culturing cells with the agent), or alternatively in vivo (e.g., by administering the agent to a subject).

[0289] Pharmaceutical compositions and methods of administration The present disclosure relates to the use of the above-mentioned agents for preventive and / or therapeutic treatments as described below. Therefore, the modulators (e.g., RNAi agents) of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically contain a nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0290] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intravitreal, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In certain exemplary embodiments, the pharmaceutical compositions of the present disclosure are administered intravenously and can cross the blood-brain barrier to enter the central nervous system. In certain exemplary embodiments, the pharmaceutical compositions of the present disclosure are delivered to the cerebrospinal fluid (CSF) by routes of administration including, but not limited to, intrastriatal (IS) administration, intracerebroventricular (ICV) administration, and intrathecal (IT) administration (e.g., via a pump, infusion, etc.).

[0291] The nucleic acid molecules of the present disclosure can be inserted into expression constructs, such as viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, using methods known in the art, such as, but not limited to, those described in Xia et al. (2002) supra. The expression constructs can be delivered to a subject, for example, by inhalation, oral administration, intravenous injection, topical administration (see U.S. Pat. No. 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). Pharmaceutical formulations of the delivery vector can include the vector in an acceptable diluent or a slow-release matrix in which the delivery vehicle is embedded. Alternatively, if the complete delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors), the pharmaceutical formulation can include one or more cells that produce the gene delivery system.

[0292] The nucleic acid molecules of the present disclosure may also include small hairpin RNAs (shRNAs) and expression constructs engineered to express shRNAs. Transcription of shRNAs is believed to begin at the polymerase III (pol III) promoter and terminate at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNAs are believed to fold into a stem-loop structure with a 3'UU overhang. The ends of these shRNAs are then processed, converting them into siRNA-like molecules of approximately 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553, Lee et al. (2002). supra, Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500, Paddison et al. (2002), supra, Paul (2002), supra, Sui (2002), supra, Yu et al. (2002), supra.

[0293] The expression construct may be any construct suitable for use in an appropriate expression system, including, but not limited to, retroviral vectors, linear expression cassettes, plasmids, and viral or virus-derived vectors known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems, such as the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. The construct may include one or both strands of the siRNA. Expression constructs that express both strands may also include a loop structure connecting both strands, or each strand may be transcribed separately from a separate promoter within the same construct. Each strand may also be transcribed from a separate expression construct (Tuschl (2002) supra).

[0294] In certain embodiments, compositions comprising compounds of the present disclosure can be delivered to the nervous system of a subject by various routes.Exemplary routes include intrathecal, parenchymal (e.g., intracerebral), nasal, and ocular delivery.Compositions can also be delivered systemically, for example, by intravenous, subcutaneous, or intramuscular injection.One route of delivery is directly to the brain, for example, to the ventricles or hypothalamus of the brain, or to the lateral or dorsal regions of the brain.Compounds for neuronal delivery can be incorporated into pharmaceutical compositions suitable for administration.

[0295] For example, a composition can include one or more species of compounds of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present disclosure can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (ophthalmic, intranasal, transdermal, etc.), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, intrathecal, or intracerebroventricular (e.g., intracerebroventricular) administration. In certain exemplary embodiments, the RNA silencing agents of the present disclosure are delivered across the blood-brain barrier (BBB) ​​using various suitable compositions and methods described herein.

[0296] The delivery route may depend on the patient's disorder. For example, for a subject diagnosed with a neurodegenerative disease, the anti-S6K1 compound of the present disclosure may be administered directly to the brain (e.g., the globus pallidus or striatum of the basal ganglia, and near the medium spiny neurons of the striatum). In addition to the compound of the present disclosure, the patient may be administered a second treatment, such as a palliative treatment and / or a disease-specific treatment. The second treatment may be, for example, symptomatic treatment (e.g., to alleviate symptoms), neuroprotection (e.g., to slow or stop disease progression), or recovery (e.g., to reverse the disease process). Other treatments may include psychotherapy, physical therapy, speech therapy, communication and memory aids, social support services, and dietary advice.

[0297] The compounds of the present disclosure can be delivered to neurons in the brain. In certain embodiments, the compounds of the present disclosure can be delivered to the brain without being administered directly to the central nervous system, i.e., the compounds can be delivered intravenously and enter the brain through the blood-brain barrier. Delivery methods that do not require the composition to cross the blood-brain barrier can be utilized. For example, a pharmaceutical composition containing a compound of the present disclosure can be delivered to a patient by direct injection into an area containing cells affected by the disease. For example, the pharmaceutical composition can be delivered by direct injection into the brain. The injection can be by stereotactic injection into a specific area of ​​the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The compound can be delivered to multiple areas of the central nervous system (e.g., multiple areas of the brain and / or to the spinal cord). The compound can be delivered to a diffuse area of ​​the brain (e.g., diffuse delivery to the cortex of the brain).

[0298] In one embodiment, the compound can be delivered by a cannula or other delivery device with one end implanted into a tissue, such as the brain, for example, the substantia nigra, cortex, hippocampus, striatum, or globus pallidus of the brain. The cannula can be connected to a reservoir containing the compound. The flow or delivery can be mediated by a pump, such as an osmotic pump or a minipump, such as an Alzet pump (Durect, Cupertino, CA). In one embodiment, the pump and reservoir are implanted in a region remote from the tissue, such as the abdomen, and delivery is achieved by a conduit leading from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in U.S. Patent Nos. 6,093,180 and 5,814,014. [Example]

