WO2020149702A1 - Arnsi asymétrique pour inhiber l'expression de la fermeture éclair à leucines de la rétine neuronale (nrl) - Google Patents

Arnsi asymétrique pour inhiber l'expression de la fermeture éclair à leucines de la rétine neuronale (nrl) Download PDF

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WO2020149702A1
WO2020149702A1 PCT/KR2020/000870 KR2020000870W WO2020149702A1 WO 2020149702 A1 WO2020149702 A1 WO 2020149702A1 KR 2020000870 W KR2020000870 W KR 2020000870W WO 2020149702 A1 WO2020149702 A1 WO 2020149702A1
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sirna
sense
antisense
antisense strand
strand
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이동기
이숙영
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올릭스 주식회사
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present invention relates to an asymmetric siRNA that suppresses the expression of a NRL (Neural retina leucine zipper) and its use, and more particularly, an antisense strand comprising a sequence complementary to an mRNA encoding NRL, and a complement 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 disease comprising the asymmetric siRNA.
  • Retinitis pigmentosa is a disease in which genes that have characteristics of rod cells are mutated, which leads to destruction of rod cells and secondary destruction of cone cells, which can eventually lead to blindness.
  • NRL neural retina leucine zipper
  • AAV adenovirus-associated virus
  • NRL plays a very important role in the differentiation process of rods and cones, and it means that the transcriptional activity of NRLs directly acts on the differentiation of rods. Since NRL functions not only in the development and differentiation of the retina, but also in the expression of photoreceptor proteins such as rhodopsin, studies have reported that genetic mutations in NRL are also associated with retinal disease.
  • CRISPR/Cas9 is a gene editing technology that regulates gene expression at the transcriptional level by causing a double strand break in DNA.
  • knockout of a specific gene through CRISPR/Cas9 cannot be restored to the state before knockout again when an unintended mutation occurs in a target gene, and serious side effects may occur.
  • Cas9 is a bacterial protein, it can cause an immune response and other unknown side effects when expressed in vivo, making it difficult to develop drugs for diseases including retinitis pigmentosa.
  • siRNA 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.
  • the conventional siRNA has a short duplex of 19-21 bp, and two nucleotides protrude on both strands 3'. siRNA enters the cell, attaches to the target mRNA, degrades the target mRNA, and inhibits expression of the target gene.
  • siRNA introduced into cells can cause side effects such as inducing an immune response and suppressing non-target genes.
  • delivery systems that introduce siRNA into cells are becoming the biggest problem in the development of therapeutic agents using siRNA.
  • the siRNA has a negative charge due to the phosphate backbone, and it has a repulsive force with the cell membrane having a negative charge, so a delivery system is required to introduce siRNA into the cell.
  • a delivery system a method of introducing negatively charged liposomes or polymers into a cell by offsetting siRNA is introduced into cells.
  • a carrier having a positive charge can cause various side effects, such as attaching to a cell membrane having a negative charge, showing unwanted toxicity or forming an unwanted complex through interaction with various types of proteins in the cell.
  • 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 in which siRNA can be safely and effectively delivered into target cells.
  • the present inventors selected NRL target siRNA capable of inhibiting the expression of NRL, delivered to cells without the help of a transporter, and conducted a rigorous research effort to develop a highly resistant siRNA-to-nuclease, as a result of NRL targeting siRNAs were designed, and siRNAs that most effectively inhibit NRL were selected through screening, and it was confirmed that the intracellular delivery problem could be overcome through modification, and the present invention was completed.
  • An object of the present invention is to provide an asymmetric siRNA (asymmetric shorter duplex siRNA, asiRNA) that specifically inhibits the expression of NRL.
  • asymmetric siRNA asymmetric shorter duplex siRNA, asiRNA
  • Another object of the present invention is to provide a pharmaceutical composition for improving or treating retinal disease including the asymmetric siRNA, or a method for improving or treating retinal disease.
  • Another object of the present invention is the use of the asymmetric siRNA to prepare a pharmaceutical composition for improving or treating retinal disease; And to provide a medicinal use of the asymmetric siRNA for improving or treating retinal disease.
  • the present invention includes an antisense strand comprising a sequence complementary to an mRNA encoding a NRL (Neural retina leucine zipper), and a sense strand forming a complementary bond with the antisense strand, and the antisense
  • the 5'end of the strand and the 3'end of the sense strand provide an siRNA characterized by forming 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 disease, comprising administering the siRNA to an individual.
