KR102321425B1 - Asymmetric siRNA Inhibiting Expression of NRL - Google Patents

Abstract

The present invention relates to an asymmetric siRNA that inhibits the expression of neural retina leucine zipper (NRL) and uses thereof, and more particularly, to an antisense strand comprising a sequence complementary to an mRNA encoding NRL, and to the antisense strand complementary to the antisense strand It relates to an asymmetric siRNA comprising a sense strand forming a bond, and a pharmaceutical composition for improving or treating retinal diseases comprising the asymmetric siRNA.

Description

Asymmetric siRNA Inhibiting Expression of NRL {Asymmetric siRNA Inhibiting Expression of NRL}

The present invention relates to an asymmetric siRNA that inhibits the expression of neural retina leucine zipper (NRL) and uses thereof, and more particularly, to an antisense strand comprising a sequence complementary to an mRNA encoding NRL, and to the antisense strand complementary to the antisense strand It relates to an asymmetric siRNA comprising a sense strand forming a bond, and a pharmaceutical composition for improving or treating retinal diseases comprising the asymmetric siRNA.

Retinitis pigmentosa is a disease that can lead to blindness due to mutations in a gene that gives characteristics of rod cells, resulting in destruction of rod cells and secondary destruction of cone cells. At this time, when the Neural retina leucine zipper (NRL), a major transcription factor that allows differentiation into rod cells, is knocked out with adenovirus-associated virus (AAV) delivered CRISPR/Cas9, rod cells acquire partial cone-like properties, and rod cells It was observed in a mouse model of retinal degeneration that, when there is a mutation in a gene that gives the characteristics of by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice." Nature communications 8 (2017): 14716). NRL plays a very important function in the differentiation process of rod and cone cells, indicating that the transcriptional activity of NRL acts directly on rod cell differentiation. Because NRL functions in the development and differentiation of retina as well as in the expression of photoreceptor proteins such as rhodopsin, a study has been reported that genetic mutations in NRL are also related to retinal diseases.

CRISPR/Cas9 is a gene editing technology that regulates gene expression at the transcriptional level by causing a double strand break in DNA. However, knockout of a specific gene through CRISPR/Cas9 cannot restore the state prior to knockout when an unintended mutation is made in a non-target gene, which may cause serious side effects. In addition, since Cas9 is a bacterial protein, when expressed in vivo, it can cause immune responses and other unknown side effects, making it difficult to develop as a drug for diseases including retinitis pigmentosa.

On the other hand, the treatment of diseases using RNA interference phenomenon can treat diseases more safely because siRNA that targets mRNA and regulates gene expression at the translational level is used. A short interfering RNA (siRNA) consists of a sense strand having the same sequence as the target mRNA and an antisense strand having a complementary sequence thereto. Conventional siRNA has a short duplex of 19-21bp, and 2 nucleotides protrude from both strands 3'. siRNA enters the cell, attaches to the target mRNA, degrades the target mRNA, and suppresses the expression of the target gene. Since it is possible to target all mRNAs by changing the sequence of oligo, it is possible to suppress the expression of proteins with complex structures, thus enabling the treatment of diseases such as cancer, viral infection, and genetic diseases that are currently difficult to treat. . However, siRNA introduced into cells can cause side effects such as induction of immune response and suppression of non-target genes.

Because siRNA is negatively charged due to the phosphate backbone, it has a repulsive force with the negatively charged cell membrane, so a delivery system is required to introduce siRNA into the cell. As an example of a delivery system, a method of introducing siRNA into a cell by offsetting the negative charge by wrapping the siRNA with a liposome or polymer having a positive charge is widely used. However, a positively charged carrier may cause various side effects, such as binding to a negatively charged cell membrane, exhibiting unwanted toxicity, or forming an unwanted complex through interaction with various types of proteins in the cell. In addition, since siRNA is rapidly degraded by nuclease in the blood, the amount of siRNA reaching the target cell may not be sufficient to significantly reduce the expression of the target gene. Therefore, there is a need for a method by which siRNA can be safely and effectively delivered into target cells.

Accordingly, the present inventors selected an NRL target siRNA capable of inhibiting the expression of NRL, and made intensive research efforts to develop an siRNA that is delivered into cells without the aid of a carrier and has high resistance to nuclease. The siRNAs were designed, the siRNA that most efficiently inhibits NRL was selected through screening, and it was confirmed that the problem of intracellular delivery could be overcome through modification, and the present invention was completed.

An object of the present invention is to provide an asymmetric shorter duplex siRNA (asiRNA) that specifically inhibits the expression of NRL.

Another object of the present invention is to provide a pharmaceutical composition for improving or treating a retinal disease, or a method for improving or treating a retinal disease, comprising the asymmetric siRNA.

In order to achieve the above object, the present invention includes an antisense strand comprising a sequence complementary to an mRNA encoding a neural retina leucine zipper (NRL), and a sense strand forming a complementary bond with the antisense strand, The 5' end of the strand and the 3' end of the sense strand form a blunt end.

The present invention also provides a pharmaceutical composition for improving or treating retinal diseases comprising the siRNA.

The present invention also provides a method for improving or treating retinal diseases comprising administering the siRNA to an individual.

According to the present invention, an asymmetric siRNA capable of efficiently inhibiting the expression of NRL, which plays a very important function in the differentiation process of rod and cone cells, is selected, and the siRNA is introduced into the cell without the aid of a transporter and is subjected to nuclease. By chemically modifying it to have resistance to cytotoxicity due to the carrier and enabling more efficient inhibition of gene expression in vivo, it can be usefully used as a therapeutic agent for retinal diseases including retinitis pigmentosa.

1 shows the structure of an asiRNA (hereinafter, NRL asiRNA or asiNRL) for suppressing NRL expression of 16-19mer.
Figure 2 shows that the siRNA was designed in consideration of the homology of Homo sapiens (H), Rattus norvegicus (R), and Mus musculus (M), and Figure 2 (A) shows the number of mismatches between H and R, and M The number of corresponding sequences is shown in a table, Figure 2 (B) is a schematic diagram of Figure 2 (A), Figure 2 (C) is after designing 73 NRL asiRNA Human NRL transcript variants information was updated and , a diagram showing that 40 NRL asiRNAs belonging to all transcript variants were screened accordingly.
3 is a graph showing the relative NRL mRNA level (n=2) obtained by transfection of 1 nM NRL asiRNA, and shows the result of normalization to the expression amount of Neo in the transfected plasmid.
4 is a qRT-PCR result of the top 7 efficiently inhibiting NRL These are the results of western blot by transfection (3nM) of NRL asiRNA.
Figure 5 is a result of western blot with different concentrations of NRL asiRNA #47 and #49 (from 0.01 nM to 3 nM) to obtain an _{approximate IC 50 value.} Of NRL asiRNA # 47 IC ₅₀ value is about 0.2nM, NRL asiRNA IC ₅₀ value is 0.6-1nM of # 49.
6 is a schematic diagram of chemical modification of NRL asiRNA of Table 4, and for the sense strand, S-ori, S-OMe/F, and SF are sequentially indicated as 1S, 2S, 3S, AS-ori, AS- OMe/F and AS-F are indicated as 1AS, 2AS, and 3AS in that order.
7 shows #47 asiNRL (sense strand-antisense strand, 16mer-19mer) without chemical modification, and #47 cp asiNRL, #49 cp asiNRL with 25mer as 25mer with the chemical modification described in FIG. 6 introduced to A549 -Stable cell line is the result of treatment without a carrier.
The 1 ^st modification will effect the results of Fig. 7 in Fig. 8 best 2S / 2AS (S-OMe / F, AS-OMe / F) will showing a, 2 ^nd modification of Figure 8 is the 2S / 2AS (S- OMe/F, AS-OMe/F) is a diagram showing additional modification. That is, an additional modification to the sense strand 2S is denoted as 2S-1, and an additional modification to the antisense strand 2AS is denoted as 2AS-1 and 2AS-2, respectively.
9 shows the effect of 2S/2AS, 2S/2AS-1, 2S/2AS-2, 2S-1/2AS-1, 2S-1/2AS-2 of #47 cp asiNRL (SS-AS: 16mer-25mer) is the result of checking
Figure 10 is #47 cp asiNRL (SS-AS: 16mer-25mer) _{to obtain the IC 50} value and maximum knockdown value of 2S/2AS and 2S/2AS-1 NRL These are the results of different concentrations of cp-asiRNA (from 30 nM to 3000 nM).
11 is a result confirming the efficacy of #47 cp-asiNRL (SS-AS: 16mer-26mer) in which the antisense strand (AS) is extended from 25mer to 26mer in #47 cp-asiNRL (SS-AS: 16mer-25mer).
12 shows the sequence and modification information of #47 cp-asiNRL 2S-2AS-1(1626) (sense strand: 16mer, antisense strand: 26mer) in FIG. 11 .
13 shows the change in the expression of NRL according to the administration of the candidate substance in the retinal tissue of the mouse eye on the 7th day after intraocular administration of the #47 cp-asiNRL 2S-2AS-1(1626) substance through western blot. is the result
14 shows the change in the expression of NRL according to the administration of the candidate substance in the retinal tissue of the mouse eye on the 14th day after intraocular administration of the #47 cp-asiNRL 2S-2AS-1(1626) substance through western blot. is the result

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

Definitions of key terms used in the detailed description of the present invention are as follows.

