Nucleic acid molecules for inducing asymmetric RNAi that inhibit expression of ROR-beta

Abstract

The present application relates to a nucleic acid molecule for inducing asymmetric RNAi for inhibiting ROR-β (RAR related orphan receptor B) expression and use thereof, in particular, to a nucleic acid molecule for inducing asymmetric RNAi comprising an antisense strand comprising a sequence complementary to an mRNA encoding ROR-β and a sense strand forming a complementary bond with the antisense strand, and a pharmaceutical composition for improving or treating a retinal disease, the composition comprising the nucleic acid molecule for inducing asymmetric RNAi.

Description

Technical Field

[0001] This invention relates to a nucleic acid molecule for inducing asymmetric RNAi by inhibiting ROR-β (Retinoid-related orphan nuclear receptor) expression and its use. Specifically, it relates to a nucleic acid molecule for inducing asymmetric RNAi and a pharmaceutical composition for improving or treating retinal diseases. The nucleic acid molecule for inducing asymmetric RNAi comprises an antisense strand containing a sequence complementary to the mRNA encoding ROR-β and a sense strand forming a complementary bond with the antisense strand. The composition comprises the nucleic acid molecule for inducing asymmetric RNAi. Background Technology

[0002] Retinitis pigmentosa is a disease caused by mutations in genes that confer characteristics on rod cells, leading to the destruction of rod cells and subsequently cone cells, ultimately resulting in blindness. ROR-β (Retinoid-related orphan nuclear receptor) is a transcription factor that plays a crucial role in the differentiation of rod and cone cells. It regulates the expression of Neural retina leucine zipper (NRL), which differentiates into rod cells, and is involved in various other retinal functions. ROR-β has two isoforms, RORβ1 and RORβ2. RORβ1 induces NRL expression upstream, while RORβ2 is a factor regulated by NRL, enhancing its expression. In other words, a positive feedback loop exists between the ROR-β and NRL genes, thereby stabilizing rod cell differentiation. (Fu et al., "Feedback Induction of a Photoreceptor-specific Isoform of Retinoid-related Orphan Nuclear Receptorβ by the Rod Transcription Factor NRL" Journal of Biological Chemistry 2014 Nov 21; 289(47):32469-80.). In particular, taking NRL, which is closely related to ROR-β, as an example, it was observed in a mouse model of retinal degeneration that when CRISPR / Cas9 knocked down by adenovirus-associated virus (AAV), rod cells acquired some of the properties of cone cells, so that rod cells were not destroyed and were preserved when the genes that confer rod cell characteristics were mutated, thereby preventing the loss of cone cells (Yu, Wenhan, et al., "Nrl knockdown by AAV-delivered CRISPR / Cas9 prevents retinal degeneration in mice." Nature Communications 8(2017):14716).This phenomenon is similar to the study of rod cells acquiring cone cell characteristics in mice with missing Ror-β (Jia et al., "Retinoid-related orphan nuclear receptor RORβ is an early-acting factor in rod photoreceptor development" Proceedings of the National Academy of Sciences of the United States of America (2009) 13; 106(41): 17534-9.). This shows that NRL and ROR-β play a very important role in the differentiation of rod cells and cone cells in the retina.

[0003] On the other hand, using RNA interference to treat diseases involves siRNA, which targets mRNA and regulates gene expression at the translational level, thus offering a safer treatment option. Small interfering RNA (siRNA) consists of a sense strand with the same sequence as the target mRNA and an antisense strand with a complementary sequence. Conventional siRNA has a short double-stranded structure of 19 to 21 bp, with two nucleotides protruding from the 3' of each strand. After entering the cell, siRNA attaches to the target mRNA and breaks it down, thereby inhibiting the expression of the target gene. Because all mRNAs can be targeted by altering oligonucleotide sequences, the expression of structurally complex proteins can be inhibited, potentially leading to the treatment of currently difficult-to-treat diseases such as cancer, viral infections, and genetic disorders. However, the introduction of siRNA into cells can cause side effects such as induction of immune responses and inhibition of off-target genes. Therefore, the delivery system for introducing siRNA into the cell is the most critical issue in the development of therapeutic drugs utilizing siRNA.

[0004] siRNA carries a negative charge due to its phosphate backbone, which repels the negatively charged cell membrane, thus requiring a delivery system to introduce it into cells. A widely used method is to encapsulate siRNA with positively charged liposomes or polymers to counteract the negative charge, thereby introducing it into cells. However, positively charged carriers can cause various side effects, such as attaching to negatively charged cell membranes and exhibiting unwanted toxicity, or forming unwanted complexes through interactions with various types of proteins in the cell. Furthermore, because siRNA is rapidly broken down by nucleases in the blood, the amount of siRNA reaching the target cell may be insufficient to significantly reduce the expression of the target gene. Therefore, a safe and efficient method for delivering siRNA to target cells is needed.

[0005] To this end, the inventors have made efforts to select siRNAs that target ROR-β and inhibit ROR-β expression, and to develop siRNAs that can be delivered to cells without delivery substances and are highly resistant to nucleases. Finally, they designed siRNAs that target ROR-β, selected the most effective siRNAs that inhibit ROR-β through screening, and confirmed that the problem of intracellular delivery can be solved through modification, thus completing the present invention.

[0006] The information described in this background section is only for enhancing the understanding of the background of the present invention, and therefore may not include information constituting prior art known to those skilled in the art to which this invention pertains. Summary of the Invention

[0007] Technical issues

[0008] The purpose of this invention is to provide a nucleic acid molecule that specifically inhibits ROR-β expression for inducing asymmetric RNAi.

[0009] Another object of the present invention is to provide a pharmaceutical composition or method for improving or treating retinal diseases.

[0010] Technical solution

[0011] To achieve the above objectives, the present invention provides a nucleic acid molecule for inducing RNAi, characterized in that it comprises an antisense strand containing a sequence complementary to an mRNA encoding ROR-β (Retinoid-related orphan nuclear receptor) and a sense strand forming a complementary bond with the antisense strand, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end.

[0012] The present invention further provides a pharmaceutical composition for improving or treating retinal diseases, comprising the nucleic acid molecule for inducing RNAi.

[0013] The present invention also provides a method for improving or treating retinal diseases, comprising administering the nucleic acid molecule used to induce RNAi to an individual.

[0014] Beneficial effects

[0015] According to the present invention, an asymmetric siRNA that can effectively inhibit the expression of ROR-β, which plays a very important role in the differentiation of rod and cone cells, is screened out. The siRNA is then chemically modified to be introduced into cells without a vector and to be resistant to nucleases, thereby removing the cytotoxicity caused by the vector and achieving more effective gene expression inhibition in vivo. This makes it effective as a therapeutic drug for retinal diseases, including retinitis pigmentosa. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the structure of ROR-βasiRNA, consisting of a 16-mer sense strand and a 19-mer antisense strand.

[0017] Figure 2 The results were obtained by treating Y-79 cells with 62 types of ROR-β asiRNAs to confirm changes in ROR-β protein expression.

[0018] Figure 3 The results confirmed the changes in ROR-β protein expression after treating Y-79 cells with 13 types of ROR-β asiRNA.

[0019] Figure 4 The results confirmed the changes in ROR-β mRNA expression after treating Y-79 cells with 13 types of ROR-β asiRNA.

[0020] Figure 5 The results confirmed the changes in ROR-β protein expression after treating Y-79 cells with 67 types of ROR-βcp-asiRNA.

[0021] Figure 6 The results confirmed the changes in ROR-β protein expression after treating Y-79 cells with 10 types of ROR-βcp-asiRNA.

[0022] Figure 7 The results were obtained by treating A549 cells that transiently expressed ROR-β with the ROR-β plasmid using 10 cp-asiRNAs, which confirmed the changes in ROR-β protein expression.

[0023] Figure 8 The results were obtained by treating A549 cells that transiently expressed ROR-β with the ROR-β plasmid using five cp-asiRNAs, and then confirming the changes in ROR-β mRNA expression.

[0024] Figure 9 The results were obtained by treating A549 cells that transiently expressed ROR-β with the ROR-β plasmid using five cp-asiRNAs, which confirmed the changes in ROR-β protein expression.

[0025] Figure 10 The sequence and modification information of ROR-βcp-asiRNA 4 (OLX304C-026-4) and ROR-βcp-asiRNA 5 (OLX304C-026-5) are shown (sense strand: 16mer, antisense strand: 19mer). Figure 11 The results confirmed changes in ROR-β protein expression after treating normal mouse eyes with two ROR-β cp-asiRNAs (OLX304C-026-4 and OLX304C-026-5). The results are shown in (A) after isolating retinal tissue from normal mice and (B) after isolating retinal pigment epithelial cells / choroidal layer from normal mice.