[0299] Materials and Methods Synthesis of lipid-functionalized solid supports Non-phosphocholine (PC) lipid moieties (except α-tocopherol succinate) were directly conjugated via peptide bonds to controlled pore glass (CPG) functionalized with a C7 linker, as previously described (Nikan M, Osborn MF, Coles AH, et al. Docosahexaenoic acid conjugation enhances distribution and safety of siRNA upon local administration in mouse brain. Mol. Ther. Nucleic Acids. 2016;5:e344). To synthesize PC derivatives, amino-C7 CPG was first functionalized with phosphocholine (Nikan M, Osborn MF, Coles AH, et al. Synthesis and evaluation of parenchymal retention and efficacy of a metabolically stable O-phosphocholine-N-docosahexaenoyl-l-serine siRNA conjugate in mouse brain. Bioconjug. Chem. 2017;28:758-1766). Briefly, Fmoc-L-serine tert-butyl ester (TCI America) was phosphitylated using 2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (ChemGenes). The resulting phosphoramidite was coupled to choline p-toluenesulfonate (Alfa Aesar) using 5-(ethylthio)-1H-tetrazole (ETT) as the activating agent. The phosphine ester was then oxidized, and the carboxylic acid and phosphate ester groups were deprotected (i.e., the tert-butyl and cyanoethyl groups were removed). The resulting intermediate was conjugated to amino C7 CPG via a peptide bond to form a phosphocholine-functionalized CPG. The Fmoc group was removed, and the selected lipid moiety was attached to the CPG via a peptide bond. All lipid-functionalized solid supports were obtained at a loading of 55 μmol / g.

[0300] Synthesis of α-tocopherol succinate-linked oligonucleotides α-Tocopherol succinate was conjugated to the amino group at the 3' end of purified oligonucleotides synthesized on amino C7 CPG or phosphocholine-functionalized amino C7 CPG. N-hydroxysuccinimide α-tocopherol succinate and purified oligonucleotides were combined in a solution of 0.1 M sodium bicarbonate and 20% (v / v) dimethylformamide and incubated overnight at room temperature. One-tenth the volume of 3 M sodium acetate (pH 5.2) was added to a final concentration of 0.3 M sodium acetate. Three volumes of 95% (v / v) ethanol were added, the mixture was vortexed, and then placed at -80°C for 1 hour. The solution was pelleted by centrifugation at 5200 x g for 30 minutes. The pellet containing the lipid-conjugated siRNA sense strand was dissolved in water, purified, and desalted as described below.

[0301] Oligonucleotide synthesis Oligonucleotides were synthesized by phosphoramidite solid-phase synthesis on a Dr Oligo 48 (Biolytic, Fremont, CA) or MerMade 12 (Biosearch Technologies, Novato, CA) using 2'-F or 2'-O-Me modified phosphoramidites with standard protecting groups. 5'-(E)-vinyltetraphosphonate (pivaloyloxymethyl) 2'-O-methyluridine 3'-CE phosphoramidite (VP) for in vivo non-ligated oligonucleotides was purchased from Hongene Biotech, USA, and Quasar 570 CE phosphoramidite (Cy3) was purchased from GenePharma, Shanghai, China. Bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite (5'P) for in vitro non-ligated oligonucleotides and all other phosphoramidites used were purchased from ChemGenes, Wilmington, MA. Phosphoramidites were prepared at 0.1 M in anhydrous acetonitrile (ACN), except for 2'-O-methyluridine phosphoramidite, which was dissolved in anhydrous ACN containing 15% dimethylformamide. 5-(benzylthio)-1H-tetrazole (BTT) was used as the activator at 0.25 M, and the coupling time for all phosphoramidites was 4 min using 10 equivalents. Detritylation was performed using 3% trichloroacetic acid in dichloromethane. The capping reagents used were CAP A (20% n-methylimidazole in ACN) and CAP B (20% acetic anhydride and 30% 2,6-lutidine in ACN). Reagents for capping and detritylation were purchased from AIC, Framingham, MA. Phosphite oxidation to convert to phosphate or phosphorothioate was carried out for 4 min using 0.05 M iodine in pyridine-HO (9:1, v / v) or a 0.1 M solution of 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine (ChemGenes). Unconjugated oligonucleotides were synthesized on 500 Å long-chain alkylamine (LCAA) controlled-pore glass (CPG) functionalized with Unylinker termini (ChemGenes).Cholesterol-conjugated oligonucleotides were synthesized on a 500Å LCAA-CPG support, where the cholesterol moiety was attached to tetraethylene glycol via a succinic acid linker (ChemGenes, Wilmington, MA). Lipid-conjugated oligonucleotides were synthesized on a modified solid support (synthesis described above). Bivalent oligonucleotides (dimers) were synthesized on a modified solid support using a previously described synthesis (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)).

[0302] Synthesis of branched oligonucleotides Branched oligonucleotide synthesis was performed by phosphoramidite solid phase on an AKTA Oligoplilot 10 (Cytiva, Marlborough, MA) using the parameters described above or otherwise specified herein. Trimeric and tetrameric branched oligonucleotides were prepared using commercially available trebler and doubler phosphoramidites, respectively, purchased from Glen Research, Sterling, VA. Trimeric linkers were generated in two steps: first, 10 equivalents of DMT-tetraethyloxy-glycol CED phosphoramidite (ChemGenes) were coupled onto 1000 Å thymidine 3'-LCAA-CPG (ChemGenes) for 8 minutes, followed by 10 equivalents of trebler phosphoramidite for 8 minutes. Trivalent oligonucleotides were then grown on this linker using 30 equivalents. Tetrameric linkers were generated in three steps: First, on a 1000 Å thymidine 3'-LCAA-CPG, 10 equivalents of DMT-tetraethyloxyglycol CED phosphoramidite were coupled for 8 minutes, followed by 10 equivalents of doubler phosphoramidite for 8 minutes, followed by 20 equivalents of doubler phosphoramidite for 8 minutes, followed by 40 equivalents of tetravalent oligonucleotide growth.