"RNAi (RNA interference; RNA interference)" refers to the introduction of double-stranded RNA (dsRNA) consisting of a strand having a sequence homologous to the mRNA of a target gene and a strand having a sequence complementary to this into a cell etc. It refers to a mechanism for suppressing the expression of a target gene by inducing degradation.

“Small interfering RNA (siRNA)” refers to a short double-stranded RNA (dsRNA) that mediates sequence-specifically efficient gene silencing.

An "antisense strand" is a polynucleotide substantially or 100% complementary to a target nucleic acid of interest, for example, messenger RNA (mRNA), non-mRNA RNA sequence (eg, microRNA, piwiRNA). , tRNA, rRNA and hnRNA) or coding or non-coding DNA sequences in whole or in part.

A "sense strand" is a polynucleotide having the same nucleic acid sequence as a target nucleic acid, and is either a messenger RNA (mRNA), a non-mRNA RNA sequence (eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA) or coding or non-coding Refers to a polynucleotide identical in whole or in part to a DNA sequence.

A “gene” is to be taken in its broadest sense and may encode a structural protein or a regulatory protein. In this case, the regulatory protein includes a transcription factor, a heat shock protein, or a protein involved in DNA/RNA replication, transcription and/or translation. In the present invention, the target gene to be suppressed is inherent in the viral genome, and may be integrated into an animal gene or exist as an extrachromosomal component. For example, the gene of interest may be a gene on the HIV genome. In this case, the siRNA molecule is useful for inactivating the translation of an HIV gene in a mammalian cell.

"Neural retina leucine zipper (NRL)" is a transcription factor that attaches to the promoter of a gene that gives rod cell characteristics, such as rhodopsin, so that photoreceptor precursors are differentiated into rod cells. NRL blocks the differentiation of photoreceptor progenitors into cones either directly or through NR2E3 downstream. Therefore, NRL is an important factor for the differentiation of photoreceptor progenitors into rod cells.

Retinitis pigmentosa (retinitis pigmentosa) is a disease in which a mutation occurs in a gene that expresses characteristics of rod cells, causing rod cells to be destroyed first and cone cells secondary to destruction. Studies have shown that the detrimental effects of mutations in genes important for photoreceptor function are greatest in a fully differentiated photoreceptor environment. Therefore, when NRL, which induces differentiation into rod cells, is knocked out, rod cells are morphologically changed to cone-like cells and rod cell functions are lost. has been shown to prevent the loss of Therefore, suppressing the expression of NRL has potential as a treatment for retinitis pigmentosa.

The object of the present invention can be broadly expressed in two ways. First, to design siRNAs that target NRL, and to select siRNAs that most efficiently inhibit NRL through screening, and second, to introduce siRNA into cells without a specific carrier. It is the introduction of chemical modifications to deliver and increase resistance to nucleases. To pass through a cell membrane composed of phospholipids, it must be small or hydrophobic. However, siRNA is negatively charged by the phosphate backbone, so it is difficult to penetrate the cell membrane. In addition, it is necessary to increase the resistance to nucleases to have a long lifetime in serum, so that the amount to reach the target is sufficient to cause effective RNAi. Therefore, by introducing a modification (modification), the delivery problem of siRNA was overcome.

In an embodiment of the present invention, NRL asiRNA with the best knockdown efficiency was selected by first designing an asiRNA targeting NRL and transfecting the asiRNA in NRL-expressing cells. The following four modifications are introduced into the selected siRNA to transform the asiRNA to have cell penetrating ability and resistance to nucleases. First, cholesterol is added to the 3' end of the sense strand, allowing siRNA to penetrate the cell membrane. Second, the phosphate backbone near the 5' end or 3' end of the sense and antisense strands is substituted with phosphorothioate to have resistance to exohydrolytic enzymes, and uptake into cells and siRNA in vivo enable the bioavailability of Third, by modifying 2' of sugar with OMethyl, it gives resistance to nucleolytic enzymes, lowers siRNA immunogenicity, and reduces off-target effects. Fourth, the 2' of the sugar is modified with fluoro to give stability to the double-stranded duplex, increase the stability in serum, and enable efficient silencing in vitro and in vivo. By adding the above modifications to the siRNA, the siRNA has the ability to penetrate cells and stays in the serum for a longer period of time, enabling more efficient gene suppression as a sufficient amount of siRNA is delivered to the target cell.

Accordingly, in one aspect, the present invention includes an antisense strand comprising a sequence complementary to an mRNA encoding a neural retina leucine zipper (NRL), and a sense strand forming a complementary bond with the antisense strand, and the antisense strand It relates to an siRNA, characterized in that the 5' end of the and the 3' end of the sense strand form a blunt end.

siRNA in the present invention is a concept including all substances having a general RNAi (RNA interference) action. RNAi is an intracellular gene regulation mechanism first discovered in Caenorthabditis elegans in 1998. It is known that the mechanism of action induces target gene degradation by complementary binding of the antisense strand among the RNA double strands injected into the cell to the mRNA of the target gene. Among them, siRNA is one of the methods of suppressing gene expression "in vitro". siRNA of 19-21bp is theoretically capable of selectively suppressing almost all genes, so it can be developed as a treatment for various gene-related diseases such as cancer and viral infection, and is the most popular new drug development candidate technology. The first attempt at in vivo treatment using siRNA in mammals was in mid 2003, and since then, many attempts have been made on in vivo treatment for applied research.

However, contrary to the possibility, side effects and disadvantages of siRNA have been continuously reported. For the development of RNAi-based therapeutics, it is necessary to overcome barriers such as 1) absence of an effective delivery system, 2) off-target effect, 3) induction of immune response, and 4) saturation of RNAi machinery in cells. Although siRNA is an effective method for directly regulating the expression of a target gene, it is difficult to develop a therapeutic agent due to these problems. In this regard, asymmetric shorter duplex siRNA (asiRNA) is an asymmetric RNAi-inducing structure having a shorter double helix length compared to the 19+2 structure of conventional siRNA. It is a technology that overcomes problems such as the off-target effect, the saturation of the RNAi mechanism, and the immune response by TLR3, which are confirmed in the existing siRNA structure technology, and thus it is possible to develop new RNAi drugs with low side effects.

Based on this, the present invention provides an asymmetric siRNA comprising a sense strand and an antisense strand complementary to the sense strand, and the siRNA according to the present invention does not cause problems such as off-target effect and saturation of the RNAi mechanism and is stable While maintaining high delivery efficiency, it is possible to effectively inhibit the expression of the NRL target gene to a desired degree.

In the present invention, the siRNA may be characterized in that the sense strand has a length of 15 to 17 nt, and the antisense strand has a length of 16 nt or more. Although not limited thereto, the antisense strand may be characterized as having a length of 16 to 31 nt, and preferably having a length of 19 to 26 nt. More preferably, the length of the sense strand is 16 nt, and the length of the antisense strand complementary thereto may be characterized in that it is 19 nt, 24 nt, 25 nt or 26 nt, but is not limited thereto.

The 3' end of the sense strand and the 5' end of the antisense strand form a blunt end. The 3' end of the antisense strand may include, for example, an overhang of 1 to 16 nt.

In one embodiment of the present invention, 73 NRL asiRNAs were designed to suppress the expression of NRL, and suppression of mRNA and protein levels was achieved in transient cells that transiently express NRL and stable cell lines that continuously express NRL. Confirmed.

In the present invention, the sense strand is 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, 123, 125, 127, 129, 131, 133, 135, 137, 139 , 141, 143, and may be characterized in that selected from the group consisting of 145.