[0026] Figure 12 The results were obtained by treating normal mouse eyes with two ROR-βcp-asiRNAs (OLX304C-026-4 and OLX304C-026-26) at different doses and confirming the changes in ROR-β mRNA expression in retinal tissue relative to different doses.

[0027] Figure 13 The results were obtained by confirming the duration of the inhibitory effect of ROR-βmRNA expression in retinal tissue relative to different doses after treating normal mouse eyes with a ROR-βcp-asiRNA (OLX304C-026-4) at various doses.

[0028] Figure 14 The results were obtained by comparing the thickness of each layer in a mouse model of retinitis pigmentosa after treatment with optimal doses of two ROR-beta cp-asiRNAs (OLX304C-026-4 and OLX304C-026-26) in ocular tissues using H&E staining.

[0029] Figure 15 The results of electroretinograms (ERGs) were obtained in a mouse model of retinitis pigmentosa after treatment with optimal doses of two ROR-beta cp-asiRNAs (OLX304C-026-4 and OLX304C-026-26). (A) Results confirming changes in a-wave amplitude, and (B) Results confirming changes in b-wave amplitude. Detailed Implementation

[0030] All technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise defined. Generally, the nomenclature used in this specification is known and commonly used in the art.

[0031] The main terms used in the detailed description of this invention are defined as follows.

[0032] "RNAi (RNA interference)" refers to the mechanism of inhibiting target gene expression by introducing double-stranded RNA (dsRNA), composed of a strand with a homologous sequence of the target gene mRNA and its complementary strand, into cells to induce the degradation of the target gene mRNA.

[0033] "Nucleic acid molecule for inducing RNAi" refers to any nucleic acid molecule capable of mediating RNA interference in a sequence-specific manner to inhibit or downregulate gene expression or viral replication. The term may refer to a single nucleic acid molecule, multiple nucleic acid molecules, or a library of nucleic acid molecules. In one specific embodiment, the nucleic acid molecule used to induce RNAi may be siRNA.

[0034] "siRNA (small interfering RNA)" refers to short double-stranded RNA (dsRNA) that mediates effective gene silencing in a sequence-specific manner.

[0035] An "antisense strand" refers to a polynucleotide that is actually or 100% complementary to the target nucleic acid of interest. For example, it may be wholly or partially complementary to mRNA (messenger RNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences.

[0036] A "sense strand" is a polynucleotide that has the same nucleic acid sequence as the target nucleic acid and is identical in whole or in part to mRNA, non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences.

[0037] The term "gene" should be considered in its broadest sense and can encode structural or regulatory proteins. In this context, regulatory proteins include transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation. In this invention, the target gene for expression repression is a gene inherent in the viral genome, which may be integrated into animal genes or exist as an extrachromosomal component. For example, the target gene could be a gene on the HIV genome. In this case, siRNA molecules can be used to inactivate the translation of HIV genes in mammalian cells.

[0038] "Retinoid-related orphan nuclear receptor"

[0039] ROR-β is an important transcription factor regulating the differentiation of rod and cone cells in the retina. It is known to act as an upstream regulator of NRL (Neural retina leucine zipper), which enables photoreceptor precursors to differentiate into rod cells, and its hypothetical gene NR2E3 (nuclear receptor subfamily 2, group E, member 3). On the other hand, reports indicate that ROR-β-deficient Rorb- / - mice show significantly reduced expression of Nrl and Nr2e3 and decreased expression of rod-related genes, while increasing expression of cone-related genes, which play an important role in the regulation of rod cell differentiation signaling induced by ROR-β and NRL.

[0040] Retinitis pigmentosa is a disease caused by mutations in genes that give rod cells their characteristics, leading to the destruction of rod cells and subsequently cone cells. A recent study showed that when genes important for photoreceptor function are mutated, the detrimental effects are strongest in fully differentiated photoreceptors. Therefore, ROR-β, the epistatic gene for NRL that induces rod cell differentiation, when knocked out, causes rod cells to morphologically resemble cone cells, thus losing their rod cell function. However, even these morphologically and functionally altered rod cells can prevent further cone cell loss, thus showing promise as a treatment for retinitis pigmentosa.

[0041] The objectives of this invention are twofold: first, to design siRNAs targeting ROR-β and select the most effective siRNAs for inhibiting ROR-β through screening; second, to deliver siRNAs into cells without a vector and introduce chemical modifications to enhance their resistance to nucleases. To cross the cell membrane, which is composed of phospholipids, siRNAs must be small or hydrophobic. However, the phosphate backbone of siRNAs carries a negative charge, making it difficult for them to penetrate the cell membrane. Furthermore, enhancing resistance to nucleases is crucial to ensure a longer lifespan in serum, thereby achieving a sufficient amount to effectively induce RNAi. Therefore, the delivery problem of siRNAs is overcome through these modifications.

[0042] In one embodiment of the present invention, firstly, an asiRNA targeting ROR-β is designed, and the asiRNA is transfected into cells expressing ROR-β to select the ROR-β asiRNA with the best knockdown efficiency. The selected siRNA is then modified with the following four modifications to enhance its cell penetration ability and resistance to nucleases: First, cholesterol is added to the 3' end of the sense strand, allowing the siRNA to penetrate the cell membrane. Second, the phosphate backbone near the 5' or 3' ends of the sense and antisense strands is replaced with phosphate thioesters to make it resistant to exonucleases, thereby enabling cellular uptake and bioavailability of the siRNA in vivo. Third, resistance to nucleases is conferred by modifying the 2' end of the sugar with an O-methyl group, and off-target effects are reduced by decreasing the immunogenicity of the siRNA. Fourth, double-stranded stability is conferred by modifying the 2' end of the sugar with fluorine, thereby improving stability in serum and achieving effective silencing in vitro and in vivo. By modifying siRNA as described above, siRNA gains cell penetration ability and stays in serum for a longer time. Therefore, sufficient siRNA is delivered to target cells, thereby effectively inhibiting genes.

[0043] Therefore, this invention relates in one aspect to a nucleic acid molecule for inducing RNAi, characterized by comprising an antisense strand containing a sequence complementary to mRNA encoding ROR-β and a sense strand forming a complementary bond with the antisense strand, wherein the 5' end of the antisense strand and the 3' end of the sense strand form a blunt end. The siRNA in this invention is a concept encompassing all substances with general RNAi (RNA interference) activity. RNAi is an intracellular gene regulation mechanism first discovered in *C. elegans* in 1998. Its mechanism of action is known to be the complementary binding of the antisense strand of an intracellular RNA double strand to the mRNA of a target gene, thereby inducing the degradation of the target gene. siRNA is a method of inhibiting gene expression "in vitro." Theoretically, 19bp to 21bp siRNA can selectively inhibit almost all genes, thus it can be developed into therapeutic drugs for various gene-related diseases such as cancer and viral infections, and is currently one of the most popular candidate technologies for new drug development. The first attempt to use siRNA for in vivo therapy in mammals was in mid-2003, and since then, numerous reports have shown attempts at in vivo therapeutic applications.

[0044] However, contrary to its usability, there are continuous reports of side effects and drawbacks of siRNA. To develop RNAi-based therapeutics, the following problems need to be overcome: 1) lack of effective delivery systems, 2) off-target effects, 3) induction of immune responses, and 4) saturation of intracellular RNAi mechanisms. Although siRNA is an effective method for directly regulating target gene expression, the above-mentioned problems have created a bottleneck in the development of therapeutics. In response, asymmetric shorter duplex siRNA (asiRNA) is an asymmetric RNAi-inducible structure with a shorter double strand than the 19+2 structure of existing siRNAs. It overcomes the off-target effects and saturation of TLR3 immune responses found in existing siRNA structural technologies, thereby enabling the development of novel RNAi drugs with fewer side effects.

[0045] Based on this, in one embodiment, an asymmetric siRNA comprising a sense strand and an antisense strand complementary to the sense strand is proposed. The siRNA according to one embodiment does not cause off-target effects, RNAi mechanism saturation, etc., thus maintaining stable and high delivery efficiency, and can effectively suppress the expression of ROR-β target genes to the desired extent.

[0046] In this invention, the nucleic acid molecule used to induce RNAi is characterized in that the sense strand has a length of 15 to 17 nt, and the antisense strand has a length greater than or equal to 18 nt. However, it is not limited to this; the antisense strand may have a length of 18 to 31 nt, preferably 18 to 23 nt. More preferably, the sense strand is 16 nt long, and its complementary antisense strand is 19 nt, 20 nt, 21 nt, or 22 nt long, but is not limited to this.

[0047] The 3' end of the sense chain and the 5' end of the antisense chain form a flat end. The 3' end of the antisense chain may include, for example, a protruding end of 1 to 16 nt.