  • the present invention also, the use of the asymmetric siRNA for preparing a pharmaceutical composition for improving or treating retinal disease; It provides a medicinal use of the asymmetric siRNA for improving or treating retinal disease.
  • asymmetric siRNA capable of efficiently suppressing the expression of NRL which plays a very important function in the differentiation process of rod and cone cells, is screened, and the siRNA is introduced into the cell without the help of a transporter and added to the nuclease.
  • the siRNA By chemically modifying it to have resistance to resistance, it can be used as a therapeutic agent for retinal diseases, including retinitis pigmentosa, by removing cytotoxicity caused by a carrier and enabling more efficient suppression of gene expression in vivo.
  • NRL asiRNA or asiNRL shows the structure of asiRNA (hereinafter, NRL asiRNA or asiNRL) for suppressing NRL expression of 16-19mer.
  • FIG. 2 shows that siRNA was designed in consideration of homology of Homo sapiens(H), Rattus norvegicus(R), and Mus musculus(M).
  • FIG. 2(A) shows the number of mismatches between H, R, and M The number of sequences corresponding thereto is shown in a table
  • FIG. 2(B) is a schematic of FIG. 2(A)
  • FIG. 2(C) is designed after 73 NRL asiRNAs are designed, and information of Human NRL transcript variants is updated.
  • This is a diagram showing that 40 NRL asiRNAs belonging to all transcript variants are screened.
  • Figure 4 is a qRT-PCR results of the top 7 to effectively suppress the NRL It is the result of western blot by transfection (3nM) of NRL asiRNA.
  • NRL asiRNA #47 and #49 (0.01 nM to 3 nM) to obtain approximate IC 50 values.
  • NRL asiRNA # 47 IC 50 value is about 0.2nM
  • NRL asiRNA IC 50 value is 0.6-1nM of # 49.
  • FIG. 6 is a diagram schematically showing chemical modification of NRL asiRNA in Table 4, and for sense strands, S-ori, S-OMe/F, and SF are sequentially shown as 1S, 2S, and 3S, and AS-ori, AS- OMe/F and AS-F are shown in order of 1AS, 2AS, and 3AS.
  • FIG. 7 is #47 asiNRL (sense strand-antisense strand, 16mer-19mer) without chemical modification, and #47 cp asiNRL, #49 cp asiNRL with antisense strand as 25mer and chemical modification described in FIG. -It is the result of processing without a carrier on the stable cell line.
  • 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). That is, 2S-1 which is an additional modification to the sense strand 2S, and 2AS-1 and 2AS-2, which are additional modifications to the antisense strand 2AS, respectively.
  • Figure 9 is the effect of #47 cp asiNRL (SS-AS: 16mer-25mer) 2S/2AS, 2S/2AS-1, 2S/2AS-2, 2S-1/2AS-1, 2S-1/2AS-2 It is the result of checking.
  • Figure 11 is the result of confirming the efficacy of #47 cp-asiNRL (SS-AS: 16mer-26mer) increased the antisense strand (AS) from 25mer to 26mer in #47 cp-asiNRL (SS-AS:16mer-25mer) .
  • FIG. 13 shows the change in expression of NRL according to the administration of the candidate substance in the retinal tissue of the mouse eye on the 7th day after administration of the #47 cp-asiNRL 2S-2AS-1(1626) substance in the eye through western blot. Is the result.
  • RNAi RNA interference
  • dsRNA double-stranded RNA
  • RNA small interfering RNA
  • dsRNA short double-stranded RNA
  • a “antisense strand” is a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest, eg, mRNA (messenger RNA), non-RNA RNA sequence (eg, microRNA, piwiRNA) , tRNA, rRNA and hnRNA) or coding or non-coding DNA sequences, in whole or in part.
  • mRNA messenger RNA
  • non-RNA RNA sequence eg, microRNA, piwiRNA
  • tRNA e.g, tRNA, rRNA and hnRNA
  • a “sense strand” is a polynucleotide having a nucleic acid sequence identical to a target nucleic acid, and is an mRNA (messenger RNA), an RNA sequence other than mRNA (eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA) or coding or noncoding. Refers to a polynucleotide that is identical to the DNA sequence as a whole or as a part.
  • mRNA messenger RNA
  • RNA sequence other than mRNA eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA
  • Gene should be considered in the broadest sense and can encode a structural or regulatory protein.