[0303] Deprotection and purification of oligonucleotides for sequence screening Prior to deprotection, the synthesis column containing the oligonucleotides was treated with 10% diethylamine (DEA) in ACN to deprotect the cyanoethyl groups. In the synthesis column, both the unbound and cholesterol-bound oligonucleotides on the solid support were deprotected with methylamine gas (Airgas) at room temperature for 1 hour. The deprotected oligonucleotides released from the solid support were precipitated onto the support by passing a mixture of (i) 0.1 M sodium acetate in 85% ethanol, followed by (ii) 85% ethanol through the synthesis column. The excess ethanol on the solid support was dried with a stream of air, and the oligonucleotides were washed out by passing water through the column. This procedure yielded pure oligonucleotides for use in in vitro experiments.

[0304] Deprotection and purification of oligonucleotides for in vivo experiments Prior to deprotection, the synthesis column containing the oligonucleotides was treated with 10% diethylamine (DEA) in ACN to deprotect the cyanoethyl groups. Cy3-labeled and lipid-conjugated oligonucleotides were cleaved and deprotected in 28–30% ammonium hydroxide, 40% methylamine (1:1, v / v) (AMA) at room temperature for 2 hours. Cy3-labeled and unlabeled unconjugated, bivalent, trivalent, and tetravalent oligonucleotides were cleaved and deprotected by AMA treatment at 45°C for 2 hours. VP-containing oligonucleotides were cleaved and deprotected as previously described without post-synthesis DEA pretreatment (O'Shea J, Theile CS, Das R, et al., An efficient deprotection method for 5'-[O,O-bis(pivaloyloxymethyl)]-(E)-vinylphosphonate containing oligonucleotides. Tetrahedron 74, 6182–6186 (2018)). Briefly, CPGs bearing VP-oligonucleotides were treated with a 3% DEA solution in 28–30% ammonium hydroxide at 35°C for 20 h.

[0305] All solutions containing cleaved oligonucleotides were filtered to remove CPG and dried under vacuum. The resulting pellet was resuspended in 5% ACN in water. Purification was performed on an Agilent 1290 Infinity II HPLC system. VP and unlabeled unconjugated, bivalent, trivalent, and tetravalent oligonucleotides were purified using a custom 25 x 150 mm column packed with Source 15Q anion exchange resin (Cytiva, Marlborough, MA). Run conditions: Eluent A, 10 mM Tris-HCl buffer (pH 9) in 7.5% ACN in water; Eluent B, 1 M sodium perchlorate in 10 mM Tris-HCl buffer (pH 9) in 7.5% ACN in water; linear gradient, 12 to 35% B in 40 min at 50 °C. Lipid-conjugated and Cy3-labeled oligonucleotides were purified using a 21.2 x 150 mm PRP-C18 column (Hamilton Co., Reno, NV). Run conditions: Eluent A, 50 mM sodium acetate in 5% ACN in water (pH 6); Eluent B, 100% ACN; linear gradient, 15–60% B in 40 min at 60 °C. The flow rate for both methods was 40 mL / min, and peaks were monitored at 260 nm for unlabeled oligonucleotides and 550 nm for labeled oligonucleotides. To avoid cross-contamination, a separate column was used for Cy3-labeled oligonucleotides. Fractions were analyzed by liquid chromatography-mass spectrometry (LC-MS), and pure fractions were combined and dried under vacuum. The oligonucleotides were resuspended in 5% ACN and desalted by size exclusion on a 25 x 250 mm custom column packed with Sephadex G-25 medium (Cytiva, Marlborough, MA) using an isocratic method with HPLC-grade water (Honeywell Chemicals, Charlotte, NC). Finally, the oligonucleotides were lyophilized.

[0306] LC-MS analysis of oligonucleotides The identity of the oligonucleotides was verified by LC-MS analysis on an Agilent 6530 accurate-mass Q-TOF using the following conditions: Buffer A: 100 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 9 mM triethylamine (TEA) in LC-MS-grade water; Buffer B: 100 mM HFIP and 9 mM TEA in LC-MS-grade methanol; Column: Agilent AdvanceBio Oligonucleotide C18; Linear gradient: 0–40% B in 5 min (unconjugated, bivalent, trivalent, and tetravalent oligonucleotides); Linear gradient: 50–100% B in 5 min (lipid-conjugated and Cy3-labeled oligonucleotides); Temperature: 60°C; Flow rate: 0.85 ml / min. LC peaks were monitored at 260 nm and 550 nm for labeled oligonucleotides. MS parameters: source, electrospray ionization; ion polarity, negative mode; range, 100–3,200 m / z; scan rate, 2 spectra / s; capillary voltage, 4,000; fragmentor, 200 V; gas temperature, 325 °C.

[0307] In vivo experiments Intravitreal injections into adult mice were performed as previously described (Venkatesh A, Ma S, Langellotto F, et al. Retinal gene delivery by rAAV and DNA electroporation. Curr Protoc Microbio: 2013; Chapter 14: Unit 14D 14). Injections were performed using a glass needle (Clunbury Scientific LLC, Catalog No. B100-58-50) with a constant pressure and injection time of 300 psi and 1.5 s, respectively, delivering approximately 2 mL of fluid into the vitreous. All concentrations were adjusted to use a 2 mL injection volume for the desired amount of siRNA. For intravitreal injections into adult pigs, 100 mL of siRNA was injected into the vitreous approximately 2–3 mm from the temporal limbus using an insulin syringe needle. Anesthesia and euthanasia of the pigs were performed by a veterinary specialist according to standard procedures. Prior to siRNA injection, the corneas were treated with proparacaine and ophthalmic betadine. After injection, the eyes were washed with saline eyewash. Enucleated pig and mouse eyes were treated as described (Venkatesh A, Ma S, Langellotto F, et al. Retinal gene delivery by rAAV and DNA electroporation. Curr Protoc Microbio: 2013; Chapter 14: Unit 14D 14.).

