Modified oligonucleotide or salt thereof

Abstract

The present disclosure provides a technique for inhibiting ubiquitin ligase RFFL function and improving the plasma-membrane expression of a mutant CFTR protein. The present disclosure also provides a novel therapeutic agent for CF.　The present inventors have found that a modified oligonucleotide, or a salt thereof, having a specific base sequence and a modified structure, can inhibit ubiquitin ligase RFFL function and improve the plasma-membrane expression of a mutant CFTR protein.

Description

Modified oligonucleotide or its salt

[0001] The present disclosure relates to modified oligonucleotides or salts thereof, as well as compositions and kits containing the modified oligonucleotides or salts thereof.

[0002] Cystic fibrosis (CF) is a monogenic disease common in Europe and the United States, affecting approximately 80,000 people worldwide. CF is caused by genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel expressed in epithelial cells. Approximately 90% of CF patients have a mutation in the CFTR amino acid sequence that results in a deletion of phenylalanine at position 508 (ΔF508-CFTR). In the ΔF508-CFTR mutant, CFTR transcription and translation are normal, but the protein is misfolded. As a result, the ΔF508-CFTR protein is ubiquitinated and degraded by the endoplasmic reticulum quality control system. As a result, the ΔF508-CFTR protein cannot be expressed on the plasma membrane.

[0003] In recent years, several CFTR-modifying drugs have been launched as curative treatments for CF. To date, the only CF treatment that has shown significant efficacy in treating CF patients with the ΔF508-CFTR mutation is Trikafta®, a triple combination of elexacaftor, ivacaftor, and tezacaftor. Trikafta corrects the misfolded ΔF508-CFTR protein, promoting its translocation to the plasma membrane and enhancing CFTR channel activity.

[0004] International Publication No. 2011 / 052436 International Publication No. 2015 / 125783

[0005] Okiyoneda T, Veit G, Sakai R, Aki M, Fujihara T, Higashi M, Susuki-Miyata S, Miyata M, Fukuda N, Yoshida A, Xu H, Apaja PM, Lukacs GL. Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase. Dev Cell. 2018 Mar 26,44(6):697-708.Shogo Taniguchi, Yukiko Ito, Hibiki Kiritani, Asuka Maruo, Ryohei Sakai, Yuji Ono, Ryosuke Fukuda, Tsukasa Okiyoneda. The ubiquitin ligase RNF34 participates in the peripheral quality control of CFTR. Front. Mol. Biosci. 9:840649. doi: 10.3389 / fmolb.2022.840649Taniguchi S, Ono Y, Doi Y, Taniguchi S, Matsuura Y, Iwasaki A, Hirata N, Fukuda R, Inoue K, Yamaguchi M, Tashiro A, Egami D, Aoki S, Kondoh Y, Honda K, Osada H, Kumeta H, Saio T, Okiyoneda T. Identification of α-Tocopherol succinate as an RFFL-substrate interaction inhibitor inducing peripheral CFTR stabilization and apoptosis. Biochem Pharmacol. 2023 Aug 4;215:115730. doi:10.1016 / j.bcp.2023.115730.Michaels WE, Bridges RJ, Hastings ML.Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells. Nucleic Acids Res. 2020 Jul 27;48(13):7454-7467. doi: 10.1093 / nar / gkaa490.Yoshida, T., Hagihara, T., Uchida, Y., Horiuchi, Y., Sasaki, K., Yamamoto, T., Yamashita, T., Goda, Y., Saito, Y., Yamaguchi, T., Obika, S., Yamamoto, S., & Inoue, T. Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation. Sci Rep. 2024 May 21;14(1):11540. doi: 10.1038 / s41598-024-61666-3.

[0006] As mentioned above, several CFTR modulating drugs are available on the market as curative treatments for CF. However, the median lifespan of CF patients remains in their 50s, and there is still room for improvement in CF treatment. It has also been reported that the ΔF508-CFTR protein is ubiquitinated and subsequently degraded, even in the presence of Trikafta. Therefore, it is possible that the efficacy of CFTR modulating drugs is not fully realized because the ΔF508-CFTR protein is degraded before it reaches the plasma membrane.