In the present invention, the antisense strand is 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, 124, 126, 128, 130, 132, 134, 136, 138, 140 , 142, 144, 146, 153, 154, and may be characterized in that selected from the group consisting of 155.

Specifically, the sense strand may be, for example, selected from the group consisting of SEQ ID NOs: 11, 93, 95, 97, 101, 111, 119, 123, 133, 135, 143, and 145, and the antisense strand The strand may be selected from the group consisting of SEQ ID NOs: 12, 94, 96, 98, 102, 112, 120, 124, 134, 136, 144, 146, 153, 154, and 155. In addition, the sense strand may be selected from the group consisting of SEQ ID NOs: 93, 95, 97, 133, 135, 143 and 145, and the antisense strand may be selected from the group consisting of SEQ ID NOs: 94, 96, 98, 134, 136, 144, It may be one selected from the group consisting of 146, 153, 154, and 155. In addition, the sense strand may be SEQ ID NO: 93, and the antisense strand may be SEQ ID NO: 153 or 155.

In the present invention, the sense strand or the antisense strand of the siRNA may include one or more chemical modifications.

General siRNA cannot pass through the cell membrane for reasons such as high negative charge and high molecular weight due to the phosphate backbone structure, and is rapidly degraded and removed from the blood, so it is difficult to deliver a sufficient amount for RNAi induction to the actual target site. Currently, in the case of in vitro delivery, many high-efficiency delivery methods using cationic lipids and cationic polymers have been developed, but in the case of in vivo, it is difficult to deliver siRNA as high as in vitro, and it is There is a problem in that the siRNA delivery efficiency is reduced by the interaction.

Accordingly, the present inventors introduced a chemical modification to the asymmetric siRNA structure to develop an asiRNA construct (cp-asiRNA) with self-delivery ability that can be effectively and intracellularly delivered without a separate carrier.

In the present invention, the chemical modification in the sense strand or the antisense strand may include one or more selected from the group consisting of: -OH group at the 2' carbon position of the sugar structure in the nucleotide -CH ₃ (methyl), - OCH ₃ (methoxy), -NH ₂ , -F (fluorine), -O-2-methoxyethyl -O-propyl (propyl), -O-2-methylthioethyl, -O-3-amino propyl, substituted with -O-3-dimethylaminopropyl; replacement of oxygen in the nucleotide structure with sulfur; nucleotide linkages are modified with phosphorothioate, boranophosphate, or methyl phosphonate; transformation into peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA) form; and a phosphate group, a lipophilic compound, or a cell penetrating peptide bond.

In the present invention, the lipophilic compound may be characterized in that it is selected from the group consisting of cholesterol, tocopherol and long-chain fatty acids having 10 or more carbon atoms. Preferably, it may be characterized as cholesterol, but is not limited thereto.

Specifically, the siRNA is a modification in which the -OH group is substituted with -OCH3(methoxy) or -F(fluorine) at the 2' carbon position of the sugar structure within two or more nucleotides of the sense strand or the antisense strand; at least 10% of the nucleotide linkages in the sense or antisense strand are modified to phosphorothioate; cholesterol binding to the 3' end of the sense strand; and a phosphate group bond to the 5' end of the antisense strand; it may be characterized in that it comprises one or more modifications selected from the group consisting of.

Preferably, the sense strand is any one selected from the group consisting of (a) to (g) of the table below, and the antisense strand is any one selected from the group consisting of (h) to (q) of the table below. can

In the above sequence, * is a phosphorothioated bond, m is 2'-O-methyl (Methyl), 2'-F- is 2'-fluorine (Fluoro), chol is cholesterol, P is 5'- It means a phosphate group.

Specifically, the sense strand may be any one of (a) to (c) of the above table, and the antisense strand may be any one of (h) to (j) of the above table; The sense strand may be any one of (d) to (f) in Tables above, and the antisense strand may be any one of (k) to (m) in Tables above.

In addition, the sense strand is (b) of the above table, and the antisense strand is any one of (i), (n) and (o) of the above table; The sense strand may be (g) in Table, and the antisense strand may be (n) or (o) in Table.

In addition, the sense strand may be (b) of the table above, and the antisense strand may be (p) or (q) of the table.

According to one embodiment, the siRNA comprises a sense strand consisting of (b) the nucleotide sequence of the table and an antisense strand consisting of the nucleotide sequence (i) of the table; a sense strand consisting of the nucleotide sequence (b) of the table above and an antisense strand consisting of the nucleotide sequence (n) of the table above; a sense strand consisting of the nucleotide sequence (b) of the table above and an antisense strand consisting of the nucleotide sequence (p) of the table above; Alternatively, the sense strand consisting of the nucleotide sequence of (b) in the table above and the antisense strand consisting of the nucleotide sequence of (q) in the table above, preferably, the siRNA is a sense strand consisting of the nucleotide sequence of (b) in the table and (q) in the table It may be an antisense strand consisting of a nucleotide sequence.

In the present invention, one to three phosphate groups may be bonded to the 5' end of the antisense strand, but the present invention is not limited thereto.

In one embodiment of the present invention, three phosphate linkages were substituted with phosphorothioate linkage at the 3' end of the sense strand and cholesterol was added. In the antisense strand, 4 phosphate linkages were substituted with phosphorothioate linkages at the 3' end in common. And 3 sense strands and 3 antisense strands were synthesized by differentiating the number and substitution positions of OMethyl and fluoro at 2' of the sugar. Therefore, by annealing in 9 cases, the modifications that best knockdown NRL without a transporter were selected, and several modifications were added. This involves the substitution of two phosphate linkages at the 5' end of the sense strand and the antisense strand with a phosphorothioate linkage and the addition of phosphate at the 5' end of the antisense antisense strand. Therefore, the NRL cp-asiRNA that knockdown the NRL most efficiently through two modifications was selected.

In another aspect, the present invention relates to a pharmaceutical composition for improving or treating retinal diseases comprising the siRNA.

In the present invention, the retinal disease is Usher syndrome, Stargardt disease, Badette-Biddle syndrome, Best's disease, choroid deficiency, choroidal atrophy (gyrate-atrophy), retinitis pigmentosa, retinal macular degeneration, Leber Congenital Amaurosis (Leber's Hereditary Optic Neuropathy), BCM (Blue-cone monochromacy), retinal interlayer separation, ML (Malattia Leventinese), Oguchi disease or Refsum disease can, but is not limited thereto.

The pharmaceutical composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredient siRNA. A pharmaceutically acceptable carrier must be compatible with the active ingredient of the present invention, and contains saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or two or more of these components. They can be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats can be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, and the like. In particular, it is preferable to provide the formulation in a lyophilized form. For the preparation of the freeze-dried formulation, a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for freeze-drying may be added.

The method of administering the pharmaceutical composition may be determined by a person skilled in the art based on the symptoms of the patient and the severity of the disease. In addition, it can be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, etc., and may be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles. .

The pharmaceutical composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited thereto, but for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual Alternatively, topical administration is possible. The dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, method, excretion rate or severity of disease, etc. can decide In addition, the composition of the present invention may be formulated into a suitable dosage form for clinical administration using known techniques.

In another aspect, the present invention relates to a method for improving or treating a retinal disease comprising administering the siRNA to a subject. Since the configuration included in the improvement or treatment method according to the present invention is the same as the configuration included in the invention described above, the above description can be equally applied to the improvement or treatment method.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Screening of 73 RNAi-induced double-stranded nucleic acid molecules targeting NRL

NRL for screening Asymmetric siRNA (asiRNA) was designed. Conventional siRNA is a duplex of 19 base pairs having 2 nucleotide overhangs on both strand 3'. asiRNA has a blunt end at the 5' end of the antisense strand and a short duplex of 15-16 base pairs, exhibiting the same inhibitory efficiency as siRNA, and reduced off-target effect due to the shortened sense length. Therefore, it was designed as an asiRNA of 16mer (sense strand)-19mer (antisense strand) ( FIG. 1 ).