[0048] In one embodiment of the present invention, 62 ROR-β asiRNAs were designed to suppress ROR-β expression, and mRNA and protein levels were confirmed in cells expressing ROR-β or transient cells that temporarily expressed ROR-β.

[0049] In this invention, the feature of the sense chain may be selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123.

[0050] Specifically, the meaningful chain can be selected from, for example, the group consisting of SEQ ID NO: 27, 29, 51, 83, 85, 91, 95, 97, 103, 105, 107, 109 and 115, or the group consisting of SEQ ID NO: 27, 29, 51 and 109, for example, SEQ ID NO: 51 or 109.

[0051] In this invention, the antisense chain can be selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, and 124.

[0052] Specifically, the antisense chain can be selected from the group consisting of, for example, SEQ ID NO: 28, 30, 52, 84, 86, 92, 96, 98, 104, 106, 108, 110 and 116, or from the group consisting of SEQ ID NO: 28, 30, 52 and 110, for example, SEQ ID NO: 52 or 110.

[0053] In this invention, the sense or antisense strand of the nucleic acid molecule used to induce RNAi may be characterized by including at least one chemical modification.

[0054] Conventional siRNAs cannot cross cell membranes due to the high negative charge of their phosphate backbone and their high molecular weight, and they are rapidly degraded and removed in the blood, making it difficult to deliver a sufficient amount to the actual target site for RNAi induction. Currently, efficient delivery methods using cationic lipids and cationic polymers have been developed for in vitro delivery, but these methods are not as efficient as in vitro delivery of siRNAs in vivo, and the delivery efficiency decreases due to various protein interactions present in the body.

[0055] Therefore, the inventors have developed a self-transmitting asiRNA construct (cp-asiRNA) by introducing chemical modifications into the asymmetric siRNA structure, which can be effectively delivered into cells without a vector.

[0056] In this invention, the chemical modifications in the sense or antisense strand may include at least one selected from the group consisting of: OH group at the 2' carbon position of the sugar structure within the nucleotide being replaced by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; oxygen in the sugar structure within the nucleotide being replaced by sulfur; nucleotide bond modification to thiophosphate, borophosphate, or methyl phosphonate; or modification to PNA (peptide nucleic acid), LNA (locked nucleic acid), or UNA (unlocked nucleic acid); and phosphate groups, lipophilic compounds, or cell-penetrating peptide bonds.

[0057] In this invention, the lipophilic compound is characterized by being selected from the group consisting of cholesterol, tocopherol, stearic acid, retinoic acid, DHA, palmitic acid, linoleic acid, linolenic acid, and long-chain fatty acids having 10 or more carbon atoms. A preferred feature is that it is cholesterol, but it is not limited thereto.

[0058] In one specific embodiment, the sense chain may include at least one chemical modification selected from the following: two to four nucleotide bonds near the 3' end are modified with thiophosphate, borophosphate, or methyl phosphonate; the OH group at the 2' carbon position of the sugar structure in two or more nucleotides is replaced by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; a lipophilic compound or cell-penetrating peptide is bound to the 3' end.

[0059] In one specific embodiment, the antisense chain may include any one or more chemical modifications selected from the following: three to five nucleotide bonds near the 3' end are modified to thiophosphate, borophosphate, or methyl phosphonate; the OH group at the 2' carbon position of the sugar structure in two or more nucleotides is replaced by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; a phosphate group or cell-penetrating peptide is bound to the 5' end. In another specific embodiment, the nucleic acid molecule used to induce RNAi may be characterized by at least one modification selected from the group consisting of: a modification in which the OH group in the 2' carbon position of the sugar structure of two or more nucleotides in the sense or antisense strand is replaced by -OCH3 (methoxy) or -F (fluorine); a modification in which 10% or more of the nucleotide bonds in the sense or antisense strand are modified to be thiophosphates; cholesterol or palmitic acid is bound to the 3' end of the sense strand; and a phosphate group is bound to the 5' end of the antisense strand.

[0060] Preferably, the sense chain is characterized by being selected from any one of the groups consisting of Table 1(a) to (l) below, and the antisense chain is characterized by being selected from any one of the groups consisting of Table 1(m) to (x) below.

[0061] Table 1

[0062] Sequence (5′>3′) (a) mCmCAACUUGUUUACC*mU*mA*chol (b) mCCmAAmCUmUGmUUmUAmCC*mU*A*chol (c) mCCmAmACUUmGUUUmACC*U*mA*chol (d) mCmCAAmCmUmUGmUmUmUAmCmC*mU*A*chol (e) mCfCmAfAmCfUmUfGmUfUmUfAmCfC*mU*fA*chol (f) mCmAGAAGCUUCAGGA*mA*mA*chol (g) mCAmGAmAGmCUmUCmAGmGA*mA*A*chol (h) mCmAmGmAmAmGCUUCmAmGmGmA*mA*mA*chol (i) mCAGAAGmCmUmUmCAGGA*A*A*chol (j) mCmAmGmAmAmGfCfUfUfCmAmGmGmA*mA*mA*chol (k) mCfAmGfAmAfGmCfUmUfCmAfGmGfA*mA*fA*chol (l) mCmCAACUUGUUUACC*mU*mA*PA (m) P-mUAmGGmUAmAAmCAmAGmUUmG*G*mG*U*mA (n) P-mUAGGUAAACmAmAmGUUmG*mG*mG*U*mA (o) P-mUAGGmUAAAmCAAGmUUG*G*G*mU*A (p) P-mUAGGfUAAAfCAAGfUfUG*G*G*fU*A (q) P-mUfAmGfGmUfAmAfAmCfAmAfGmUfUmG*fG*mG*fU*mA (r) P-mUUmUCmCUmGAmAGmCUmUCmU*G*mG*A*mC (s) P-mUUUCCUGAmAmGmCmUmUCmU*mG*mG*mA*mC (t) P-mUUUCCUmGmAmAmGCUUCU*mG*mG*mA*C (u) P-mUUUCCUGAAGmCmUmUCmU*G*G*A*mC (v) P-mUfUfUfCfCfUmGmAmAmGfCfUfUfCfU*mG*mG*mA*fC (w) P-mUfUfUfCfCfUGAAGfCfUfUfCfU*G*G*A*fC (x) P-mUfUmUfCmCfUmGfAmAfGmCfUmUfCmU*fG*mG*fA*mC

[0063] In the sequence, * indicates a thiophosphate bond, m indicates 2'-O-methyl, 2'-F- indicates 2'-fluoro(Fluoro), chole indicates cholesterol, PA indicates palmitic acid, and P indicates a 5'-phosphate group.

[0064] Specifically, the meaningful chain can be any one of (a) to (e) and (1) in Table 1 above, and the antisense chain can be any one of (m) to (q) in Table 1 above; the meaningful chain can be any one of (f) to (k) in Table 1 above, and the antisense chain can be any one of (r) to (x) in Table 1 above; the meaningful chain can be (a) or (l) in Table 1 above, and the antisense chain can be (p) or (q) in Table 1 above.

[0065] In this invention, one to three phosphate groups may be attached to the 5' end of the antisense chain, but are not limited thereto.

[0066] In one embodiment of the invention, the sense strand uniformly has three phosphate bonds at its 3' end replaced by thiophosphate bonds, and cholesterol or palmitic acid is added. The antisense strand uniformly has four phosphate bonds at its 3' end replaced by thiophosphate bonds. Furthermore, by varying the number and position of substitutions for the O-methyl and fluoro groups at the 2' end of the sugar, 12 sense strands and 12 antisense strands were synthesized. Annealing was performed in 68 different cases to select the modification that best knocked down ROR-β without a vector, and the ROR-βcp-asiRNA that most effectively knocked down ROR-β was selected.

[0067] Another aspect of the present invention relates to a pharmaceutical composition for improving or treating retinal diseases, comprising the nucleic acid molecule for inducing RNAi.

[0068] In this invention, the retinal disease may be characterized by Usher syndrome, Stargardt disease, Balde-Bede syndrome, Best disease, choroidal defect, choroidal-retinal atrophy, retinitis pigmentosa, macular degeneration, congenital amaurosis, BCM (Blue-cone monochromacy), retinoschisis, ML (Malattia Leventinese), microcheilosis, or Rifsum disease, but is not limited thereto.