  • the regulatory protein includes a transcription factor, a heat shock protein or a protein involved in DNA/RNA replication, transcription and/or translation.
  • the target gene targeted for suppression of expression is inherent in the viral genome, and may be integrated into an animal gene or exist as an extrachromosomal component.
  • the gene of interest may be a gene on the HIV genome.
  • siRNA molecules are useful for inactivating the translation of HIV genes in mammalian cells.
  • NRL Neuronal retina leucine zipper
  • Retinitis pigmentosa is a disease in which genes that exhibit characteristics of rod cells are mutated, so rod cells are destroyed first, and cone cells are subsequently destroyed. Studies have shown that the detrimental effects of mutations in genes important for photoreceptor function are maximized in a fully differentiated photoreceptor environment. Therefore, when knocking out NRL that induces differentiation into rod cells, rod cells are transformed into cells similar to cone cells, and the function of rod cells is lost, but even if the rod cells change shape and function, their existence is secondary cone cells. It was found to prevent the loss of. Therefore, suppressing the expression of NRL has potential as a treatment for retinitis pigmentosa.
  • siRNAs targeting NRLs are designed, and siRNAs that suppress NRLs are most efficiently selected through screening, and secondly, siRNAs can be introduced into cells without any specific transporter.
  • Chemical modifications are introduced to deliver and increase resistance to nucleases.
  • siRNA is negatively charged by the phosphate backbone, which makes it difficult to penetrate the cell membrane.
  • the resistance to nucleic acid hydrolase should be increased to have a long lifetime in serum, so that the amount reached to the target should be sufficient to cause effective RNAi. Therefore, modification was introduced to overcome the siRNA delivery problem.
  • NRL asiRNA having the best knockdown efficiency was selected by first designing asiRNA targeting NRL and transfection of asiRNA in cells expressing NRL. The following four modifications are introduced into the selected siRNA to modify asiRNA to have cell penetrating ability and to be resistant to nucleic acid hydrolase. First, cholesterol is added to the 3'end of the sense strand to allow siRNA to penetrate the cell membrane. Second, the phosphate backbone near the 5'end or the 3'end of the sense strand and the antisense strand is substituted with phosphorothioate to have resistance to external hydrolysis enzymes, and it can be absorbed into cells and siRNA in vivo. It enables the bioavailability of.
  • 2'of sugar is modified with OMethyl to impart resistance to nucleic acid hydrolase, lower siRNA immunogenicity, and reduce off-target effects.
  • the fourth is to modify the 2'of sugar with fluoro to give stability to the double strand duplex, increase the stability in serum, and enable efficient silencing in vitro and in vivo.
  • the siRNA has a cell penetrating ability and stays longer in the serum, allowing more efficient gene suppression as a sufficient amount of siRNA is delivered to the target cell.
  • the present invention in one aspect, comprises an antisense strand comprising a sequence complementary to an mRNA encoding a NRL (Neural retina leucine zipper), a sense strand forming a complementary bond with the antisense strand, and the antisense strand
  • NRL Neurological retina leucine zipper
  • the 5'end of and the 3'end of the sense strand relates to siRNA, which is characterized by forming a blunt end.
  • RNAi RNA interference
  • siRNAi is an intracellular gene regulation mechanism first discovered in Caenorthabditis elegans in 1998, and its mechanism of action is known to induce target gene degradation by complementarily binding the antisense strand to the mRNA of the target gene among RNA double strands introduced into the cell.
  • siRNA is one of methods for suppressing gene expression in "in vitro".
  • the 19-21 bp siRNA can theoretically selectively inhibit almost all genes, so it can be developed as a therapeutic agent for various gene-related diseases such as cancer and viral infection, and is the most popular candidate for new drug development.
  • the first attempt to in vivo treatment with siRNA in mammals was in mid-2003, and since then many reports have been made on in vivo treatment with many attempts at applied research.
  • siRNA is an effective way to directly regulate the expression of a target gene, these problems have made it difficult to develop therapeutic agents.
  • an asymmetric siRNA (asymmetric shorter duplex siRNA, asiRNA) is an asymmetric RNAi-inducing structure having a short double helix length compared to the 19+2 structure of a conventional siRNA. It is a technology that overcomes the problems of off-target effect, RNAi mechanism saturation, and immune response by TLR3, which is confirmed in the existing siRNA structure technology, and thus, it is possible to develop a new RNAi drug with low side effects.