NRL asiRNA was designed considering the homology of Homo sapiens (H), Rattus norvegicus (R), and Mus musculus (M) for animal experiments. These are sequences that are 100% identical to Homo sapiens, and even sequences with one or two nucleotide mismatches with Rattus norvegicus and Mus musculus are allowed. In this case, the seed region (from the 2nd nucleotide to the 8th nucleotide), which plays an important role in RNA interference, contained only those without a mismatched sequence. Therefore, 6 sequences in which both H and R and M match, H and R match, 12 sequences in which M matches one or two nucleotides mismatch, H and M match, and R matches one nucleotide A total of 73 sequences were designed, with 8 mismatched sequences, 47 sequences in which H and R have one nucleotide mismatch, and M and one or two nucleotide mismatches. Later, information on human transcript variants was updated, and among them, 40 NRLs included in all transcript variants asiRNA was selected and screened. Table 1 below shows the 73 NRLs. The sequence of asiRNA is shown.

No. sequence (5'→ 3') SEQ ID NO: One sense CGGCUGAAGCAGAGGC One antisense GCCUCUGCUUCAGCCGCAG 2 2 sense GGCUGAAGCAGAGGCG 3 antisense CGCCUCUGCUUCAGCCGCA 4 3 sense AAGCAGAGGCGCCGCA 5 antisense UGCGGCGCCUCUGCUUCAG 6 4 sense AGCAGAGGCGCCGCAC 7 antisense GUGCGGCGCCUCUGCUUCA 8 5 sense CGCUCCAAGCGGCUGC 9 antisense GCAGCCGCUUGGAGCGACA 10 6 sense GCUCCAAGCGGCUGCA 11 antisense UGCAGCCGCUUGGAGCGAC 12 7 sense ACUUUGACUUGAUGAA 13 antisense UUCAUCAAGUCAAAGUCAU 14 8 sense CUUUGACUUGAUGAAG 15 antisense CUUCAUCAAGUCAAAGUCA 16 9 sense UUUGACUUGAUGAAGU 17 antisense ACUUCAUCAAGUCAAAGUC 18 10 sense UUGACUUGAUGAAGUU 19 antisense AACUUCAUCAAGUCAAAAGU 20 11 sense GCCUGUCGCUCCAAGC 21 antisense GCUUGGAGCGACAGGCCUG 22 12 sense CCUGUCGCUCCAAGCG 23 antisense CGCUUGGAGCGACAGGCCU 24 13 sense GUCGCUCCAAGCGGCU 25 antisense AGCCGCUUGGAGCGACAGG 26 14 sense UCGCUCCAAGCGGCUG 27 antisense CAGCCGCUUGGAGCGACAG 28 15 sense CUCCAAGCGGCUGCAG 29 antisense CUGCAGCCGCUUGGAGCGA 30 16 sense UGACUUGAUGAAGUUU 31 antisense AAACUUCAUCAAGUCAAAG 32 17 sense AGGCCUGUCGCUCCAA 33 antisense UUGGAGCGACAGGCCUGCG 34 18 sense GGCCUGUCGCUCCAAG 35 antisense CUUGGAGCGACAGGCCUGC 36 19 sense CCUCCUUCACCCACCU 37 antisense AGGUGGGUGAAGGAGGCAC 38 20 sense CUCCUUCACCCACCUU 39 antisense AAGGUGGGUGAAGGAGGCA 40 21 sense UCCUUCACCCACCUUC 41 antisense GAAGGUGGGUGAAGGAGGC 42 22 sense CCUUCACCCACCUUCA 43 antisense UGAAGGUGGGUGAAGGAGG 44 23 sense CUUCACCCACCUUCAG 45 antisense CUGAAGGUGGGUGAAGGAG 46 24 sense UUCACCCACCUUCAGU 47 antisense ACUGAAGGUGGGUGAAGGA 48 25 sense UCACCCACCUUCAGUG 49 antisense CACUGAAGGUGGGUGAAGG 50 26 sense CACCCACCUUCAGUGA 51 antisense UCACUGAAGGUGGGUGAAG 52 27 sense AAUAUGUCAAUGACUU 53 antisense AAGUCAUUGACAUAUUCCA 54 28 sense AUAUGUCAAUGACUUU 55 antisense AAAGUCAUUGACAUAUUCC 56 29 sense UAUGUCAAUGACUUUG 57 antisense CAAAGUCAUUGACAUAUUC 58 30 sense AUGUCAAUGACUUUGA 59 antisense UCAAAGUCAUUGACAUAUU 60 31 sense UGUCAAUGACUUUGAC 61 antisense GUCAAAGUCAUUGACAUAU 62 32 sense UGCCUCCUUCACCCAC 63 antisense GUGGGUGAAGGAGGCACUG 64 33 sense GCCUCCUUCACCCACC 65 antisense GGUGGGUGAAGGAGGCACU 66 34 sense UCAGUGAACCAGGCAU 67 antisense AUGCCUGGUUCACUGAAGG 68 35 sense CAGUGAACCAGGCAUG 69 antisense CAUGCCUGGUUCACUGAAG 70 36 sense AGUGAACCAGGCAUGG 71 antisense CCAUGCCUGGUUCACUGAA 72 37 sense GUGAACCAGGCAUGGU 73 antisense ACCAUGCCUGGUUCACUGA 74 38 sense AGCUGUACUGGCUGGC 75 antisense GCCAGCCAGUACAGCUCCU 76 39 sense GGCUGGCUACCCUGCA 77 antisense UGCAGGGUAGCCAGCCAGU 78 40 sense GCUGGCUACCCUGCAG 79 antisense CUGCAGGGUAGCCAGCCAG 80 41 sense CUGGCUACCCUGCAGC 81 antisense GCUGCAGGGUAGCCAGCCA 82 42 sense UGGCUACCCUGCAGCA 83 antisense UGCUGCAGGGUAGCCAGCC 84 43 sense GGCUACCCUGCAGGCAG 85 antisense CUGCUGCAGGGUAGCCAGC 86 44 sense GCUACCCUGCAGCAGC 87 antisense GCUGCUGCAGGGUAGCCAG 88 45 sense CUACCCUGCAGGCAGCA 89 antisense UGCUGCUGCAGGGUAGCCA 90 46 sense UACCCUGCAGCAGCAG 91 antisense CUGCUGCUGCAGGGUAGCC 92 47 sense CGGCGCUGGUCUCGAU 93 antisense AUCGAGACCAGCGCCGCGU 94 48 sense GCGCUGGUCUCGAUGU 95 antisense ACAUCGAGACCAGCGCCGC 96 49 sense CGCUGGUCUCGAUGUC 97 antisense GACAUCGAGACCAGCGCCG 98 50 sense ACCGGCAGCUGCGGGG 99 antisense CCCCGCAGCUGCCGGUUUA 100 51 sense AGGCGCUGCGGCUGAA 101 antisense UUCAGCCGCAGCGCCUCGU 102 52 sense CGCUGCGGCUGAAGCA 103 antisense UGCUUCAGCCGCAGGCGCCU 104 53 sense CUGCGGCUGAAGCAGA 105 antisense UCUGCUUCAGCCGCAGGCGC 106 54 sense UGCGGCUGAAGCAGAG 107 antisense CUCUGCUUCAGCCGCAGCG 108 55 sense GCGGCUGAAGCAGAGG 109 antisense CCUCUGCUUCAGCCGCAGC 110 56 sense GCACGCUGAAGAACCG 111 antisense CGGUUCUUCAGCGUGCGGC 112 57 sense CACGCUGAAGAACCGC 113 antisense GCGGUUCUUCAGCGUGCGG 114 58 sense ACGCUGAAGAACCGCG 115 antisense CGCGGUUCUUCAGCGUGCG 116 59 sense CGCUGAAGAACCGCGG 117 antisense CCCGGGUUCUUCAGGCGUGC 118 60 sense GCUGAAGAACCGCGGC 119 antisense GCCGCGGUUCUUCAGCGUG 120 61 sense CUGAAGAACCGCGGCU 121 antisense AGCCCGCGGUUCUUCAGCGU 122 62 sense UGAAGAACCGCGGCUA 123 antisense UAGCCGCGGUUCUUCAGCG 124 63 sense GAAGAACCGCGGCUAC 125 antisense GUAGCCCGCGGUUCUCAGC 126 64 sense AAGAACCGCGGCUACG 127 antisense CGUAGCCGCGGUUCUUCAG 128 65 sense AGAACCGCGGCUACGC 129 antisense GCGUAGCCCGCGGUUCUUCA 130 66 sense GAACCGCGGCUACGCG 131 antisense CGCGUAGCCGCGGUUCUUC 132 67 sense ACAAGGCUCGCUGUGA 133 antisense UCACAGCGAGCCUUGUAGA 134 68 sense CAAGGCUCGCUGUGAC 135 antisense GUCACAGCGAGCCUUGUAG 136 69 sense AAGGCUCGCUGUGACC 137 antisense GGUCACAGCGAGCCUUGUA 138 70 sense AGGCUCGCUGUGACCG 139 antisense CGGUCACAGCGAGCCUUGU 140 71 sense GGCUCGCUGUGACCGG 141 antisense CCGGUCCAGCGAGCCUUG 142 72 sense GCUCGCUGUGACCGGC 143 antisense GCCGGUCACAGCGAGCCUU 144 73 sense CUCGCUGUGACCGGCU 145 antisense AGCCGGUCACAGCGAGCCU 146

Example 2: Screening of RNAi-induced double-stranded nucleic acid molecules targeting NRL

In order to find the asiRNA with the highest knockdown efficiency among synthesized NRL asiRNAs, a cell line expressing NRL to some extent is required.