[0069] The pharmaceutical composition can be prepared by further comprising at least one pharmaceutically acceptable carrier, in addition to the nucleic acid molecule for inducing RNAi as the active ingredient. The pharmaceutically acceptable carrier should be compatible with the active ingredient of the invention and can be used by mixing with saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and may add other conventional additives such as antioxidants, buffers, and antibacterial agents as needed. Furthermore, it can be formulated into injectable preparations such as aqueous solutions, suspensions, and emulsions by adding diluents, dispersants, surfactants, binders, and lubricants. In particular, it is preferred to provide the preparation as a lyophilized form. To prepare the lyophilized formulation, methods conventionally known in the art to which this invention pertains can be used, and stabilizers for lyophilization can be added.

[0070] Those skilled in the art can determine the method of administration of the pharmaceutical composition based on the symptoms and severity of the disease in a typical patient. Furthermore, it can be formulated into various preparations such as powders, tablets, capsules, solutions, injections, ointments, and syrups, and can be provided in single-dose or multi-dose containers, such as sealed ampoules and bottles.

[0071] The pharmaceutical compositions of the present invention can be administered orally or parenterally. The routes of administration of the compositions according to the present invention are not limited thereto, but may include, for example, intravitreal injection (IVT), oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intraintestinal, sublingual, or local administration. The dosage of the compositions according to the present invention varies depending on the patient's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate, or severity of disease, and can be readily determined by those skilled in the art. Furthermore, the compositions of the present invention can be formulated into suitable formulations for clinical use using known techniques.

[0072] Another aspect of the present invention relates to a method for improving or treating retinal diseases, comprising administering the nucleic acid molecule used to induce RNAi to an individual. The constituent elements included in the improved or treated methods according to the present invention are the same as those included in the invention described above; therefore, the above description can also be applied equally to the improved or treated methods.

[0073] The term "application" can refer to a method of at least partially localizing a nucleic acid molecule for inducing RNAi according to a specific embodiment to a desired site, or to administering a nucleic acid molecule for inducing RNAi according to a specific embodiment into an individual via a pathway.

[0074] The “individual” can be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, monkey, or cat. The individual can be one who needs improvement for retinal diseases such as retinitis pigmentosa, for example, one who requires inhibition of ROR-β gene expression.

[0075] Another aspect of the present invention relates to the use of the aforementioned nucleic acid molecule for inducing RNAi, wherein the nucleic acid molecule for inducing RNAi is used in the preparation of a medicament for improving or treating retinal diseases. In the stated use, the nucleic acid molecule for retinal diseases, improvement, treatment, and inducing RNAi is as described above.

[0076] The present invention will be further described in detail below through embodiments. These embodiments are for illustrative purposes only, and those skilled in the art will understand that the scope of the invention should not be construed as limited to these embodiments.

[0077] Example 1: 62 RNAi-induced double-stranded nucleic acid molecules targeting ROR-β

[0078] In this embodiment, ROR-β asymmetric siRNA (asiRNA) was designed for screening. Conventional siRNA is a 19-base-pair duplex with two nucleotide overhangs at the 3' end of each of its two strands. The antisense strand of asiRNA has a blunt 5' end and is a short duplex of 15 to 16 base pairs, thus exhibiting the same inhibitory efficiency as siRNA, and a reduced off-target effect due to the shorter sense strand length. Therefore, asiRNAs designed to be 16-mer (sense strand) to 19-mer (antisense strand) were used. Figure 1 ).

[0079] For animal testing, ROR-βasiRNAs were designed with homology in mind for Homo sapiens (humans), house mice (mice), rabbits (rabbits), and macaques (monkeys), and contain sequences that are 100% identical to those of these species. Table 2 below shows the sequences of 62 ROR-βasiRNAs.

[0080] Table 2

[0081]

[0082]

[0083]

[0084]

[0085] Example 2: Screening for RNAi-induced double-stranded nucleic acid molecules targeting ROR-β

[0086] In this embodiment, to verify the expression inhibition effect of ROR-β asiRNA in Example 1, 10 nM of asymmetric siRNA was transfected into Y-79 cells (ATCC), and the expression level of ROR-β protein was detected by Western blotting. Specifically, Y-79 cells were transfected at a concentration of 1 × 10⁻⁶ m² / h². 5Cells / wells were seeded into 12-well plates and then transfected with 10 nM asiRNA for 48 hours using LipofectamineRNAiMax Transfection Reagent (Invitrogen, 13778030) according to the Invitrogen protocol. Afterward, transfected cells were lysed to obtain cell lysates, which were then subjected to Western blotting. The expression level of ROR-β protein in the obtained cell lysates was detected using 12% SDS-polyacrylamide gel electrophoresis, ROR-β antibody (Proteintech, Cat. #17635-1), and Vinculin antibody (Santa Cruz Biotechonology, Cat. #sc-73614) according to each manufacturer's protocol. In this example, the negative control group utilized the untreated group.

[0087] The results are as follows Figure 2 As shown, the expression inhibition effect of ROR-β protein treated with ROR-βasiRNA was confirmed. Specifically, among 62 ROR-βasiRNAs, 13 were identified (#14, #15, #26, #42, #43, ...).

[0088] Asymmetric siRNAs (#46, #48, #49, #52, #53, #54, #55, #58) exhibit excellent inhibitory effects on ROR-β protein expression.

[0089] Example 3: Confirmation of ROR-β expression inhibition efficiency against the derived asiRNA

[0090] 3.1. Confirm the inhibitory effect of ROR-β protein expression.

[0091] In this embodiment, in order to verify the ROR-βasiRNA (#14, #15, ...) derived from Example 2,

[0092] The inhibitory effect of protein expression of #26, #42, #43, #46, #48, #49, #52, #53, #54, #55, and #58 was investigated. After transfecting Y-79 cells (ATCC) with 1 nM or 5 nM of asymmetric siRNA, the expression level of ROR-β protein was detected by Western blotting. Specifically, Y-79 cells were transfected with 1 × 10⁻⁶ siRNA. 5Cells / wells were seeded into 12-well plates and then transfected with 1 nM or 5 nM asiRNA for 48 hours using Lipofectamine RNAiMax Transfection Reagent (Invitrogen, 13778030) according to the Invitrogen protocol. Subsequently, transfected cells were lysed to obtain cell lysates, which were then subjected to Western blotting. ROR-β protein expression levels were detected in the obtained cell lysates using the same method as in Example 2, employing 12% SDS-polyacrylamide gel electrophoresis, ROR-β antibody (Proteintech, Cat. #17635-1), and Vinculin antibody (Santa Cruz Biotechonology, Cat. #sc-73614) according to each manufacturer's protocol. In this example, the negative control group utilized the untreated group.

[0093] The results are as follows Figure 3 As shown, the concentration-dependent inhibition of ROR-β protein expression by ROR-β asiRNA treatment was confirmed. Specifically, based on the results of 1 nM treatment, approximately 50% or higher inhibition of protein expression was observed in #14, #26, and #55.

[0094] 3.2. Confirm the inhibitory effect of ROR-β mRNA expression.

[0095] In this embodiment, to verify the inhibitory effect of ROR-β asiRNA (#14, #15, #26, #42, #43, #46, #48, #49, #52, #53, #54, #55, #58) mRNA expression derived from Example 2, 1 nM or 10 nM of asymmetric siRNA was transfected into Y-79 cells (ATCC), and the expression level of ROR-β mRNA was detected by real-time quantitative PCR. Specifically, Y-79 cells were transfected at a concentration of 6 × 10⁻⁶ mRNAs. 4 Cells / wells were seeded into 24-well plates, and then transfected with 1 nM or 10 nM asaRNA for 24 hours using Lipofectamine RNAiMaxTransfection Reagent (Invitrogen, 13778030) according to the Invitrogen protocol. Total RNA was then extracted using Tri-RNA reagent (FAVORGEN, FATRR001), and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813). Then, using… ROR-β (Hs00199445_m1) and RN18S1 (Hs03928985_g1) from Gene Expression Assays (Applied Biosystems) ROR-β mRNA expression levels were detected using the Probe qPCR Mix (TOYOBO, QPS-101) and the CFXConnect real-time quantitative PCR detection system (BioRad). ROR-β mRNA expression levels were normalized using RN18S1, and the negative control group consisted of the untreated group.

[0096] like Figure 4 As shown, the inhibitory effect of ROR-β mRNA expression based on ROR-β asaRNA is confirmed. Specifically, based on the results of 1 nM treatment, approximately 60% or higher mRNA expression inhibition was observed in #15, #26, and #55. Summarizing the results of this Example 3, it is deduced that the top three sequences with excellent ROR-β mRNA and protein expression inhibitory effects all include two sequences (#26, #55).

[0097] Example 4: Design and preparation of chemically modified asymmetric siRNA

[0098] In this embodiment, chemically modified asymmetric siRNAs (cell penetrating-asymmetric siRNAs: cp-asiRNAs) were designed for two of the ROR-βasiRNAs (#26 and #55) that were shown to have inhibitory effects in Example 3. Compared to the aforementioned asiRNAs, the cp-asiRNA designed in this embodiment is chemically modified (2'OMe, PS, Fluoro), resulting in an asymmetric siRNA with enhanced intracellular delivery.