  • the present invention presents an asymmetric siRNA comprising a sense strand and an antisense strand complementary to the sense strand, and the siRNA according to the present invention stably does not cause problems such as off-target effect, saturation of RNAi mechanism, etc. While maintaining high delivery efficiency, it is possible to effectively suppress the expression of the NRL target gene to a desired degree.
  • the siRNA may be characterized in that the sense strand has a length of 15 to 17nt, and the antisense strand has a length of 16nt or more.
  • the antisense strand may be characterized as having a length of 16 to 31nt, and preferably having a length of 19 to 26nt. More preferably, the length of the sense strand is 16nt, and the length of the complementary antisense strand is 19nt, 24nt, 25nt or 26nt, 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 16nt.
  • 73 NRL asiRNAs were designed to suppress the expression of NRL, and suppression of mRNA level and protein level in transient cell expressing NRL temporarily and stable cell line continuously expressing NRL. Confirmed.
  • 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 145.
  • 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 155.
  • the sense strand may be, for example, those 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 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.
  • the sense strand may be selected from the group consisting of SEQ ID NO: 93, 95, 97, 133, 135, 143 and 145, and the antisense strand may have SEQ ID NO: 94, 96, 98, 134, 136, 144, It may be selected from the group consisting of 146, 153, 154, and 155.
  • the sense strand is SEQ ID NO: 93, and the antisense strand may be SEQ ID NO: 153 or 155.
  • the sense strand or the antisense strand of the siRNA may be characterized by including one or more chemical modifications (chemical modification).
  • 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 it is difficult to deliver sufficient amounts for RNAi induction to actual target sites due to rapid decomposition and removal in blood.
  • in vitro delivery a high-efficiency delivery method using cationic lipids and cationic polymers has been developed, but in vivo, it is difficult to deliver siRNA with inefficiency as high as in vitro. There is a problem in that siRNA delivery efficiency is reduced by interaction.
  • the present inventors have developed an asiRNA construct (cp-asiRNA) having an autonomous delivery ability capable of effective and intracellular delivery without a separate carrier by introducing a chemical modification into an asymmetric siRNA structure.
  • the chemical modification in the sense strand or the antisense strand may include one or more selected from the group consisting of: -OH group -CH 3 (methyl) at the 2'carbon position of the sugar structure in the nucleotide,- OCH 3 (methoxy), -NH 2 , -F (fluorine), -O-2-methoxyethyl -O-propyl, -O-2-methylthioethyl, -O-3-amino Substituted with propyl, -O-3-dimethylaminopropyl; Oxygen in the sugar structure in the nucleotide is substituted with sulfur; Nucleotide bonds are modified with phosphorothioate, boranophosphate, or methyl phosphonate; Modifications to peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA) form; And a phosphate group, a lipophilic compound, or
  • the lipophilic compound may be 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 being cholesterol, but is not limited thereto.
  • 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 in two or more nucleotides of the sense strand or the antisense strand; 10% or more nucleotide bonds in the sense or antisense strand are modified with phosphorothioate; Cholesterol binding to the 3'end of the sense strand; And it may be characterized in that it comprises at least one modification selected from the group consisting of; phosphate group (phosphate group) bond to the 5'end of the antisense strand.
  • phosphate group phosphate group
  • the sense strand is any one selected from the group consisting of (a) to (g) in the following table
  • the antisense strand is any one selected from the group consisting of (h) to (q) in the following table.
  • * is a phosphorothioate bond
  • m is 2'-O-methyl (Methyl)
  • 2'-F- is 2'-Fluoro
  • chol is cholesterol
  • P is 5'- It means Phosphate group.
  • the sense strand is any one of (a) to (c) in the table, and the antisense strand may be any one of (h) to (j) in the table;
  • the sense strand may be any one of (d) to (f) in the table, and the antisense strand may be any of (k) to (m) in the table.
  • the sense strand is (b) in the table, and the antisense strand is any of (i), (n) and (o) in the table;
  • the sense strand may be (g) of the table, and the antisense strand may be (n) or (o) of the table.
  • the sense strand may be (b) of the table, and the antisense strand may be (p) or (q) of the table.