In addition, NRL plasmid and NRL asiRNA were sequentially transfected to conduct an experiment first in a cell (transient cell line) expressing transiently NRL. More specifically, HeLa cells were cultured in DMEM (Dulbecco's modified Eagle's medium-corning) supplemented with 10% fetal bovine serum-gibco (FBS) in a 100 mm petri dish and grown, and then the cells were grown in a 12-well plate at 5× per well. 10⁴ was dispensed. After 24 hours, the NRL plasmid was incubated with Lipofectamine 2000 (Invitrogene) and Opti-MEM reduced serum media (gibco), treated with cells, and the culture medium was changed to DMEM (10% FBS) after three hours. One day later, siRNA was transfected using Lipofectamine RNAimax Transfection Reagent (Invitrogene). Each strand of siRNA was dilution in siRNA duplex buffer (bioneer) and annealed by incubation at 95°C for 5 minutes and 37°C for 1 hour. The annealed siRNA was electrophoresed on 12% polyacrylamide gel, followed by staining in EtBr for 10 minutes to confirm annealing through a UV transiluminator. Twenty-four hours after transfection of siRNA, total RNA was extracted by treatment with Tri-RNA Reagent (FAVOGEN), and cDNA was synthesized according to the provided protocol using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Thereafter, qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction) was performed on cDNA with Step One real-time PCR system (Applied Biosystems) to analyze the expression level of the target gene, NRL. Since the transfection efficiency may be different for each well because the cells were temporarily allowed to express NRL through transfection of the NRL plasmid, the expression level of NRL was normalized with the expression amount of the Neo gene in the NRL plasmid. Table 2 below shows the NRL primer and Neo primer sequences used for qRT-PCR.

gene direction sequence(5'→3') SEQ ID NO: NRL Forward GTCCTGAAGAGGCCATGGAG 147 Reverse GTTTAGCTCCCGCACAGACA 148 Neo Forward TTGCGCAGCTGTGCTCGACG 149 Reverse AAGGCGATGCGCTGCGAATC 150

3 shows the relative mRNA level (n=2) of NRL obtained by transfection of 1 nM NRL asiRNA. At first, 3nM of NRL asiRNA was transfected, and 12 sequences that did not perform well in knockdown were primarily excluded from a total of 40 sequences. PO stands for plasmid only, and only NRL plasmid was transfected, and NRL asiRNA was not transfected.

Example 3: Confirmation of NRL expression inhibition efficiency for the selected asiRNA

The effects of 7 NRL asiRNAs, #47, #48, #49, #67, #68, #72, and #73, which performed the most knockdown, were confirmed at the protein level by western blot.

After dispensing 14×10⁴ cells per well in a 6-well plate, follow the same procedure as when checking the RNA level with NRL plasmid (Origene-RG202237) and NRL After transfection with asiRNA (3 nM), 48 hours later, the cells were broken with RIPA buffer and western blot was performed. Primary antibodies were NRL antibody (AVIVA-OAAB19847) and β-Tubulin (Santa Cruz Biotechnology-sc-5274), and secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology-sc-2030) and goat anti- Mouse IgG-HRP (Santa Cruz Biotechnology-sc-2005) was used. si-Oct4 was used as a negative control. The si-Oct sequence is sense: 5'-AUGAUGCUCUUGAUUUUUUTT-3' (SEQ ID NO: 151), antisense: 5'-AAAAAAUCAAGAGCAUCAUTT-3' (SEQ ID NO: 152).

As a result of Western blot, #47 had the best knockdown efficiency, followed by #49, #67, and #68 with similar knockdown efficiency (Fig. 4). Therefore, considering the mRNA level and protein level, it was decided to make #47 and #49 in the form of cell penetrating asiRNA.

The results of subdividing the transfection concentration of asiRNA from 0.01 nM to 3 nM in order to obtain _{IC 50} of #47 and #49 are shown in FIG. 5 . As a control, GAPDH antibody (Santa Cruz Biotechnology-sc-47724) was used. As a result, #47 NRL It was confirmed that the IC _{50 value of asiRNA was about 0.2 nM, and the IC 50} value of #49 NRL asiRNA was about 0.6 nM ~ 1 nM.

Example 4: cp-asiRNA screening with cell-penetrating ability to target NRL

Based on the two types of asiRNA nucleotide sequences derived in Example 3, the length of the 3' end of the antisense strand was increased to synthesize 16mer (sense strand)-25mer (antisense strand). At this time, the extended antisense strand is human It is a sequence complementary to NRL. Table 3 below shows asiRNA nucleotide sequences targeting NRL.

No. Sequence(5'->3') SEQ ID NO: 47 S(16mer) CGGCGCUGGUCUCGAU 93 AS (25mer) AUCGAGACCAGCGCCGCGUCGGAAA 153 49 S(16mer) CGCUGGUCUCGAUGUC 97 AS (25mer) GACAUCGAGACCAGCGCCGCGUCGG 154

Modifications were carried out to make NRL asiRNAs #47 and #49 into asiRNAs with cell penetrating ability and resistance to nucleolytic enzymes. Modifications of NRL asiRNA #47 and 49 having cell penetrating ability are shown in Table 4 below.

siRNA No sequence(5'→3') cp-asiNRL #47 sense ori
(hereinafter, 1S) mCGmGCmGCmUGmGUmCUmCG*mA*U*-Chol OMe/F
(hereinafter referred to as 2S) mC(2'-FG)mG(2'-FC)mG(2'-FC)mU(2'-FG)mG(2'-FU)mC(2'-FU)mC(2'-FG)* mA*(2'-FU)*-Chol F
(hereinafter referred to as 3S) mCmGmGmCGmCmUGGmUmCmUmCmG*mA*mU*-Chol antisense ori
(hereinafter, 1AS) AUCGAGACCAGCGCmCmGmCmGmUmCmG*mG*mA*mA*mA OMe/F
(hereinafter referred to as 2AS) mA(2'-FU)mC(2'-FG)mA(2'-FG)mA(2'-FC)mC(2'-FA)mG(2'-FC)mG(2'-FC)mC (2'-FG)mC(2'-FG)mU(2'-FC)mG*(2'-FG)*mA*(2'-FA)*mA F
(hereinafter referred to as 3AS) mA(2'-F-U)(2'-F-C)GAGA(2'-F-C)(2'-F-C)AG(2'-F-C)GCCGmCmGmUmCmG*mG*mA*mA*mA #49 sense ori
(hereinafter, 1S) mCGmCUmGGmUCmUCmGAmUG*mU*C*-Chol OMe/F
(hereinafter referred to as 2S) mC(2'-FG)mC(2'-FU)mG(2'-FG)mU(2'-FC)mU(2'-FC)mG(2'-FA)mU(2'-FG)* mU*(2'-FC)*-Chol F
(hereinafter referred to as 3S) mCmGmCmUGGmUmCmUmCGAmUmG*mU*mC*-Chol antisense ori
(hereinafter, 1AS) GACAUCGAGACCAGmCmGmCmCmGmCmG*mU*mC*mG*mG OMe/F
(hereinafter referred to as 2AS) mG(2'-FA)mC(2'-FA)mU(2'-FC)mG(2'-FA)mG(2'-FA)mC(2'-FC)mA(2'-FG)mC (2'-FG)mC(2'-FC)mG(2'-FC)mG*(2'-FU)*mC*(2'-FG)*mG F
(hereinafter referred to as 3AS) mGA(2'-F-C)A(2'-F-U)(2'-F-C)GAGA(2'-F-C)(2'-F-C)AGCGmCmCmGmCmG*mU*mC*mG*mG

On the other hand, chemical modifications indicated by “*”, “m”, “2’-F-“, and “chol” in Table 4 are as shown in Table 5 below.

notation introduced chemical modifications * phosphorothioate bond m 2'-O-methyl (Methyl) 2'-F- 2'-Fluoro chol cholesterol

Specifically, in Table 5, “*” means a form in which an existing phosphodiester bond is substituted with a phosphorothioate bond, and “m” indicates that the existing 2'-OH is 2'-O- It means a form substituted with methyl. In addition, "2'-F-" means, for example, in the case of 2'-FG, the existing 2'-OH of G (guanine) is substituted with fluorine, and "Chol" is 3'- It means a form in which cholesterol is added at the end.