[0099] The sequence information of the 67 cp-asiRNAs prepared in this embodiment is shown in Table 3 below.

[0100] Table 3

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] On the other hand, the chemical modifications represented by “*”, “m”, “f” and “chol” in Table 3 are shown in Table 4 below.

[0107] Table 4

[0108] Marking methods Chemical modification * Thiophosphate bond m 2'-O-methyl f 2' Fluorine chol cholesterol

[0109] Specifically, in Table 3, "*" indicates a form in which the existing phosphodiester bond is replaced by a thiophosphate bond, and "m" indicates a form in which the existing 2'-OH is replaced by a 2'-O-methyl. Additionally, "f" refers to, for example, fG, a form in which the 2'-OH of the existing G (guanine) is replaced by fluorine, and "Chol" refers to a form in which cholesterol is added to the 3' end.

[0110] Example 5: Screening for chemically modified asymmetric siRNAs

[0111] 5.1 Confirming the inhibitory effect of ROR-β protein expression

[0112] In this embodiment, to confirm the expression inhibition effect of ROR-βcp-asiRNA in Example 4, each cp-asiRNA was treated into Y-79 cells and incubated (free uptake), and the expression level of ROR-β protein was detected by Western blotting. Specifically, Y-79 cells were incubated at 1×10⁻⁶ cells per cell line. 5 Cells / wells were seeded into 12-well plates, and after 24 hours, 1 μM ξácp-asiRNA was added. Cells were then incubated in RPMI (Gibco, 11875-093) medium containing 10% FBS (fetal bovine serum, Gibco, 16000-044) (free uptake). After 48 hours, transfected cells were lysed to obtain cell lysates, which were then subjected to Western blotting. The expression level of ROR-β protein in the obtained cell lysates was detected using 12% SDS-polyacrylamide gel electrophoresis, ROR-β antibody (Proteintech, Cat. #17635-1), and Vinculin antibody (Santa Cruz Biotechonology, Cat. #sc-73614). In this embodiment, the negative control group used the untreated group, and the positive control group used the group transfected with unmodified ROR-β asimaRNA at a concentration of 10 nM using lipofectamine RNAiMAX.

[0113] The results are as follows Figure 5As shown, the inhibitory effect of ROR-β protein expression treatment with ROR-βcp-asiRNA was confirmed. In particular, ten ROR-βcp-asiRNAs (4, 5, 6, 10, 24, 25, 32, 43, 52, and 53) that showed excellent inhibitory effects on ROR-β protein expression were identified.

[0114] To further confirm the inhibitory effect of the 10 cp-asiRNAs on ROR-β protein, the expression level of ROR-β protein was detected after treatment with the same procedure at a concentration of 2 μM. The results are as follows: Figure 6 As shown, the inhibitory effect of ROR-β protein expression treatment with ROR-βcp-asiRNA was confirmed. In particular, the reproducibility of the inhibitory effect on ROR-β protein expression was confirmed in cp-asiRNAs 4, 5, 10, 24, 25, 32, and 52.

[0115] 5.2. Confirm the inhibitory effect of ROR-β protein under ROR-β conditions.

[0116] In this embodiment, the protein-inhibiting effect of cp-asiRNA in transient cell lines expressing ROR-β was confirmed using the ROR-β plasmid (Origene, RC208666). More specifically, A549 cells were cultured at 5 × 10⁶ cells per well using Ham's F-12K (Kaighn's) medium (Gibco, 21127022) supplemented with 10% FBS. 4Cells were seeded into 12-well plates and incubated with 200 ng of ROR-β plasmid in Lipofectamine 2000 (Invitrogen, 11668-019) and Opti-MEM reduced serum medium (gibco, 31985-070) for 24 hours before cell treatment. After 6 hours, the plates were washed with PBS and then incubated for 24 hours with 1 μM cp-asiRNA in Opti-MEM reduced serum medium (free uptake). The next day, the medium was changed to Ham's F-12K (Kaighn's) medium (10% FBS). After one day, the cells were lysed to obtain cell lysates. The expression level of ROR-β protein was then detected using 12% SDS-polyacrylamide gel electrophoresis, ROR-β antibody (Proteintech, Cat. #17635-1), Vinculin antibody (Santa Cruz Biotechonology, Cat. #sc-73614), and neomycin phosphotransferase 2 antibody (Invitrogen, Cat. MA5-15275). In this example, neomycin phosphotransferase 2 antibody was used as a control group for plasmid transfection, and the negative control group consisted of the untreated cp-asiRNA group treated with the ROR-β plasmid.

[0117] The results are as follows Figure 7 As shown, the protein expression inhibition effect was higher than that of the untreated group. In particular, the results of this example showed a similar trend to the ROR-β protein inhibition effect in the Y-79 cell line of Example 5.1. The results of Examples 5.1 and 5.2 were summarized to identify five cp-asiRNAs (4, 5, 10, 24, 25) with excellent ROR-β protein expression inhibition effect.

[0118] Example 6: Confirmation of ROR-β expression inhibition efficiency against the derived cp-asiRNA

[0119] 6.1. Confirm the inhibitory effect of ROR-β mRNA expression.

[0120] In this embodiment, to confirm the inhibitory effect of the five ROR-β cp-asiRNAs (4, 5, 10, 24, 25) derived in Example 5 on ROR-β mRNA expression, 200 nM or 500 nM cp-asiRNA were applied to A549 cell lines that temporarily expressed ROR-β, and the expression level of ROR-β mRNA was detected by real-time quantitative PCR. Specifically, A549 cells were treated with 8 × 10⁻⁶ cp-asiRNAs. 3Cells / wells were seeded into 96-well plates. The next day, after incubating 10 ng of ROR-β plasmid in Lipofectamine 2000 (Invitrogen, 11668-019) and Opti-MEM reduced serum medium (gibco, 31985-070), the cells were treated. After 6 hours, the plates were washed with PBS and then incubated for 24 hours in Opti-MEM reduced serum medium with 200 nM or 500 nM cp-asiRNA (free uptake). cDNA was then synthesized from the cell lysates using the SuperPrep II Celllysis & RT Kit for qPCR (TOYOBO, SCQ-101) according to the manufacturer's instructions. Gene Expression Assays (Applied Biosystems, ROR-beta; Hs00199445_m1, RN18S1; s03928985_g1), The expression level of ROR-β, a target mRNA for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), was analyzed using Probe qPCR Mix (TOYOBO, QPS-101) and the CFXConnect real-time quantitative PCR detection system (Bio-Rad). In this embodiment, the negative control group used untreated cp-asiRNA containing the ROR-β plasmid, while the positive control group used a group transfected with unmodified ROR-β asiRNA at a concentration of 10 nM using lipofectamine RNAiMAX.

[0121] The results are as follows Figure 8 As shown, it was confirmed that all five cp-asiRNAs exhibited greater than or equal to 80% mRNA expression inhibition when treated at a concentration of 500 nM, and greater than or equal to 70% mRNA expression inhibition when treated at a concentration of 200 nM.

[0122] 6.2. Confirm the inhibitory effect of ROR-β protein expression.

[0123] In this embodiment, to confirm the inhibitory effect of the five ROR-β cp-asiRNAs (4, 5, 10, 24, 25) derived in Example 5 on ROR-β protein expression, 200 nM or 500 nM of cp-asiRNA were applied to A549 cell lines that temporarily expressed ROR-β, and the expression level of ROR-β protein was detected by Western blotting. Specifically, A549 cells were cultured in Ham's F-12K (Kaighn's) medium (Gibco, 21127022) supplemented with 10% FBS at a density of 5 × 10⁶ cells per well. 4 Cells were seeded into 12-well plates and incubated with 200 ng of ROR-β plasmid in Lipofectamine 2000 (Invitrogen, 11668-019) and Opti-MEM reduced serum medium (gibco, 31985-070) for 24 hours before cell treatment. After 6 hours, the plates were washed with PBS and then incubated for 24 hours in Opti-MEM reduced serum medium with 200 nM or 500 nM cp-asiRNA (free uptake). The next day, the medium was replaced with Ham's F-12K (Kaighn's) medium (10% FBS), and ROR-β protein expression levels were detected one day later using the same method as described in Example 5.2. In this example, the negative control group used untreated cp-asiRNA containing the ROR-β plasmid, and the positive control group used cells transfected with unmodified ROR-β asiRNA at a concentration of 10 nM using Lipofectamine RNAiMAX.