  • the siRNA comprises (b) a sense strand consisting of the base sequence of the table and (i) an antisense strand consisting of the base sequence of the table; (B) the sense strand consisting of the nucleotide sequence in the table and the (n) antisense strand consisting of the nucleotide sequence in the table; (B) the sense strand consisting of the base sequence of the table and the antisense strand consisting of the base sequence of (p) of the table; Or it may be a sense strand consisting of the base sequence (b) of the table and an antisense strand consisting of the base sequence (q) of the table, preferably, the siRNA is a sense strand consisting of the base sequence (b) of the table and (q) of the table. It may be an antisense strand consisting of a base sequence.
  • one to three phosphate groups may be attached to the 5'end of the antisense strand, but is not limited thereto.
  • the sense strands commonly substituted 3 phosphate linkages at the 3'end with phosphorothioate linkage and cholesterol was added.
  • the antisense strand four phosphate linkages were commonly substituted at the 3'end with phosphorothioate linkages.
  • 3 sense strands and 3 antisense strands were synthesized by changing the number of OMethyl and fluoro substitutions on the 2'sugars and the substitution positions. Therefore, in 9 cases, the annealing was performed to select the modification that knocked down the NRL best without a carrier, and several modifications were added thereto.
  • NRL cp-asiRNA was selected to knock down NRL most efficiently through two modifications.
  • the present invention relates to a pharmaceutical composition for improving or treating retinal disease comprising the siRNA.
  • the retinal disease is Usher syndrome, Stargardt disease (Stargardt disease), Badet-Biddle syndrome, Best disease, choroidal deficiency, chorioretinal atrophy (gyrate-atrophy), retinitis pigmentosa, retinal macular degeneration, Leber Congenital Amaurosis; Leber's Hereditary Optic Neuropathy (BCM), Blue-cone monochromacy (BCM), Interretinal Separation, ML (Malattia Leventinese), Oguchi disease or Refsum disease It can, but is not limited to this.
  • the pharmaceutical composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to siRNA as an active ingredient.
  • the pharmaceutically acceptable carrier should be compatible with the active ingredient of the present invention, and may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these ingredients. It can be used in combination, and if necessary, other conventional additives such as antioxidants, buffers, bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders and lubricants can be added in addition to formulated into injectable formulations such as aqueous solutions, suspensions and emulsions.
  • a formulation in a lyophilized form it is preferable to provide a formulation in a lyophilized form.
  • a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for lyophilization may be added.
  • the method of administration of the pharmaceutical composition can be determined by a person skilled in the art based on the symptoms and severity of the disease in a typical patient.
  • it can be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, and may be provided in unit-dose or multi-dose containers, for example, 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 to these, for example, oral cavity, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual Or topical administration is possible.
  • the dosage amount of the composition according to the present invention varies in its range according to the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and is easy for a person skilled in the art. Can decide.
  • the compositions of the present invention can be formulated into suitable formulations using known techniques for clinical administration.
  • the present invention relates to a method for improving or treating retinal disease, comprising administering the siRNA to an individual. 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 applied to the improvement or treatment method.
  • Example 1 Screening of 73 RNAi-induced double-stranded nucleic acid molecules targeting NRL
  • siRNA Asymmetric siRNA (asiRNA) was designed.
  • Conventional siRNA is a duplex of 19 base pairs with two nucleotide overhangs on both strands 3'.
  • the asiRNA has a reduced du-target effect due to the shortened sense length while the 5'of the antisense strand is blunt end and has a short duplex of 15-16 base pairs, showing the same inhibitory efficiency as siRNA. Therefore, it was designed with 16mer (sense strand)-19mer (antisense strand) asiRNA (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 allow up to one or two nucleotide mismatches with Rattus norvegicus and Mus musculus. At this time, the seed region (second nucleotide to eighth nucleotide), which plays an important role in RNA interference, contains only those that do not have a mismatched sequence.
  • NRL plasmid and NRL asiRNA were continuously transfected, and experiments were first performed on cells expressing NRL temporarily (transient cell line). More specifically, after culturing and growing HeLa cells in Dulbecco's modified Eagle's medium-corning (DMEM) with 10% fetal bovine serum-gibco (FBS) added to a 100 mm petri dish, the cells are grown in 12 well plates and 5 ⁇ per well 104 pieces were dispensed. After 24 hours, NRL plasmid was incubated in Lipofectamine 2000 (Invitrogene) and Opti-MEM reduced serum media (gibco), treated with cells, and cultured medium was changed to DMEM (10% FBS) after 3 hours.