[0308] Example 1: Delivery of siRNA for ocular diseases. An initial screen of different siRNAs targeting the Htt gene, all with the same sequence, was performed to examine their distribution and knockdown efficiency in the eye.

[0309] It is difficult to determine cellular distribution from Cy3 labeling alone. Therefore, we injected a lower dose of siRNA compound (0.1 nmol) (Cy3-labeled siRNA is shown in red in Figure 1). After 3 days, tissues were dissociated, Cy3-positive cells were sorted by FACS, and cell-type-specific antibodies were used to determine which cell types were enriched by which siRNA modifications.

[0310] In Figure 1, all siRNAs are labeled with Cy3 and shown in red, while Müller glial cell-specific glutamine synthetase (GS) expression is shown in green and nuclear DAPI is shown in blue. All siRNAs can be observed throughout the retinal cross section, although their cellular distribution varies slightly. The right side of Figure 1 shows one example per group, showing the entire retinal cross section with its distribution throughout the retina. Half of the cross section shows nuclear DAPI, GS, and siRNA, while the other half shows only siRNA.

[0311] The overall goal was to A) determine the cellular distribution and B) identify modifications that allow optimal cellular entry into cone and rod photoreceptors and Müller glia cells, as these three cell types are the most important targets for many retinal diseases. Figures 2 and 3 show the enrichment results for siRNAs in different retinal cell types, arranged by cell type (Figure 2) and modification (Figure 3). The results suggest that monomeric configurations are optimal for cone photoreceptors, tetramers for rod photoreceptors, and trimers for Müller glia cells. Other excellent Müller glia cell-stimulating compounds are PC-TS, PC-DHA, and DCA.

[0312] Repeated injections of a subset of these compounds and high-magnification observations show that PC-TS accumulates primarily in Müller glial cells, and trimers and tetramers accumulate primarily in rod photoreceptors (Figures 4 and 5).

[0313] To determine whether the tetramer could knockdown HTT protein in photoreceptors more effectively than other configurations, antibody staining for HTT protein was performed 2 weeks after intravitreal injection of 0.3 nmoles of siRNA, as shown in Figure 6. The first column in Figure 6 shows antibody staining in control mice injected with NTC-siRNA. HTT protein exhibits pan-retinal expression, particularly concentrated in the photoreceptor inner segment (IS) and the outer plexiform layer (OPL), where photoreceptors form synaptic connections with bipolar and horizontal cells; moderately concentrated in the inner nuclear layer (INL), where bipolar, amacrine, and horizontal cells, and Müller glial cell bodies are present; and strongly concentrated in the inner plexiform layer (IPL), where synaptic connections with amacrine, bipolar, and ganglion cells are present. The second column shows HTT protein expression after knockdown with PC-RA-Htt siRNA. This siRNA tends to preferentially accumulate in bipolar and amacrine cells, as shown in Figures 2 and 3. Therefore, expression in the OPL and IPL is more efficiently reduced by this siRNA. The third column shows HTT protein expression in two different mice, each injected with approximately 0.3 nmoles of Htt-siRNA, after knockdown with tetrameric Htt siRNA. This configuration tends to efficiently accumulate in rod photoreceptors and bipolar cells. Consistently, expression in the photoreceptor IS is much reduced than in the case of PC-RA-Htt siRNA.

[0314] Two weeks after Htt-siRNA injection, we also performed quantification of remaining total HTT protein from whole retinal extracts by Western blotting using the same experimental setup as in Figure 6, using different mice from the same injection batch. Note that when quantifying total HTT protein, knockdown is approximately 50% relative expression at two weeks post-injection. The two configurations targeted different cell populations in the retina with different efficiencies. Nevertheless, due to the ubiquitous expression of HTT in the retina, the overall knockdown is similar.

[0315] Example 2: Evaluation of Htt mRNA knockdown by bDNA assay. Two weeks after Htt-siRNA injection, using 0.1 nmoles per injection of the indicated siRNA modifications, total Htt mRNA levels were quantified using a bDNA assay. As shown in Figure 8, quantification of Htt mRNA levels revealed that 0.1 nmoles resulted in approximately 20%–30% knockdown two weeks after injection.

[0316] We also quantified total Htt mRNA levels using a bDNA assay 3 days after Htt-siRNA injection using 0.3 nanomoles per injection of the described siRNA modifications. When quantifying Htt mRNA levels, note that at 0.3 nanomoles, approximately 30%–60% knockdown occurred 3 days after injection. Furthermore, PC-RA showed a similar knockdown rate when compared to total protein measurements at 2 weeks after injection (Figure 7: 60% knockdown), indicating that both quantification methods are similar for the Htt gene in the retina and that there is a direct correlation between mRNA and protein levels of this gene (each dot represents one retina).

[0317] Example 3: Long-term evaluation of Htt mRNA knockdown by bDNA assay Figure 10 shows the results of a bDNA assay performed to quantify total Htt mRNA levels 100 days after Htt-siRNA injection using 0.3 nmoles per injection of the indicated siRNA modifications. Note that, compared to Figure 9, the knockdown effect only changed by approximately 10% (from 60% to 50% knockdown) over a time frame of approximately 100 days. This indicates that the knockdown is highly stable (each dot in Figure 10 represents one retina). Figure 11 shows representative fundus images over time of an eye injected with Cy3-labeled siRNA with the modifications indicated. The fluorescent signal exposure is the same for all four siRNAs at any given time point, but not over time. Figure 11 supplements Figure 10 and shows fundus images of the mice used in Figure 10. All mice were intravitreally injected with 0.3 nmoles of siRNA.