[0124] The results are as follows Figure 9 As shown, the inhibitory effect of ROR-β protein expression treatment with ROR-βcp-asiRNA was confirmed. In particular, cp-asiRNAs 4 and 5, which exhibited excellent inhibitory effects on ROR-β protein, were ultimately derived from this study. Figure 10 The images show OLX304C-026-4 and OLX304C-026-5, with cp-asiRNA values ​​of 4 and 5, respectively.

[0125] Example 7: Confirmation of the inhibitory effect of ROR-β protein expression in normal mouse eyes

[0126] In this embodiment, to confirm whether the two ROR-βcp-asiRNAs (OLX304C-026-4, OLX304C-026-5) derived in Example 6 also have a high inhibitory effect on ROR-β protein expression in vivo, OLX304C-026-4 or OLX304C-026-5 was administered to the eyes of normal mice, and the expression level of ROR-β protein was detected by Western blotting. Specifically, 1 μl of OLX304C-026-4 or OLX304C-026-5 was administered intravitreally to both eyes of 8-week-old male C57B16 mice at a dose of 0.6 μg / μl. The procedure was as follows: After instilling Mydrin-P eye drops (Santen, E00170121), anesthesia was administered via intraperitoneal injection of anesthetic using a 0.3 ml 31G insulin syringe (BD, 328822). Following this, ocular anesthesia was achieved by instilling Alcaine eye drops (Alcon, E07370271). While confirming the presence of mydriasis and anesthesia in the mouse eye using a surgical microscope (Leica, M844 F40), a small hole was created in the sclera of the eye using a 0.3 ml 31G insulin syringe. 10 μl of OLX304C-026-4 or OLX304C-026-5 was administered via a 10 μl Exmire microsyringe (Ito co, MS*NE10U) with a Microthin Dual Gauge Needle with tapeper 90° (35G) (Ito co., DWG.No.5-0608). Maxitrol eye ointment (Alcon, E07370151) was applied to prevent inflammation, and the patient was placed on a heating pad while the anesthesia wore off to maintain body temperature. On day 7 of treatment, the eyeball was enucleated, and proteins were extracted after separating the retina and retinal pigment epithelial (RPE) / choroidal layer. ROR-β (17635-1-AP, 1:500) and Vinculin (sc-73614, 1:2000) were identified by electrophoresis of the extracted proteins at 20 μg per well on an 8% SDS-polyacrylamide gel.

[0127] The results are as follows Figure 11 As shown, when treated with a dose of 0.6 μg, both OLX304C-026-4 and OLX304C-026-5 exhibited 60% to 78% inhibition of ROR-β protein expression in normal mouse retinal tissue, and similarly, approximately 40% inhibition of protein expression was observed in the RPE / choroidal layer.

[0128] Example 8: Confirmation of the inhibitory effect of ROR-β mRNA expression relative to different dosages in normal mouse eyes

[0129] In this embodiment, to confirm whether OLX304C-026-4, one of the two ROR-βcp-asiRNAs derived in Example 6, and OLX304C-026-26, which is synthesized by adding palmitic acid to the 3' end of the sense strand of OLX304C-026-4 to replace cholesterol, have a high inhibitory effect on ROR-β mRNA expression in vivo, OLX304C-026-4 or OLX304C-026-26 were applied to the eyes of normal mice, and the expression level of ROR-β mRNA was detected by real-time quantitative PCR. Specifically, the procedure was the same as in Example 7, except that OLX304C-026-4 was administered to both eyes of 7-week-old male C57Bl6 via intravitreal injection (IVT) at doses of 0.4 μg / μl, 0.4 μg / μl, 1.0 μg / μl, and a total of 1 μl of OLX304C-026-26 at doses of 0.4 μg / μl, 1.0 μg / μl, 4.0 μg / μl. RNA was extracted from the retinal tissue after enucleation on day 7 of administration. RNA extraction was performed using the RNeasy Plue Mini Kit (QIAGEN, 74136) according to the manufacturer's protocol. The extracted RNA was analyzed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TM (4368814) cNDA was synthesized according to the manufacturer's plan, and then TB was used. Premix Ex Taq TM The expression level of ROR-β, a target mRNA for qRT-PCR, was analyzed using the II (TaKaRa, RR820B) product and the CFX Connect Real-Time PCR Detection System (Bio-Rad). qRT-PCR was performed using primers for ROR-β (F: 5'-TGCCCAAGTCCGAAGGTTATT-3' (SEQ ID NO: 125), R: 5'-CCATGCCAGCTGATGGAGTT-3' (SEQ ID NO: 126), and GAPDH (F: 5'-GGGTGTGAACCACGAGAAAT-3' (SEQ ID NO: 127), R: 5'-GTCATGAGCCCTTCCACAAT-3' (SEQ ID NO: 128)).

[0130] The results are as follows Figure 12As shown, both cp-asiRNAs demonstrated a dose-dependent inhibitory effect on ROR-β mRNA expression, with OLX304C-026-4 showing approximately 97% inhibition at 1.0 μg and OLX304C-026-26 showing approximately 87% inhibition at 4.0 μg.

[0131] Table 5

[0132]

[0133] In Table 5, the chemical modifications represented by “*”, “m”, “f” and “PA” are shown in Table 6 below.

[0134] Table 6

[0135] Marking methods Chemical modification * Thiophosphate bond m 2'-O-methyl f 2' Fluorine PA Palmitic acid

[0136] Example 9: Confirmation of the Duration of ROR-β mRNA Expression Inhibition in Normal Mouse Eyes In this example, to confirm the duration of the ROR-β mRNA inhibition effect of OLX304C-026-4, one of the two ROR-βcp-asiRNAs derived in Example 7, the expression level of ROR-β mRNA was detected by real-time quantitative PCR after OLX304C-026-4 was administered to the eyes of normal mice. Specifically, except that 1 μl of OLX304C-026-4 was injected intravitreally into the eyes of 7-week-old male C57B16 mice at doses of 1.0 μg / μl or 2.0 μg / μl, and RNA was extracted from the retinal tissue after enucleation on days 14, 21, or 28 after administration, the experiment was performed using the same method as in Example 8. The results are as follows: Figure 13 As shown, OLX304C-026-4 maintained approximately 44% inhibition of ROR-β mRNA expression up to day 28 after administration of 2 μg. It was confirmed that the efficacy maintained for a longer period was more significant with 2 μg administration compared to 1 μg administration, and similar inhibition of ROR-β mRNA expression was observed at both doses after day 28. Furthermore, it was confirmed that administration of 1 μg resulted in approximately 50% or higher inhibition of ROR-β mRNA expression up to week 3.

[0137] Example 10: Confirmation of therapeutic efficacy in a mouse model of retinitis pigmentosa (RP) disease.

[0138] In this embodiment, efficacy was tested in a mouse model of retinitis pigmentosa by administering the optimal dose of ROR-β mRNA expression inhibitor validated in Example 8. Specifically, heterozygous RhoP23H knock-in mice, known as representative mouse models of retinitis pigmentosa, were used in the experiment. Based on the results of Example 8, ocular tissue analysis and electroretinography (ERG) were performed on day 21 after administration of 1.0 μg of OLX304C-026-4 and 4.0 μg of OLX304C-026-26 to confirm the therapeutic effect. As in Example 8, OLX304C-026-4 or OLX304C-026-5 was injected intraocularly, and tissue sections obtained from paraffin sections were stained with H&E using an automated slide staining machine. Mice were placed in a dark room for dark adaptation one day before imaging, and ERG imaging was performed under dark conditions. Mydriasis and anesthesia were performed in the same manner as in Example 8. The ground electrode was then fixed to the skin directly above the tail, and the reference electrode was fixed to the forehead. 2% Hicell sterile solution (samil-pharm, A05050401) was applied to the eyeball, and the position was adjusted to ensure good contact between the eyeball and the Ganzfeld ERG (Phoenix, MICRON Ganzfeld ERG) instrument. Imaging was then performed sequentially from low-intensity light stimulation to high-intensity light stimulation.

[0139] The results are as follows Figure 14 As shown, the results of comparing the thickness of each layer in ocular tissue using H&E staining in OLX304C-026-4 and OLX304C-026-26 confirmed that the thickness of ocular tissue significantly increased after application of OLX304C-026-4 and OLX304C-026-26. The increased thickness, particularly in the ONL (outer nuclear layer) where photoreceptors are primarily located, compared to the control group, may be related to the recovery of visual function. Therefore, application of OLX304C-026-4 or OLX304C-026-26 can protect the ONL layer from disintegration.