Except for the sequence in Table 4, the modifications were schematically shown in FIG. 6 (the same modifications were performed on #47 and #49). Three sense strands and three antisense strands are capable of binding a total of 9 types, and each strand was dilution in Opti-MEM reduced serum media and annealed by incubation at 95°C for 5 minutes and 37°C for 20 minutes.

Cholesterol attached to the 3' end of the sense strand allows it to penetrate the cell membrane. In addition, the substitution of phosphorothioate for the phosphate backbone close to the 3' ends of the sense and antisense strands increases resistance to exohydrolase, cellular uptake and bioavailability in vivo. do. In addition, if 2' of some sugars are modified with OMethyl, resistance to nucleases is increased, and siRNA immunogenicity and off-target effect are reduced. Finally, the 2' modification of some sugars with fluoro stabilizes the double-stranded RNA duplex, increases stability in the seurm, and enables efficient silencing in vitro and in vivo.

Example 5: Confirmation of NRL expression inhibition efficiency for cp-asiRNA targeting NRL

Example 5-1: Confirmation of the effect of modified cp-asiRNA in stable cell line

The effects of modified #47 and #49 cp-asiRNA were confirmed in the prepared A549- NRL stable cell line. The A549- NRL stable cell line was prepared by transfecting A549 cells with an NRL-expressing plasmid (Origene-RG202237), selecting and growing cells resistant to the G418 antibiotic (Goldbio).

The A549- NRL stable cell line was cultured in Ham's F-12K (Kaighn's) Medium (gibco) supplemented with 10% FBS (fetal bovine serum) and G418 antibiotic (1mg/ml) in a 100mm petri dish and grown, and then propagated in 6 wells. 150,000 cells were aliquoted and changed to Ham's F-12K (Kaighn's) Medium without FBS (fetal bovine serum) added after 24 hours. This serum starvation process aims to maximize the response to treat by homogenizing the cells in each cell cycle by causing G1 arrest and starving the cells. After 24 hours, it was changed to Opti-MEM reduced serum media, and Cp-asiRNA was treated without transfection reagent. After 24 hours, it was changed to Ham's F-12K (Kaighn's) Medium supplemented with 10% fetal bovine serum (FBS). After 24 hours, the cells were awakened with RIPA and western blot was performed.

The western blot results are shown in Figure 7, and in order to mark each strand more conveniently, S-ori, S-OMe/F, and SF were marked as 1S, 2S, and 3S in that order, and AS-ori, AS-OMe/ F and AS-F are indicated as 1AS, 2AS, and 3AS in order.

Similar results were obtained in the replicate experiment, and therefore, an additional, i.e., second Modification was carried out (Table 6).

siRNA No sequence (5'→3') cp-asiNRL #47 sense OMe/F-PS2
(hereinafter referred to as 2S-1) mC*(2'-FG)*mG(2'-FC)mG(2'-FC)mU(2'-FG)mG(2'-FU)mC(2'-FU)mC(2'-FG )*mA*(2'-FU)*-Chol antisense OMe/FP-PS1
(hereinafter referred to as 2AS-1) PmA(2'-FU)mC(2'-FG)mA(2'-FG)mA(2'-FC)mC(2'-FA)mG(2'-FC)mG(2'-FC)mC (2'-FG)mC(2'-FG)mU(2'-FC)mG*(2'-FG)*mA*(2'-FA)*mA OMe/FP-PS2
(hereinafter referred to as 2AS-2) PmA*(2'-FU)*mC(2'-FG)mA(2'-FG)mA(2'-FC)mC(2'-FA)mG(2'-FC)mG(2'-FC )mC(2'-FG)mC(2'-FG)mU(2'-FC)mG*(2'-FG)*mA*(2'-FA)*mA

The meanings of *, m, 2'-F- and chol are the same as those described in Table 5, and P means 5'-Phosphate.

The second modification is an additional chemical modification to the sense strand and 2S of #47 in Table 4, specifically, a phosphorothioate modification added to the 5' end of the sense strand (hereinafter referred to as 2S-1), An additional chemical modification was made to the antisense strand of #47 of 4, 2AS, and specifically, phosphate modification or phosphate and phosphorothioate modifications were added to the 5' end of the antisense strand (hereinafter, 2AS-1 and 2AS-2). called). 5'-phosphorylation of the antisense strand is required for the strand complementary to the target mRNA in the siRNA duplex for loading into the RNA-induced silencing complex (RISC), which is essential for gene silencing. Therefore, in most cases, when synthetic siRNA enters the cell, phosphorylation occurs. However, phosphate modification was performed at 5' of the antisense strand for more reliable loading into RISC (FIG. 8).

Using #47 2S of Table 4 and 2S-1 of Table 5 as the sense strand, and #47 2AS of Table 4 and 2AS-1 and 2AS-2 of Table 5 as the antisense strand, the same method as in the previous process. After performing the experiment, western blot was performed. The western blot results are shown in FIG. 9 .

Example 5-2: Confirmation of knockdown efficiency of selected NRL cp-asiRNA

According to the results of Example 5-1, 2S-2AS and 2S-2AS-1 having the best knockdown efficiency were selected. The difference between 2S-2AS and 2S-2AS-1 is the presence or absence of phosphate at 5' of the antisense strand.

_{NRL to obtain IC 50} values and maximum knockdown levels of the selected 2S-2AS and 2S-2AS-1 Western blot was performed in the same manner as before by treating different concentrations of cp-asiRNA (from 30 nM to 3000 nM). 2S-2AS-1 to which 5' phosphate is added has better knockdown efficiency than 2S- _{2AS, and it has been confirmed that the IC 50} value of 2AS-1 is approximately 300 nM and can knockdown up to 67% ( FIG. 10 ).

Meanwhile, according to the human NRL sequence, the same experiment was performed by increasing the length of the antisense strand from 25mer to 26mer by 1mer. Specifically, Table 7 shows the nucleotide sequence of the asiRNA prepared for this experiment, and #47 cp-asiNRL 2S-2AS (sense:16mer/antisense:26mer, hereinafter, 1626) and #47 cp-asiNRL of FIG. 11 The sequence and chemical modifications of 2S-2AS-1 (sense:16mer/antisense:26mer, hereinafter, 1626) are shown in Table 8 below.

No. Sequence(5'->3') SEQ ID NO: 74 S(16mer) CGGCGCUGGUCUCGAU 93 AS (26mer) AUCGAGACCAGCGCCGCGUCGGAAAA 155

siRNA No sequence (5'→3') cp-asiNRL #47 sense
(16mer) 2S mC(2'-FG)mG(2'-FC)mG(2'-FC)mU(2'-FG)mG(2'-FU)mC(2'-FU)mC(2'-FG)* mA*(2'-FU)*-Chol antisense
(26mer) 2AS mA(2'-FU)mC(2'-FG)mA(2'-FG)mA(2'-FC)mC(2'-FA)mG(2'-FC)mG(2'-FC)mC (2'-FG)mC(2'-FG)mU(2'-FC)mG(2'-FG)*mA*(2'-FA)*mA*(2'-FA) 2AS-1 PmA(2'-FU)mC(2'-FG)mA(2'-FG)mA(2'-FC)mC(2'-FA)mG(2'-FC)mG(2'-FC)mC (2'-FG)mC(2'-FG)mU(2'-FC)mG(2'-FG)*mA*(2'-FA)*mA*(2'-FA)

The meanings of *, m, 2'-F-, chol, and P are the same as described in Table 6.

As described above, the NRL cp-asiRNA in which the antisense strand is modified with the 26mer exhibited knockdown efficiency similar to that of the 25mer antisense strand, and even when the antisense strand is 26mer, the 2S-2AS -1 showed better effect than 2S-2AS. According to FIG. 11, even when the antisense strand is 26mer, the IC ₅₀ value of 2S-2AS-1 (antisense: 26mer) is between 300 nM and 600 nM, and it was confirmed that it can knockdown up to 62% ( FIG. 11 ).