[0318] Example 4: Dose-escalating study of HTT knockdown in mice with tetramer configuration Figure 12 shows the results of a dose-escalation study of HTT knockdown in the retina. Mice were injected with the indicated amounts (Cy3-labeled tetramer 1–60 carrying Htt-siRNA) in a total volume of 2 microliters. Five mice were injected per siRNA amount. Tissues were harvested two weeks post-injection, and residual HTT protein in the retina was quantified by Western blotting. Injections of 15–30 micrograms roughly correspond to the same knockdown seen in previous experiments with approximately 0.3 nanomoles.

[0319] Figure 14 shows retinal cross sections of eyes from a dose-escalation study of HTT knockdown in mice with the tetramer configuration, the results of which are shown in Figures 12 and 13. The images show Cy3 distribution throughout the retinal section, indicating uniform uptake of the siRNA throughout the eye.

[0320] To assess toxicity, antibody staining was performed on retinal sections from the eye shown in Figure 14 to identify Iba1-positive cells and changes in GFAP. Figure 15 shows retinal sections from an eye stained with Iba1 (green) to identify Iba1-positive cells migrating to the outer nuclear layer (ONL), where photoreceptors reside. Iba1-positive cells in the ONL were observed at 60 micrograms per injection and occasionally at 30 micrograms, indicating an inflammatory response at 60 micrograms and a mild response at 30 micrograms. Half of each panel (dotted lines in Figure 15) shows only the Iba1 signal to better visualize the signal. Blue indicates nuclear DAPI. Figure 16 shows retinal sections from an eye from the dose-escalation study shown in Figure 14 stained with GFAP (red) to identify reactive gliosis in Müller glial cells. Although a slight increase in GFAP expression is observed at the level of the ganglion cell layer (GCL), where astrocytes reside, the expression does not extend upward to Müller glia. GFAP expression in astrocytes is normal. Expression is increased at 60 micrograms, consistent with the results seen with Iba1. However, the absence of reactive gliosis indicates the absence of severe retinal degenerative events induced by siRNA. Because these sections are from the same eye as shown in Figure 15, siRNA is not shown. In Figure 16, blue indicates nuclear DAPI, and green marks cone photoreceptor segments with peanut agglutinin lectin (PNA). Figure 17 shows measurements of photoreceptor and retinal function by electroretinography under scotopic (0.01 cd.s / m² to 1 cd.s / m²) and photopic conditions (3 and 10 flashes). A-wave and b-wave recordings demonstrate normal photoreceptor and inner retinal function, respectively, for all injected doses. As can be seen in the top two graphs, there are no statistically significant differences between the recordings (n=5 mice per siRNA dose). The implicit times of the a-wave and b-wave (bottom two graphs) are also not significantly different between the different groups of injected mice.

[0321] Example 5: Large Animal Model: siRNA in Pig Eyes (All data shown below is generated using tetramer-Htt-siRNA-Cy3 and its NTC in pigs) To further advance the application of siRNA technology, we tested its suitability in a large animal model to determine distribution, knockdown efficiency, and toxicity. For this purpose, we chose a pig model (using a 35 kg pig) because the pig eye is approximately the same size as a human eye. The only difference is the absence of a fovea. For the initial test run, three pigs were injected with five different amounts of siRNA of the same chemical configuration, with the injection volume kept constant at 100 microliters. The following data summarize the results of injecting pigs with siRNA against HTT in a tetrameric configuration. All siRNA molecules were also labeled with Cy3. The pigs were euthanized 10 days after intravitreal injection. Figure 18 shows the fluorescence intensity of tetramer-Htt-Cy3 after intravitreal delivery in pig eyes. The amount of siRNA delivered is indicated at the top of each panel in Figure 18 (100–1500 micrograms of tetramer). The fluorescence intensity is well distributed throughout the eye. The top row of Figure 18 shows Cy3 fluorescence in unfixed tissue immediately after opening the eye, while the bottom panel is a high magnification of the area from the top panel.

[0322] Huntington protein knockdown in pigs was measured by Western blot analysis from the pig eyes shown in Figure 18. Knockdown was compared to huntingtin protein levels in NTCs injected with 250 μg of tetramer-siRNA-Cy3. In Figure 19, the top panel shows the knockdown observed in the four major retinal quadrants (DT: dorsal-temporal, DN: dorsal-nasal, VT: temporo-nasal, VN: ventral-nasal) as a bar graph, with error bars generated by technical replicates. Knockdown efficiency in each quadrant depends on the needle position and insertion angle. The needle was generally inserted from the temporal side and aimed toward the center of the eye. The middle panel of Figure 19 shows the knockdown in a flat-mount view, with the corresponding values ​​of regional knockdown shown as a bar graph. The bottom panel of Figure 19 shows the average knockdown of huntingtin protein across the entire retina, calculated by averaging the knockdown observed in each quadrant per retina; error bars are generated by averaging the four data points for each quadrant per retina. The data shown in the bottom panel of Figure 19 represent one biological sample per amount of siRNA delivered.

[0323] Figure 20 shows antibody staining for Huntington protein in sections from eyes injected with different doses as shown in Figure 19. Regions of the eye sections are shown in the center panel of Figure 19. Huntington knockdown is seen throughout all retinal layers, particularly in the inner and outer plexiform layers (IPL, OPL) and where the photoreceptor segments (PS) are located.