  • DMEM Dulbecco's modified Eagle's medium-corning
  • FBS fetal bovine serum-gibco
  • siRNA was transfected using Lipofectamine RNAimax Transfection Reagent (Invitrogene). Each strand of siRNA was dilution in siRNA duplex buffer (bioneer) and incubated at 95°C for 5 minutes and 37°C for 1 hour. The annealing siRNA was electrophoresed on a 12% polyacrylamide gel, and stained for 10 minutes in EtBr to confirm annealing through a UV transiluminator. After transfection of siRNA, total RNA was extracted by treatment with Tri-RNA Reagent (FAVOGEN) 24 hours later, and cDNA was synthesized according to the protocol provided using High-capacity cDNA reverse transcription kit (Applied Biosystems).
  • Tri-RNA Reagent FAVOGEN
  • cDNA was analyzed by qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction) using a Step One real-time PCR system (Applied Biosystems) to analyze the expression level of the target gene, NRL. Since the transfection efficiency of each well may be different because the cells temporarily express NRL through transfection of NRL plasmid, the expression level of NRL was expressed as the expression level of the Neo gene in the NRL plasmid. Table 2 below shows the NRL primer and Neo primer sequences used for qRT-PCR.
  • NRL plasmid (Origene-RG202237) and NRL are performed in the same process as when checking RNA level.
  • western blot was performed by breaking the cells with RIPA buffer 48 hours later.
  • the primary antibody is NRL antibody (AVIVA-OAAB19847) and ⁇ -Tubulin (Santa Cruz Biotechnology-sc-5274), and the secondary antibody is 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 negative control.
  • si-Oct sequence is sense: 5'-AUGAUGCUCUUGAUUUUUTT-3' (SEQ ID NO: 151), antisense: 5'-AAAAAAUCAAGAGCAUCAUTT-3' (SEQ ID NO: 152).
  • #47 showed the best knockdown efficiency, and then #49, #67, and #68 showed similar knockdown efficiency (FIG. 4). Therefore, considering the mRNA level and the protein level, it was decided to make #47 and #49 into cell penetrating asiRNA.
  • Example 4 cp-asiRNA screening with cell-penetrating ability targeting NRL
  • the length of the 3'end of the antisense strand was increased to synthesize 16mer (sense strand)-25mer (antisense strand).
  • the increased antisense strand is human It is a sequence complementary to NRL. Table 3 below shows the asiRNA nucleotide sequences targeting NRL.
  • NRL asiRNAs of #47 and #49 were modified into asiRNAs having cell penetrating ability and resistant to nucleic acid hydrolase.
  • Table 4 below shows the modification of NRL asiRNA #47, 49 having cell penetrating ability.
  • siRNA No sequence(5' ⁇ 3') cp-asiNRL #47 sense ori hereinafter, 1S) mCGmGCmGCmUGmGUmCUmCG*mA*U*-Chol OMe/F (hereinafter, 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, 3S) mCmGmGmCGmCmUGGmUmCmUmCmG*mA*mU*-Chol antisense ori (hereinafter, 1AS) AUCGAGACCAGCGCmCmGmCmGmUmCmG*mG*mA*mA*mA OMe/F (hereinafter, 2AS) mA(2'-FU)mC(
  • “*” refers to a form in which an existing phosphodiester bond is substituted with a phosphorothioate bond
  • “m” indicates that the existing 2'-OH is 2'-O-. It means the form substituted with methyl.
  • “2'-F-” means, for example, in the case of 2'-FG, 2'-OH of the existing G (guanine) is substituted with fluorine, and "Chol” is 3'- It means the form in which cholesterol is added to the terminal. Except for the sequence of Table 4, modification was schematically shown in FIG. 6 (the same modification was performed in #47 and #49).
  • Cholesterol attached to the 3'end of the sense strand allows it to penetrate the cell membrane.
  • substitution of the phosphate backbone close to the 3'end of the sense strand and antisense strand with phosphorothioate increases the resistance to external nucleic acid hydrolase, enabling cellular uptake and biological use in vivo . do.
  • resistance to nucleic acid hydrolase increases, and siRNA immunogenicity and off-target effects decrease.
  • modification of 2'of sugars with fluoro stabilizes double strand RNA duplex, increases stability in seurm, and enables efficient silencing in vitro and in vivo .
  • Example 5-1 Check the effect of modified cp-asiRNA in stable cell line
  • the effect of modified #47 and #49 cp-asiRNA in the prepared A549- NRL stable cell line was confirmed.