[0140] Similarly, in showing the ERG detection results Figure 15 In the study, in the groups treated with OLX304C-026-4 or OLX304C-026-26, damaged a-waves recovered by approximately 25% and 25%, respectively, and b-waves recovered by approximately 42% and 65%, respectively, showing significant recovery compared to the control group. This is a strategy for converting rod cells with various genetic defects into cone cells in patients with retinitis pigmentosa, suggesting that visual impairment can be delayed or treated by intravitreal injection of OLX304C-026-4 or OLX304C-026-26 targeting ROR-β.

[0141] The foregoing has described specific aspects of the present invention in detail. It will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the invention is not limited thereto. Therefore, the actual scope of the invention is defined by the appended claims and their equivalents. Sequence Listing Information: DTD Version: V1_3 File Name: PX067013PCT.xml Software Name: WIPO Sequence Software Version: 2.1.0 Production Date: 2023‑06‑01 General Information: Current application / Applicant file reference: PX067013PCT Earliest priority application / IP Office: KR Earliest priority application / Application number: KR 10‑2020‑0169855 Earliest priority application / filing date: 2020‑12‑07 Applicant name: OliX Pharmaceuticals, Inc. Applicant name / Language: en Invention title: Asymmetric nucleic acid molecules inducing RNAinterference that Inhibits Expression of ROR‑beta ( en ) Sequence Total Quantity: 128 Sequences: Sequence Number (ID): 1 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑001 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aaatttgtgg cgataa 16 Sequence Number (ID): 2 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑001 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttatcgccac aaattttgc 19 Sequence Number (ID): 3 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑002 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aaaatttgtg gcgata 16 Sequence Number (ID): 4 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑002 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tatcgccaca aattttgca 19 Sequence Number (ID): 5 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑003 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: accatgcaaa atttgt 16 Sequence Number (ID): 6 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑003 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: acaaattttg catggtatc 19 Sequence Number (ID): 7 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑004 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gataccatgc aaaatt 16 Sequence Number (ID): 8 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑004 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aattttgcat ggtatcact 19 Sequence Number (ID): 9 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑005 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tgataccatg caaaat 16 Sequence Number (ID): 10 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑005 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: attttgcatg gtatcactt 19 Sequence Number (ID): 11 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑006 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gtgataccat gcaaaa 16 Sequence Number (ID): 12 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑006 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttttgcatgg tatcacttc 19 Sequence Number (ID): 13 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑007 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: agtgatacca tgcaaa 16 Sequence Number (ID): 14 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑007 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tttgcatggt atcacttca 19 Sequence Number (ID): 15 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑008 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aagtgatacc atgcaa 16 Sequence Number (ID): 16 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑008 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttgcatggta tcacttcaa 19 Sequence Number (ID): 17 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑009 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: attgaagtga taccat 16 Sequence Number (ID): 18 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑009 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atggtatcac ttcaatttg 19 Sequence Number (ID): 19 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑010 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: cactacggag tcatca 16 Sequence Number (ID): 20 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑010 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tgatgactcc gtagtggat 19 Sequence Number (ID): 21 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑011 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tccactacgg agtcat 16 Sequence Number (ID): 22 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑011 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atgactccgt agtggatcc 19 Sequence Number (ID): 23 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑012 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: atccactacg gagtca 16 Sequence Number (ID): 24 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑012 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tgactccgta gtggatccc 19 Sequence Number (ID): 25 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑013 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gagccagcag aacaat 16 Sequence Number (ID): 26 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑013 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: attgttctgc tggctcctc 19 Sequence Number (ID): 27 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑014 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ggagccagca gaacaa 16 Sequence Number (ID): 28 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑014 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttgttctgct ggctcctcc 19 Sequence Number (ID): 29 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑015 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aggagccagc agaaca 16 Sequence Number (ID): 30 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑015 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tgttctgctg gctcctcct 19 Sequence Number (ID): 31 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑016 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tgtttaattg acagaa 16 Sequence Number (ID): 32 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑016 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttctgtcaat taaacagtt 19 Sequence Number (ID): 33 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑017 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ctgtttaatt gacaga 16 Sequence Number (ID): 34 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑017 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tctgtcaatt aaacagttt 19 Sequence Number (ID): 35 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑018 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ctgaggtgca gaagca 16 Sequence Number (ID): 36 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑018 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tgcttctgca cctcagcat 19 Sequence Number (ID): 37 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑019 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: atgctgaggt gcagaa 16 Sequence Number (ID): 38 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑019 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttctgcacct cagcataca 19 Sequence Number (ID): 39 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑020 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: agcagcagag tgggga 16 Sequence Number (ID): 40 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑020 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tccccactct gctgctgcc 19 Sequence Number (ID): 41 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑021 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tgtacagcag cagcat 16 Sequence Number (ID): 42 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑021 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atgctgctgc tgtacaccc 19 Sequence Number (ID): 43 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑022 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: attgacctgc ccaagt 16 Sequence Number (ID): 44 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑022 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: acttgggcag gtcaatgac 19 Sequence Number (ID): 45 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑023 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: acgggcacgt cattga 16 Sequence Number (ID): 46 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑023 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tcaatgacgt gcccgttgg 19 Sequence Number (ID): 47 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑024 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ttgtttacct atagct 16 Sequence Number (ID): 48 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑024 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: agctataggt aaacaagtt 19 Sequence Number (ID): 49 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑025 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: caacttgttt acctat 16 Sequence Number (ID): 50 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑025 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ataggtaaac aagttgggt 19 Sequence Number (ID): 51 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑026 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ccaacttgtt taccta 16 Sequence Number (ID): 52 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑026 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: taggtaaaca agttgggta 19 Sequence Number (ID): 53 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑027 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ttgcacagaa catcat 16 Sequence Number (ID): 54 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑027 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atgatgttct gtgcaattc 19 Sequence Number (ID): 55 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑028 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: attgcacaga acatca 16 Sequence Number (ID): 56 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑028 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tgatgttctg tgcaattcg 19 Sequence Number (ID): 57 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑029 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aaagcaagtc caggga 16 Sequence Number (ID): 58 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑029 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tccctggact tgctttgat 19 Sequence Number (ID): 59 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑030 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: taacaggctt catgga 16 Sequence Number (ID): 60 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑030 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tccatgaagc ctgttatcc 19 Sequence Number (ID): 61 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑031 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ggataacagg cttcat 16 Sequence Number (ID): 62 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑031 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atgaagcctg ttatccgct 19 Sequence Number (ID): 63 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑032 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: cggataacag gcttca 16 Sequence Number (ID): 64 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑032 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tgaagcctgt tatccgctt 19 Sequence Number (ID): 65 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑033 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gaaaatatgg aggaat 16 Sequence Number (ID): 66 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑033 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: attcctccat attttcctt 19 Sequence Number (ID): 67 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑034 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ggaaaatatg gaggaa 16 Sequence Number (ID): 68 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑034 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttcctccata ttttccttc 19 Sequence Number (ID): 69 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑035 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tgaaggaaaa tatgga 16 Sequence Number (ID): 70 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑035 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tccatatttt ccttcaaac 19 Sequence Number (ID): 71 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑036 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ctgtttgaag gaaaat 16 Sequence Number (ID): 72 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑036 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: attttccttc aaacagaac 19 Sequence Number (ID): 73 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑037 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tctgtttgaa ggaaaa 16 Sequence Number (ID): 74 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑037 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttttccttca aacagaaca 19 Sequence Number (ID): 75 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑038 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ttctgtttga aggaaa 16 Sequence Number (ID): 76 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑038 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tttccttcaa acagaacag 19 Sequence Number (ID): 77 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑039 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gttctgtttg aaggaa 16 Sequence Number (ID): 78 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑039 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttccttcaaa cagaacagt 19 Sequence Number (ID): 79 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑040 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: cactgttctg tttgaa 16 Sequence Number (ID): 80 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑040 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttcaaacaga acagtgttg 19 Sequence Number (ID): 81 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑041 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: acactgttct gtttga 16 Sequence Number (ID): 82 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑041 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tcaaacagaa cagtgttgt 19 Sequence Number (ID): 83 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑042 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: caacactgtt ctgttt 16 Sequence Number (ID): 84 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑042 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aaacagaaca gtgttgttt 19 Sequence Number (ID): 85 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑043 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: acaacactgt tctgtt 16 Sequence Number (ID): 86 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑043 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aacagaacag tgttgttta 19 Sequence Number (ID): 87 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑044 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: taaacaacac tgttct 16 Sequence Number (ID): 88 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑044 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: agaacagtgt tgtttaatg 19 Sequence Number (ID): 89 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑045 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ttcaaagcct taggtt 16 Sequence Number (ID): 90 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑045 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aacctaaggc tttgaacat 19 Sequence Number (ID): 91 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑046 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tgaagcattt gacttt 16 Sequence Number (ID): 92 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑046 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aaagtcaaat gcttcattc 19 Sequence Number (ID): 93 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑047 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: atgaagcatt tgactt 16 Sequence Number (ID): 94 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑047 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aagtcaaatg cttcattca 19 Sequence Number (ID): 95 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑048 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aatgaagcat ttgact 16 Sequence Number (ID): 96 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑048 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: agtcaaatgc ttcattcac 19 Sequence Number (ID): 97 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑049 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tgaatgaagc atttga 16 Sequence Number (ID): 98 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑049 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tcaaatgctt cattcacta 19 Sequence Number (ID): 99 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑050 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: agtgaatgaa gcattt 16 Sequence Number (ID): 100 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑050 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aaatgcttca ttcactagg 19 Sequence Number (ID): 101 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑051 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: tagtgaatga agcatt 16 Sequence Number (ID): 102 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑051 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aatgcttcat tcactaggt 19 Sequence Number (ID): 103 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑052 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: ctagtgaatg aagcat 16 Sequence Number (ID): 104 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑052 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atgcttcatt cactaggtc 19 Sequence Number (ID): 105 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑053 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gttccttgca gctgac 16 Sequence Number (ID): 106 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑053 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: gtcagctgca aggaacaca 19 Sequence Number (ID): 107 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑054 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: agaagcttca ggaaaa 16 Sequence Number (ID): 108 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑054 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttttcctgaa gcttctgga 19 Sequence Number (ID): 109 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑055 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: cagaagcttc aggaaa 16 Sequence Number (ID): 110 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑055 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tttcctgaag cttctggac 19 Sequence Number (ID): 111 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑056 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: atgtgattca gaagaa 16 Sequence Number (ID): 112 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑056 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttcttctgaa tcacatgtt 19 Sequence Number (ID): 113 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑057 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: catgtgattc agaaga 16 Sequence Number (ID): 114 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑057 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tcttctgaat cacatgttg 19 Sequence Number (ID): 115 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑058 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: atagccaaga taccaa 16 Sequence Number (ID): 116 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑058 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: ttggtatctt ggctattaa 19 Sequence Number (ID): 117 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑059 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gttaatagcc aagata 16 Sequence Number (ID): 118 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑059 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tatcttggct attaacttt 19 Sequence Number (ID): 119 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑060 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: agttaatagc caagat 16 Sequence Number (ID): 120 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑060 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: atcttggcta ttaactttg 19 Sequence Number (ID): 121 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑061 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: aagttaatag ccaaga 16 Sequence Number (ID): 122 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑061 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: tcttggctat taactttgc 19 Sequence Number (ID): 123 Length: 16 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..16 > note, asiRORB‑062 sense ‑ source, 1..16 > mol_type, other RNA > organism, synthetic construct Residues: gaatacactg tttcct 16 Sequence Number (ID): 124 Length: 19 Molecule Type: RNA Features Location / Qualifiers: ‑ misc_feature, 1..19 > note, asiRORB‑062 antisense ‑ source, 1..19 > mol_type, other RNA > organism, synthetic construct Residues: aggaaacagt gtattcact 19 Sequence Number (ID): 125 Length: 21 Molecule Type: DNA Features Location / Qualifiers: ‑ misc_feature, 1..21 > note, ROR‑beta Forward primer ‑ source, 1..21 > mol_type, other DNA > organism, synthetic construct Residues: tgcccaagtc cgaaggttat t 21 Sequence Number (ID): 126 Length: 20 Molecule Type: DNA Features Location / Qualifiers: ‑ misc_feature, 1..20 > note, ROR‑beta Reverse primer ‑ source, 1..20 > mol_type, other DNA > organism, synthetic construct Residues: ccatgccagc tgatggagtt 20 Sequence Number (ID): 127 Length: 20 Molecule Type: DNA Features Location / Qualifiers: ‑ misc_feature, 1..20 > note, GAPDH Forward primer ‑ source, 1..20 > mol_type, other DNA > organism, synthetic construct Residues: gggtgtgaac cacgagaaat 20 Sequence Number (ID): 128 Length: 20 Molecule Type: DNA Features Location / Qualifiers: ‑ misc_feature, 1..20 > note, GAPDH Reverse primer ‑ source, 1..20 > mol_type, other DNA > organism, synthetic construct Residues: gtcatgagcc cttccacaat 20 END