[0324] Figure 21 shows antibody staining for GFAP (glial fibrillary acidic protein) and Iba1 (ionized calcium-binding adaptor protein 1) expression (as shown for mice in Figures 15 and 16) in retinal sections from eyes injected with different doses, as shown in Figures 18 and 19, to determine dose-dependent toxicity. Both GFAP and Iba1 are shown in green in Figure 21, as indicated on the left side of each row. Red staining in Figure 21 indicates the distribution of siRNA throughout the retinal section, while nuclei are marked with nuclear DAPI. GFAP and Iba1 expression clearly increases in a dose-dependent manner. Up to 500 μg of siRNA expression for Iba1 and GFAP rarely progresses to the outer nuclear layer (ONL), where photoreceptors reside. At 1000 μg and 1500 μg, GFAP and Iba1 expression in the ONL increases appreciably. Furthermore, much of the siRNA appears to be taken up by Iba1-positive cells, likely reflecting macrophages taking up excess extracellular material. Half of each panel in Figure 21 shows only the signal of interest (siRNA, GFAP, or Iba1) to better visualize the signal.

[0325] Summary of porcine data: Tetramer-Htt-siRNA distributes well throughout the retina after a single intravitreal delivery in large eyes such as pigs. This is particularly important because pig eyes are approximately the same size as human eyes. Except for the lack of a fovea, the pig eye is the closest animal model to the human eye. For distribution studies, its similar size compared to most experimental NHPs makes it more relevant. The dose-response in Figure 19 and toxicity in Figure 21 indicate that for this particular compound, doses in the 100-500 μg range could be used for further studies. This should result in approximately 50% knockdown of HTT, similar to that seen in mice with tetramers. Toxicity can be reduced by removing the Cy3 molecule (which was still attached in this study).

[0326] Example 6: Development of siRNA against S6K1 (RPS6KB1: ribosomal protein S6 kinase B1) An initial bioinformatics screen was performed to identify potential siRNA sequences for S6K1. The sequences identified in the initial bioinformatics screen are listed in Tables 1 and 2 below. [Table 1-1] [Table 1-2]

[0327] For the 45 nucleotide gene region listed above, the sequence corresponds to the DNA gene sequence, but the mRNA encoded by the S6K1 gene has the same sequence, with T nucleotide replaced by U nucleotide.Therefore, as an example, the siRNA with antisense strand targeting SEQ ID NO: 1 targets the mRNA sequence corresponding to the gene region of SEQ ID NO: 1. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0328] For the sense and antisense sequences in Table 3 and Table 3, "m" corresponds to a 2'-O-methyl modified nucleotide, "f" corresponds to a 2'-fluoro modified nucleotide, "#" corresponds to a phosphorothioate internucleotide linkage, "P" corresponds to a 5' phosphate, and "TegChol" corresponds to a tri- or tetraethylene glycol-linked cholesterol moiety.

[0329] Figure 22 shows the initial in vitro knockdown efficiency of duplexes formed from the sense and antisense strands shown in Tables 3 and 4. The candidates that showed the best knockdown results were duplexes 2, 3, 7, 9, 10, and 19. Figure 23 shows the dose-response curves of the four sequences highlighted in red in Figure 22. Duplex 2 (Rps6k1b_459) showed the most consistent response and was therefore selected for further in vivo studies.

[0330] The primary purpose of siRNA against S6K1 is to knockdown S6K1 in photoreceptors for the treatment of AMD. Based on data generated by different HTT-siRNA conjugates, we first developed siRNA in a tetrameric configuration without any Cy3 label to reduce toxicity. Next, we generated in vivo data from mice and NHPs based on the tetrameric configuration of duplex 2 (Rps6k1b_459).

[0331] Figure 24 shows RNA-Scope in situ hybridization in mouse retinal cross sections to detect siRNA tetramers against S6K1. The top row of Figure 24 shows sections from three mice injected with NTC against S6K1 in tetrameric configuration, the middle row shows sections from three mice injected with siRNA against S6K1 in tetrameric configuration at 3 μg / eye, and the last row shows sections from three 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.

[0332] Figures 25A and 25B show knockdown of S6K1 in mice after intravitreal injection of 6 μg of tetramer-configured siRNA. Both graphs in Figure 25 use rodTSC1- / - mice, which have been shown to develop pathology similar to age-related macular degeneration. rodTSC1+ / + mice are Cre-negative littermate controls that do not develop pathology. Figure 25A shows S6K1 protein levels detected by Western blot 2 weeks after injection. There is a slight trend toward a decrease in S6K1 protein compared to uninjected littermates or NTC mice. Figure 25B shows similar data 2 months after injection. Strong knockdown (40-45%) is observed with 6 μg of siRNA. Each dot in the graphs in Figure 25 represents one biological sample (retina) from one animal.

[0333] Figure 26 shows knockdown of S6K1 protein in non-human primates (NHPs). Western blot data are shown using retinal protein extracts from the superior temporal (ST) region (also known as the dorsal-temporal region) of one NHP intravitreally injected with 225 μg of S6K1-tetramer (in 75 μL) and six naive NHP retinas from the same region. The first set of bar graphs shows a comparison between the uninjected contralateral eye and the S6K1 siRNA-injected eye to allow for direct intraanimal comparison between the two eyes. The second set of bar graphs shows a comparison between six naive NHPs and NHPs injected with S6K1 siRNA. S6K1 knockdown efficiency is approximately 50% in both cases. NHP eyes were harvested one month after injection. Also shown is a reduction in phosphorylation of the ribosomal protein S6, the canonical target of S6K1. As with the S6K1 knockdown data, within-animal comparisons are shown on the left and comparisons with multiple NHPs are shown on the right.

[0334] Figure 27 shows knockdown of S6K1 protein in retinal cross sections from non-human primates (NHPs) after siRNA treatment. Data were generated from one injected eye (see also Figure 26) and the contralateral uninjected eye. Sections were obtained from the central region, as shown for the pig in Figure 19. The entire cross section, including the fovea, is shown on the left side of Figure 27. A magnified view of the temporal and nasal regions and the fovea is shown on the right side of Figure 27. The top row of Figure 27 shows an uninjected eye, and the bottom row shows an eye intravitreally injected with 225 μg of S6K1 tetramer (in 75 μL). Consistent with the Western blot data generated in the superior temporal region of the same eye shown in Figure 26, there is a clear decrease in the signal for S6K1 protein expression. This decrease is also evident in the fovea, which contains only cones, indicating that knockdown in cones is as efficient as knockdown in rods.