  • the A549- NRL stable cell line was prepared by transfection of A549 cell-expressing NRL plasmid (Origene-RG202237) and selecting and growing cells resistant to G418 antibiotic (Goldbio).
  • A549- NRL stable cell line was cultured in Ham's F-12K (Kaighn's) Medium (gibco) with 10% FBS (fetal bovine serum) and G418 antibiotic (1mg/ml) added to a 100mm petri dish, and then propagated.
  • the cells were dispensed into 150,000 cells, 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 cells circulating each cell cycle by causing G1 arrest and starving the cells.
  • the cells were switched to Opti-MEM reduced serum media, and Cp-asiRNA was treated without transfection reagent.
  • 10% FBS fetal bovine serum
  • was added to Ham's F-12K (Kaighn's) Medium was broken with RIPA to perform western blot.
  • the western blot results are shown in FIG. 7, and S-ori, S-OMe/F, and SF are displayed in order of 1S, 2S, and 3S in order to more easily display each strand.
  • AS-ori, AS-OMe/ F and AS-F are shown in order of 1AS, 2AS, and 3AS.
  • siRNA No sequence (5' ⁇ 3') cp-asiNRL #47 sense OMe/F-PS2 (hereinafter, 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/F-P-PS1 (hereinafter, 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/F-P-PS2 (hereinafter, 2AS
  • the second modification is an additional chemical modification to the sense strand, 2S of #47 in Table 4, specifically, a phosphorothioate modification is added to the 5'end of the sense strand (hereinafter referred to as 2S-1). It is an additional chemical modification to the antisense strand of #47 of #47, 2AS. Specifically, phosphate modification or phosphate and phosphorothioate modification is 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 in the process of gene silencing. Therefore, in most cases, when the synthetic siRNA enters the cell, phosphorylation occurs. However, phosphate modification was performed on the 5'of the antisense strand for loading into the more reliable RISC (FIG. 8).
  • RISC RNA
  • Example 5-2 Check knockdown efficiency of selected NRL cp-asiRNA
  • 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 on the 5'of the antisense strand.
  • Table 7 shows the base 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 modification of 2S-2AS-1 are shown in Table 8 below.
  • the NRL cp-asiRNA having the antisense strand modified to 26mer showed a knockdown efficiency similar to that of the antisense strand of 25mer, and 2S-2AS as in the case of antisense strand of 25mer even when the antisense strand was 26mer. -1 showed better effect than 2S-2AS. According to FIG. 11, it was confirmed that even when the antisense strand was 26mer, the IC 50 value of 2S-2AS-1 (antisense: 26mer) was between 300nM and 600nM and could knockdown up to 62% (FIG. 11 ).
  • Figure 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
  • mice in each group were sacrificed and curved forcep (FSC) was used. The eyeball was removed.
  • 13 and 14 are the results confirming the change in expression of NRL according to the administration of #47 cp-asiNRL 2S-2AS-1 (1626) in the retinal tissue of the mouse eyeball.
  • administration of the candidate substance suppressed the expression of NRL protein in retinal tissue.
  • this effect was found to remain effective not only on the 7th day after administration, but also on the 14th day after administration, and showed a tendency to depend on the administration concentration.
  • the #47 cp-asiNRL 2S-2AS-1 (1626) substance can inhibit the expression of the NRL protein even under in vivo conditions. Accordingly, the candidate substance is NRL-mediated eye or retina It can be used as an active ingredient of a pharmaceutical composition for disease improvement or treatment.

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Abstract

La présente invention concerne un ARNsi asymétrique permettant d'inhiber l'expression de la fermeture éclair à leucines de la rétine neuronale (NRL), ainsi qu'une utilisation de cet ARNsi asymétrique, et plus particulièrement un ARNsi asymétrique qui comporte un brin antisens comprenant une séquence complémentaire d'un ARNm codant pour NRL, ainsi qu'un brin sens formant une liaison complémentaire avec le brin antisens ; et une composition pharmaceutique contenant l'ARNsi asymétrique destinée à atténuer les symptômes de maladies de la rétine ou à traiter ces maladies.
PCT/KR2020/000870 2019-01-18 2020-01-17 Arnsi asymétrique pour inhiber l'expression de la fermeture éclair à leucines de la rétine neuronale (nrl) WO2020149702A1 (fr)

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WO2023049716A1 (fr) * 2021-09-21 2023-03-30 Dtx Pharma, Inc. Composés ciblant nrl pour le traitement de la rétinite pigmentaire

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