12 shows the sequence and modification information of #47 cp-asiNRL 2S-2AS-1 (1626).

Example 6: Confirmation of NRL Expression Inhibition Efficiency of cp-asiRNA Using an Animal Model

#47 cp-asiNRL 2S-2AS-1 (1626) was confirmed to inhibit the target protein in the retinal tissue of the mouse eye.

Specifically, this experiment was performed on 7-week-old mice acclimatized to C57BL/6 (7-week-old, male, OrientBio) mice for 1 week. #47 cp-asiNRL 2S-2AS-1 (1626) was prepared as a 10 mg/ml stock solution using 0.5x PBS as a vehicle solvent, which was diluted to concentrations of 60, 120, 180 and 210 μg/30 μl, respectively. Therefore, intraocular administration of 1 μl to the mouse was performed once (n=3/group). On the 7th and 14th days after administration of the candidate substance, the mice of each group were sacrificed and curved forceps (FSC) were used. and the eyeball was removed. Thereafter, after making a hole in the cornea obtained from the mouse, an incision was made along the limbus line using a micro scissor (FSC), the lens was removed, and the retina was collected. The collected retinal tissue was placed in RIPA buffer (Thermo Fisher Scientific) and tissue lysis was performed by pipetting, and then NRL protein was quantified with a BCA protein assay kit (Invitrogen). For protein analysis by Western blotting, 10 μg of protein was separated from each sample for 30 minutes at 80 V and 1 hour and 30 minutes at 100 V using 10% SDS-PAGE, followed by a PVDF membrane (Bio-Rad) at 230 mA was transferred for 2 hours. After blocking the transferred membrane at 3% BSA, 4°C for 2 hours, NRL antibody (Santa cruz) 1:500 for 16 hours, Anti-mouse IgG, HRP-linked Antibody (Santacruz) 1:10,000 for 1 hour After the reaction, the expression level of the NRL protein was checked using ChemiDoc (Thermo). Specifically, the expression level of NRL protein was quantified as a ratio of NRL/Vinculin protein, and a 0.5X PBS administration group was used as a control group.

13 and 14 are results of confirming the change in NRL expression according to the administration of #47 cp-asiNRL 2S-2AS-1 (1626) in the retinal tissue of the mouse eye. As a result, it was confirmed that the administration of the candidate material suppressed the expression of the NRL protein in the retinal tissue. Specifically, it was found that this effect was maintained effectively not only on the 7th day after administration, but also on the 14th day after administration, and showed a tendency dependent on the administration concentration.

These experimental results indicate that the #47 cp-asiNRL 2S-2AS-1(1626) substance can inhibit the expression of NRL protein even under in vivo conditions. It shows that it can be used as an active ingredient of a pharmaceutical composition for improving or treating diseases.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> OliX Pharmaceuticals, Inc. <120> Asymmetric siRNA Inhibiting Expression of NRL <130> PN131281 <150> KR 2019-0006750 <151> 2019-01-18 <160> 155 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 1 cggcugaagc agaggc 16 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 2 gccucugcuu cagccgcag 19 <210> 3 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 3 ggcugaagca gaggcg 16 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 4 cgccucugcu ucagccgca 19 <210> 5 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 5 aagcagaggc gccgca 16 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 6 ugcggcgccu cugcuucag 19 <210> 7 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 7 agcagaggcg ccgcac 16 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 8 gugcggcgcc ucugcuuca 19 <210> 9 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 9 cgcuccaagc ggcugc 16 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 10 gcagccgcuu ggagcgaca 19 <210> 11 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 11 gcuccaagcg gcugca 16 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 12 ugcagccgcu uggagcgac 19 <210> 13 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 13 acuuugacuu gaugaa 16 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 14 uucaucaagu caaagucau 19 <210> 15 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 15 cuuugacuug augaag 16 <210> 16 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 16 cuucaucaag ucaaaguca 19 <210> 17 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 17 uuugacuuga ugaagu 16 <210> 18 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 18 acuucaucaa gucaaaguc 19 <210> 19 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 19 uugacuugau gaaguu 16 <210> 20 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 20 aacuucauca agucaaagu 19 <210> 21 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 21 gccugucgcu ccaagc 16 <210> 22 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 22 gcuuggagcg acaggccug 19 <210> 23 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 23 ccugucgcuc caagcg 16 <210> 24 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 24 cgcuuggagc gacaggccu 19 <210> 25 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 25 gucgcuccaa gcggcu 16 <210> 26 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 26 agccgcuugg agcgacagg 19 <210> 27 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 27 ucgcuccaag cggcug 16 <210> 28 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 28 cagccgcuug gagcgacag 19 <210> 29 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 29 cuccaagcgg cugcag 16 <210> 30 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 30 cugcagccgc uuggagcga 19 <210> 31 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 31 ugacuugaug aaguuu 16 <210> 32 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 32 aaacuucauc aagucaaag 19 <210> 33 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 33 aggccugucg cuccaa 16 <210> 34 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 34 uuggagcgac aggccugcg 19 <210> 35 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 35 ggccugucgc uccaag 16 <210> 36 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 36 cuuggagcga caggccugc 19 <210> 37 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 37 ccuccuucac ccaccu 16 <210> 38 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 38 agguggguga aggaggcac 19 <210> 39 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 39 cuccuucacc caccuu 16 <210> 40 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 40 aaggugggug aaggaggca 19 <210> 41 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 41 uccuucaccc accuuc 16 <210> 42 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 42 gaaggugggu gaaggaggc 19 <210> 43 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 43 ccuucaccca ccuuca 16 <210> 44 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 44 ugaagguggg ugaaggagg 19 <210> 45 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 45 cuucacccac cuucag 16 <210> 46 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 46 cugaaggugg gugaaggag 19 <210> 47 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 47 uucacccacc uucagu 16 <210> 48 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 48 acugaaggug ggugaagga 19 <210> 49 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 49 ucacccaccu ucagug 16 <210> 50 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 50 cacugaaggu gggugaagg 19 <210> 51 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 51 cacccaccuu caguga 16 <210> 52 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 52 ucacugaagg ugggugaag 19 <210> 53 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 53 aauaugucaa ugacuu 16 <210> 54 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 54 aagucauuga cauauucca 19 <210> 55 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 55 auaugucaau gacuuu 16 <210> 56 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 56 aaagucauug acauauucc 19 <210> 57 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 57 uaugucaaug acuuug 16 <210> 58 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 58 caaagucauu gacauauuc 19 <210> 59 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 59 augucaauga cuuuga 16 <210> 60 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 60 ucaaagucau ugacauauu 19 <210> 61 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 61 uuugac uuugac 16 <210> 62 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 62 gucaaaguca uugacauau 19 <210> 63 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 63 ugccuccuuc acccac 16 <210> 64 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 64 gugggugaag gaggcacug 19 <210> 65 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 65 gccuccuuca cccacc 16 <210> 66 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 66 ggugggugaa ggaggcacu 19 <210> 67 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 67 ucagugaacc aggcau 16 <210> 68 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 68 augccugguu cacugaagg 19 <210> 69 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 69 cagugaacca ggcaug 16 <210> 70 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 70 caugccuggu ucacugaag 19 <210> 71 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 71 agugaaccag gcaugg 16 <210> 72 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 72 ccugccugg uucacugaa 19 <210> 73 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 73 gugaaccagg cauggu 16 <210> 74 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 74 accaugccug guucacuga 19 <210> 75 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 75 agcuguacug gcuggc 16 <210> 76 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 76 gccagccagu acagcuccu 19 <210> 77 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 77 ggcuggcuac ccugca 16 <210> 78 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 78 ugcaggguag ccagccagu 19 <210> 79 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 79 gcuggcuacc cugcag 16 <210> 80 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 80 cugcagggua gccagccag 19 <210> 81 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 81 cuggcuaccc ugcagc 16 <210> 82 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 82 gcugcagggu agccagcca 19 <210> 83 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 83 uggcuacccu gcagca 16 <210> 84 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 84 ugcugcaggg uagccagcc 19 <210> 85 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 85 ggcuacccug cagcag 16 <210> 86 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 86 cuggugcagg guagccagc 19 <210> 87 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 87 gcuacccugc agcagc 16 <210> 88 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 88 gcugcugcag gguagccag 19 <210> 89 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 89 cuacccugca gcagca 16 <210> 90 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 90 ugcugcugca ggguagcca 19 <210> 91 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 91 uacccugcag cagcag 16 <210> 92 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 92 cugcugcugc aggguagcc 19 <210> 93 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 93 cggcgcuggu cucgau 16 <210> 94 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 94 aucgagacca gcgccgcgu 19 <210> 95 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 95 gcgcuggucu cgaugu 16 <210> 96 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 96 acaucgagac cagcgccgc 19 <210> 97 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 97 cgcuggucuc gauguc 16 <210> 98 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 98 gacaucgaga ccagcgccg 19 <210> 99 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 99 accggcagcu gcgggg 16 <210> 100 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 100 ccccgcagcu gccgguuua 19 <210> 101 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 101 aggcgcugcg gcugaa 16 <210> 102 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 102 uucagccgca gcgccucgu 19 <210> 103 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 103 cgcugcggcu gaagca 16 <210> 104 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 104 ugcuucagcc gcagcgccu 19 <210> 105 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 105 cugcggcuga agcaga 16 <210> 106 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 106 ucugcuucag ccgcagcgc 19 <210> 107 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 107 ugcggcugaa gcagag 16 <210> 108 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 108 cucugcuuca gccgcagcg 19 <210> 109 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 109 gcggcugaag cagagg 16 <210> 110 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 110 ccucugcuuc agccgcagc 19 <210> 111 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 111 gcacgcugaa gaaccg 16 <210> 112 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 112 cgguucuuca gcgugcggc 19 <210> 113 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 113 cacgcugaag aaccgc 16 <210> 114 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 114 gcgguucuuc agcgugcgg 19 <210> 115 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 115 acgcugaaga accgcg 16 <210> 116 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 116 cgcgguucuu cagcgugcg 19 <210> 117 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 117 cgcugaagaa ccgcgg 16 <210> 118 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 118 ccgcgguucu ucagcgugc 19 <210> 119 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 119 gcugaagaac cgcggc 16 <210> 120 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 120 gccgcgguuc uucagcgug 19 <210> 121 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 121 cugaagaacc gcggcu 16 <210> 122 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 122 agccgcgguu cuucagcgu 19 <210> 123 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 123 ugaagaaccg cggcua 16 <210> 124 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 124 uagccgcggu ucuucagcg 19 <210> 125 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 125 gaagaaccgc ggcuac 16 <210> 126 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 126 guagccgcgg uucuucagc 19 <210> 127 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 127 aagaaccgcg gcuacg 16 <210> 128 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 128 cguagccgcg guucuucag 19 <210> 129 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 129 agaaccgcgg cuacgc 16 <210> 130 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 130 gcguagccgc gguucuuca 19 <210> 131 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 131 gaaccgcggc uacgcg 16 <210> 132 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 132 cgcguagccg cgguucuuc 19 <210> 133 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 133 acaaggcucg cuguga 16 <210> 134 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 134 ucacagcgag ccuuguaga 19 <210> 135 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 135 caaggcucgc ugugac 16 <210> 136 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 136 gucacagcga gccuuguag 19 <210> 137 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 137 aaggcucgcu gugacc 16 <210> 138 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 138 ggucacagcg agccuugua 19 <210> 139 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 139 aggcucccug ugaccg 16 <210> 140 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 140 cggucacagc gagccuugu 19 <210> 141 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 141 ggcucgcugu gaccgg 16 <210> 142 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 142 ccggucacag cgagccuug 19 <210> 143 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 143 gcuggcugug accggc 16 <210> 144 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 144 gccggucaca gcgagccuu 19 <210> 145 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 145 cucgcuguga ccggcu 16 <210> 146 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 146 agccggucac agcgagccu 19 <210> 147 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRL primer <400> 147 gtcctgaaga ggccatggag 20 <210> 148 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRL primer <400> 148 gtttagctcc cgcacagaca 20 <210> 149 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Neo primer <400> 149 ttgcgcagct gtgctcgacg 20 <210> 150 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Neo primer <400> 150 aaggcgatgc gctgcgaatc 20 <210> 151 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> si-Oct <400> 151 augaugcucu ugauuuuuut t 21 <210> 152 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> si-Oct <400> 152 aaaaaaucaa gagcaucaut t 21 <210> 153 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 153 aucgagacca gcgccgcguc ggaaa 25 <210> 154 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 154 gacaucgaga ccagcgccgc gucgg 25 <210> 155 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> NRL asiRNA <400> 155 aucgagacca gcgccgcguc ggaaaa 26