[0335] Figure 28 shows the reduction of phosphorylated S6 protein (pS6) in retinal cross sections from non-human primates (NHPs) after siRNA treatment. The data in Figure 28 are the same as those shown in Figure 27, except for the staining probe for pS6 expression (red signal). A clear reduction in pS6 is seen throughout the retina, particularly in photoreceptors, including foveal cones. In each panel of Figure 28, the green and blue signals have been removed from half of the panel (dotted lines) to better visualize pS6 knockdown. Blue in Figure 28 indicates nuclear DAPI, while green indicates cone segments marked with peanut agglutinin lectin (PNA).

[0336] Figure 29 shows the expression of inflammatory markers in NHPs after siRNA treatment with S6K1 siRNA (75 μL, 225 μg of tetramer-configured siRNA). The data in Figure 29 are the same as those shown in Figures 27 and 28, except for the staining probes for 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 eye is in the second row. In the siRNA-treated eyes, there is a slight increase in Iba1-positive cells migrating toward the photoreceptor layer. However, there is no reactive gliosis, as seen with GFAP staining. When astrocytes are present in the ganglion cell layer, there is only a slight increase in staining. There is no increase in the fovea. The data shown in Figure 29 suggest that siRNA treatment for S6K1 knockdown does not have any severe adverse effects. In each panel of Figure 29, the green and blue signals have been removed from half of the panel (dotted lines) to better visualize the Iba1 and GFAP signals. In FIG. 29, blue indicates nuclear DAPI, while green indicates cone segments marked with peanut agglutinin lectin (PNA).

[0337] Summary of S6K1-siRNA Data. Tetramer-S6K1-siRNA distributes well throughout the retina after a single intravitreal delivery of 6 μg into mouse eyes. Knockdown efficiency appears slow initially but becomes very robust over time. A therapeutic target of approximately 50% knockdown of S6K1 protein in photoreceptors is required, and this appears achievable. Duplex 2, selected from initial screening, functions very efficiently in vivo, indicating that the S6K1 target site in SEQ ID NO: 1 is a useful target for S6K1 knockdown. Based on the dose-response curve of HTT-tetramer in mice, a subset of mice will be injected with 25 μg / eye and analyzed in the near future for S6K1 knockdown and markers of age-related macular degeneration to determine whether disease progression is ameliorated. Injections into NHPs confirm that knockdown functions equally efficiently in large eyes, distribution is widespread, a therapeutic window is achievable, and there is no significant inflammatory response to treatment. Overall, the data indicate that gene knockdown in humans is feasible for the treatment of a variety of retinal diseases.

Claims

1. 1. An siRNA comprising a sense strand and an antisense strand, The siRNA, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1 to 6.

2. The siRNA of claim 1, wherein the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 7 to 12.

3. 2. The siRNA of claim 1, comprising complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1-6.

4. 4. The siRNA of claim 1 or 3, which comprises no more than three mismatches with the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1 to 6.

5. The siRNA of claim 1, comprising perfect complementarity to the S6K1 nucleic acid sequence of any one of SEQ ID NOs: 1 to 6.

6. The siRNA according to any one of claims 1 to 5, wherein the length of the antisense strand is about 15 to 25 nucleotides.

7. The siRNA according to any one of claims 1 to 6, wherein the length of the sense strand is about 15 to 25 nucleotides.

8. The siRNA according to any one of claims 1 to 7, wherein the antisense strand is 20 nucleotides in length.

9. The siRNA according to any one of claims 1 to 7, wherein the antisense strand is 21 nucleotides in length.

10. The siRNA according to any one of claims 1 to 7, wherein the antisense strand is 22 nucleotides in length.

11. The siRNA according to any one of claims 1 to 10, wherein the sense strand is 15 nucleotides in length.

12. The siRNA according to any one of claims 1 to 10, wherein the sense strand is 16 nucleotides in length.

13. The siRNA according to any one of claims 1 to 10, wherein the sense strand is 18 nucleotides in length.

14. The siRNA according to any one of claims 1 to 10, wherein the sense strand is 20 nucleotides in length.

15. The siRNA of any one of claims 1 to 14, comprising a double-stranded region of 15 to 20 base pairs.

16. The siRNA of any one of claims 1 to 15, comprising a double-stranded region of 15 base pairs.

17. The siRNA of any one of claims 1 to 15, comprising a double-stranded region of 16 base pairs.

18. The siRNA of any one of claims 1 to 15, comprising a double-stranded region of 18 base pairs.

19. The siRNA of any one of claims 1 to 15, comprising a double-stranded region of 20 base pairs.

20. The siRNA of any one of claims 1 to 19, wherein the siRNA comprises at least one blunt end.

21. The siRNA of any one of claims 1 to 20, wherein the siRNA comprises at least one single-stranded nucleotide overhang.

22. 22. The siRNA of claim 21, wherein the siRNA comprises a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

23. 22. The siRNA of claim 21, wherein the siRNA comprises a single-stranded nucleotide overhang of 2 nucleotides.

24. 22. The siRNA of claim 21, wherein the siRNA comprises a single-stranded nucleotide overhang of 5 nucleotides.

25. The siRNA of any one of claims 1 to 24, wherein the siRNA comprises naturally occurring nucleotides.

26. The siRNA of any one of claims 1 to 24, wherein the siRNA comprises at least one modified nucleotide.

27. 27. The siRNA of claim 26, 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, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing nucleotide, or a mixture thereof.