Claims

1. A nucleic acid molecule for inducing RNAi, comprising: The antisense strand is 19 nt in length and contains a sequence complementary to the mRNA encoding ROR-β; And a sense chain of 16 nt in length, which forms a complementary bond with the antisense chain, wherein the 5' end of the antisense chain and the 3' end of the sense chain form a flat end. (a) The antisense strand consists of the nucleotide sequence of SEQ ID NO: 52, and the sense strand consists of the nucleotide sequence of SEQ ID NO: 51; or (b) The antisense strand consists of the nucleotide sequence of SEQ ID NO: 110, and the sense strand consists of the nucleotide sequence of SEQ ID NO:

109.

2. The nucleic acid molecule for inducing RNAi according to claim 1, wherein, The sense or antisense strand of the nucleic acid molecule used to induce RNAi includes at least one chemical modification selected from the group consisting of: In the nucleotide, the -OH group at the 2' carbon position of the sugar structure is replaced by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl. Oxygen in the sugar structure of a nucleotide is replaced by sulfur. Nucleotide bonds are modified to form thiophosphate, borophosphate, or methyl phosphonate; or modified to form PNA, LNA, or UNA; and Phosphate groups, lipophilic compounds, or cell-penetrating peptide bonds.

3. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The lipophilic compound is selected from the group consisting of cholesterol, tocopherol, stearic acid, retinoic acid, DHA, palmitic acid, linoleic acid, linolenic acid, and long-chain fatty acids having 10 or more carbon atoms.

4. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The sense chain includes at least one chemical modification selected from the group consisting of: Two to four nucleotide bonds near the 3' end are modified with thiophosphate, borophosphate, or methyl phosphonate; In two or more nucleotides, the -OH group at the 2' carbon position of the sugar structure is replaced by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl. Lipophilic compounds or cell-penetrating peptides bind to the 3' end.

5. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The antisense chain includes any one or more chemical modifications selected from the group consisting of: The three to five nucleotide bonds near the 3' end are modified with thiophosphate, borophosphate, or methyl phosphonate; In two or more nucleotides, at the 2' carbon position of the sugar structure, the -OH group is replaced by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, or -O-3-dimethylaminopropyl; and Phosphate groups or cell-penetrating peptides bind to the 5' end.

6. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The nucleic acid molecule used to induce RNAi includes at least one modification selected from the group consisting of: A modification in which the -OH group in the 2' carbon position of the sugar structure of two or more nucleotides in the sense or antisense strand is replaced by -OCH3 (methoxy) or -F (fluorine); In the sense or antisense strand, 10% or more of the nucleotide bonds are modified to thiophosphates. Cholesterol or palmitic acid binds to the 3' end of the sense chain; and The phosphate group binds to the 5' end of the antisense chain.

7. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The nucleic acid molecules used to induce RNAi include any antisense strand selected from the group consisting of, (a) P-mUAGGfUAAAfCAAGfUfUG G G fU A; and (b)P-mUfAmGfGmUfAmAfAmCfAmAfGmUfUmG fG mG fU mA, in, The terms refer to modifications of the thiophosphate bond, where m indicates substitution by 2'-OCH3, f indicates substitution by 2'-F, and P indicates binding with a 5'-phosphate group.

8. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The nucleic acid molecules used to induce RNAi include any sense strand selected from the group consisting of, (A) mCm HOLD ON mU No chol: (B) mCCmAAmCUmUGmUUmUAmCC mU A chol: (C)mCfCmAfAmCfUmUfGmUfUmUfAmCfC in the fA chol; as well as (D)mCmHOLD UP mU No BREAK, in, The terms refer to modifications of the thiophosphate bond, where m indicates substitution by 2'-OCH3, chole indicates binding to 3'-cholesterol, and PA indicates binding to 3'-palmitic acid.

9. The nucleic acid molecule for inducing RNAi according to claim 2, wherein, The nucleic acid molecule used to induce RNAi has cell-penetrating ability.

10. A pharmaceutical composition for improving or treating retinal diseases, comprising, as an active ingredient, the nucleic acid molecule for inducing RNAi as described in any one of claims 1 to 9.

11. The pharmaceutical composition according to claim 10, wherein, The retinal disease is selected from any one of the following groups: Usher syndrome, Stargardt disease, Baldr-Bede syndrome, Best disease, choroidal defect, choroidal-retinal atrophy, retinitis pigmentosa, macular degeneration, congenital amaurosis, BCM, retinoschisis, ML, microstomia, or Rifsum disease.

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