Claims (17)

An antisense strand comprising a sequence complementary to an mRNA encoding a neural retina leucine zipper (NRL), and a sense strand forming a complementary bond with the antisense strand, the 5' end of the antisense strand and 3 of the sense strand ' The end is an siRNA, characterized in that it forms a blunt end,
siRNA, wherein the sense strand includes the nucleotide sequence of SEQ ID NO: 93, and the antisense strand includes the nucleotide sequence of SEQ ID NO: 94, 153, or 155. The siRNA according to claim 1, wherein the sense strand has a length of 15 to 17 nt, and the antisense strand has a length of 16 nt or more. The siRNA according to claim 2, wherein the antisense strand has a length of 16 to 31 nt. The siRNA according to claim 2, wherein the antisense strand has a length of 19 to 26 nt. delete delete delete delete The siRNA according to claim 1, wherein the sense strand or the antisense strand of the siRNA comprises one or more chemical modifications. 10. The siRNA of claim 9, wherein the chemical modification comprises at least one selected from the group consisting of:
-OH group at the 2' carbon position of the sugar structure in the nucleotide is -CH ₃ (methyl), -OCH ₃ (methoxy), -NH ₂ , -F (fluorine), -O-2-methoxyethyl -O-propyl ( propyl), -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl;
replacement of oxygen in the nucleotide structure with sulfur;
nucleotide linkages are modified with phosphorothioate, boranophosphate, or methyl phosphonate;
transformation into peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA) form; and
A phosphate group, a lipophilic compound, or a cell penetrating peptide bond. The siRNA according to claim 10, wherein the lipophilic compound is selected from the group consisting of cholesterol, tocopherol, and long-chain fatty acids having 10 or more carbon atoms. 10. The method according to claim 9, wherein the -OH group at the 2' carbon position of the sugar structure in two or more nucleotides of the sense strand or the antisense strand is substituted with -OCH ₃ (methoxy) or -F (fluorine);
at least 10% of the nucleotide linkages in the sense or antisense strand are modified to phosphorothioate;
cholesterol binding to the 3' end of the sense strand; and
siRNA comprising one or more modifications selected from the group consisting of; phosphate group binding to the 5' end of the antisense strand. The method of claim 12, wherein the sense strand is any one selected from the group consisting of (a) to (c) and (g) of the table below, and
The antisense strand is an siRNA, characterized in that any one selected from the group consisting of (h) to (j) and (n) to (q) of the table below:

(In the above sequence, * is a phosphorothioate bond, m is 2'-O-methyl (Methyl), 2'-F- is 2'-fluorine (Fluoro), chol is cholesterol, P is 5' -means a phosphate group). The method according to claim 13, wherein the siRNA comprises: a sense strand consisting of (b) the nucleotide sequence of the table, and an antisense strand consisting of the nucleotide sequence (i) of the table;
a sense strand consisting of the nucleotide sequence (b) of the table above and an antisense strand consisting of the nucleotide sequence (n) of the table above;
a sense strand consisting of the nucleotide sequence (b) of the table above and an antisense strand consisting of the nucleotide sequence (p) of the table above; or
siRNA that is an antisense strand consisting of a sense strand consisting of the nucleotide sequence of (b) in the above table and an antisense strand consisting of the nucleotide sequence of (q) in the above table. The siRNA according to claim 1, wherein the siRNA has a cell-penetrating ability. A pharmaceutical composition for improving or treating retinal diseases comprising the siRNA according to any one of claims 1 to 4, 9 to 15. The method of claim 16, wherein the retinal disease is Usher's syndrome, Stargardt disease, Badette-Biddle syndrome, Best's disease, choroid deficiency, choroidal atrophy (gyrate-atrophy), retinitis pigmentosa, retinal macular degeneration. , Leber Congenital Amaurosis (Leber's Hereditary Optic Neuropathy), BCM (Blue-cone monochromacy), retinal interlayer separation, ML (Malattia Leventinese), Oguchi disease or Refsum disease A pharmaceutical composition for improving or treating retinal diseases.

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