28. The siRNA of any one of claims 1 to 27, wherein the siRNA comprises at least one modified internucleotide bond.

29. 29. The siRNA of claim 28, wherein the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage.

30. 30. The siRNA of any one of claims 1 to 29, comprising 4 to 16 phosphorothioate internucleotide linkages.

31. 30. The siRNA of any one of claims 1 to 29, comprising 8 to 13 phosphorothioate internucleotide linkages.

32. 30. The siRNA of any one of claims 1 to 29, wherein the antisense strand comprises 2 to 10 phosphorothioate internucleotide linkages.

33. The siRNA of any one of claims 1 to 32, wherein the siRNA comprises at least 80% chemically modified nucleotides.

34. The siRNA of any one of claims 1 to 33, wherein the siRNA is fully chemically modified.

35. The siRNA of any one of claims 1 to 33, wherein the siRNA comprises at least 70% 2'-O-methyl nucleotide modifications.

36. The siRNA of any one of claims 1 to 33, wherein the antisense strand comprises at least 70% 2'-O-methyl nucleotide modifications.

37. The siRNA of claim 36, wherein the antisense strand comprises about 70% to 90% 2'-O-methyl nucleotide modifications.

38. The siRNA of any one of claims 1 to 33, wherein the sense strand comprises at least 65% 2'-O-methyl nucleotide modifications.

39. 39. The siRNA of claim 38, wherein the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

40. The siRNA of any one of claims 1 to 39, wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

41. The siRNA of claim 40, wherein the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.

42. 41. The siRNA of claim 40, wherein the nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.

43. The siRNA of any one of claims 1 to 42, wherein the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate.

44. The siRNA of claim 43, wherein the antisense strand comprises a 5' vinyl phosphonate.

45. The siRNA of any one of claims 1 to 44, wherein a functional moiety is linked to the 5'-end and / or the 3'-end of the antisense strand.

46. The siRNA of any one of claims 1 to 44, wherein the functional moiety is linked to the 5'-end and / or the 3'-end of the sense strand.

47. The siRNA of any one of claims 1 to 44, wherein the functional moiety is linked to the 3' end of the sense strand.

48. The siRNA of any one of claims 45 to 47, wherein the functional moiety comprises a hydrophobic moiety.

49. 49. The siRNA of claim 48, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

50. 50. The siRNA of claim 49, wherein the steroid is selected from the group consisting of cholesterol and lithocholic acid (LA).

51. 50. The siRNA of claim 49, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

52. 50. The siRNA of claim 49, wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof.

53. 53. The siRNA of claim 52, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

54. 49. The siRNA of claim 48, wherein the functional moiety comprises any one of a triple amine, retinoic acid, docosahexaenoic acid (DHA), docosanoic acid (DCA), α-tocopherol succinate, or lithocholic acid (LA).

55. The siRNA of any one of claims 45 to 54, wherein the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

56. 56. The siRNA of claim 55, wherein the linker comprises a bivalent or trivalent linker.

57. the bivalent or trivalent linker is selected from the group consisting of: 【Chemical 1】 57. The siRNA of claim 56, wherein n is 1, 2, 3, 4, or 5.

58. 57. The siRNA of claim 55 or 56, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

59. 58. The siRNA of claim 56 or 57, wherein when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

60. the phosphodiester or phosphodiester derivative is selected from the group consisting of: 【Chemistry 2】 wherein X is O, S or BH ３ The siRNA of claim 59,

61. 61. The siRNA of any one of claims 1 to 60, wherein the nucleotides at positions 1 and 2 from the 3' end of the sense strand and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate bonds.

62. A pharmaceutical composition for inhibiting the expression of the S6K1 gene in an organism, comprising the siRNA according to any one of claims 1 to 61 and a pharmaceutically acceptable carrier.

63. 63. The pharmaceutical composition of claim 62, wherein the siRNA inhibits expression of the S6K1 gene by at least 20%.

64. 63. The pharmaceutical composition of claim 62, wherein the siRNA inhibits expression of the S6K1 gene by at least 50%.

65. 1. A method for inhibiting expression of the S6K1 gene in a cell, comprising: (a) introducing the siRNA according to any one of claims 1 to 61 into the cell; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the S6K1 gene, thereby inhibiting expression of the S6K1 gene in the cells.

66. 62. A method of treating or managing an ocular disease, comprising administering to a patient in need of such treatment a therapeutically effective amount of the siRNA of any one of claims 1 to 61.

67. 67. The method of claim 66, wherein the siRNA is administered to the eye of the patient.

68. 67. The method of claim 66, wherein the siRNA is administered by intravitreal injection.

69. The method of any one of claims 65 to 68, wherein the siRNA inhibits expression of the S6K1 gene by at least 20%.

70. The method of any one of claims 65 to 68, wherein the dsRNA inhibits expression of the S6K1 gene by at least 50%.

71. A vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding a dsRNA substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NOs: 1-6.

72. 72. The vector of claim 71, wherein the dsRNA inhibits expression of the S6K1 gene by at least 20%.

73. 72. The vector of claim 71, wherein the dsRNA inhibits expression of the S6K1 gene by at least 50%.

74. The vector of claim 71, wherein the dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a sequence substantially complementary to the S6K1 nucleic acid sequence of SEQ ID NOs: 1 to -6.

75. A cell comprising the vector according to any one of claims 71 to 74.

76. A recombinant adeno-associated virus (rAAV) comprising the vector of any one of claims 71 to 74 and an AAV capsid.

77. A branched RNA compound comprising two or more siRNAs according to any one of claims 1 to 61 covalently linked to each other.

78. 78. The branched RNA compound of claim 77, wherein the siRNAs are covalently linked to each other via a linker, spacer, or branch point.

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