[0007] The present inventors have previously demonstrated that a type of ubiquitin ligase (Ring Finger and FYVE-like Domain Containing E3 Ubiquitin Protein Ligase, RFFL) is involved in CFTR degradation (Non-Patent Document 1). RFFL knockdown suppresses ΔF508-CFTR degradation and enhances the ΔF508-CFTR ameliorating effects of CFTR modifiers such as Trikafta (Non-Patent Documents 1 and 2). Therefore, combining CFTR modifiers with RFFL knockdown is expected to provide more effective CF treatment than previously possible.

[0008] Furthermore, we previously reported that RFFL knockdown alone, without the concomitant use of CFTR-modifying drugs, improved plasma membrane expression of CFTR class 6 mutants, which are rare in CF patients (Non-Patent Document 1). Therefore, RFFL knockdown may be effective in treating CF patients with CFTR class 6 mutations, even without the concomitant use of CFTR-modifying drugs.

[0009] Therefore, the main objective of the present inventors was to provide a technology for inhibiting the function of ubiquitin ligase RFFL and improving the plasma membrane expression of mutant CFTR protein, as well as to provide a novel therapeutic agent for CF.

[0010] The present inventors have discovered that modified oligonucleotides or salts thereof having specific base sequences and modified structures can inhibit the function of ubiquitin ligase RFFL and improve the plasma membrane expression of mutant CFTR proteins. Through further improvements, the present disclosure has been completed.

[0011] The present disclosure includes, for example, the subject matter described in the following sections. Note that in the present specification, when describing the structure of an oligonucleotide, it is described from left to right in the direction from the 5' end to the 3' end. Section 1. A modified oligonucleotide or a salt thereof, wherein the modified oligonucleotide has a ubiquitin ligase RFFL knockdown activity and is selected from the group consisting of TAGTGGTTG, GGGTTGGAATAGGC (SEQ ID NO: 2), CCTTGTATAGCCGG (SEQ ID NO: 3), CCTGATGGTACTG (SEQ ID NO: 4), TAGGCACC, GCCGGGTCACTCTCT (SEQ ID NO: 6), AGTCCTTTCTGATCC (SEQ ID NO: 7), CCGTTTTGGTCTTCG (SEQ ID NO: 8), TTGGGTACAGGAGGA (SEQ ID NO: 9), GCGTTTAGGGTTTGC (SEQ ID NO: 10), AAAGGCTGTGGTTCA (SEQ ID NO: 11), ATTCCCCCAACTCCT (SEQ ID NO: 12), ACCATTCAGCCTTGC (SEQ ID NO: 13), AAAGGGGTTTAGTGGTT (SEQ ID NO: 14), TTTCTGAGCCATAGTCCA (SEQ ID NO: 15), GACTTAACAAACAGGCAC (SEQ ID NO: 16), CTGACCTACATAGATTTC (SEQ ID NO: 17), GCCTTGCTTCAGTATTAG (SEQ ID NO: 18), The nucleic acid sequence of the present invention is 21 bases or less in length and contains GTCAGTTCTCACCTTATG (SEQ ID NO: 19) or GTCCCAATCCTTCCAGCT (SEQ ID NO: 20), and has at least two modified nucleosides within five bases from the 5'-end and at least one modified nucleoside within five bases from the 3'-end, wherein the modified nucleosides are each independently represented by one of the general formulas (I) to (III): (wherein Base represents a base). Item 2. A modified oligonucleotide or a salt thereof according to Item 1, wherein internucleoside linkages contained in the modified oligonucleotide are, independently of one another, a phosphodiester linkage or a phosphorothioate linkage. Item 3. A modified oligonucleotide or a salt thereof according to Item 1 or 2, wherein the modified nucleoside is a modified nucleoside represented by general formula (I). Item 4. A modified oligonucleotide or a salt thereof, wherein the modified oligonucleotide is a modified oligonucleotide consisting of [G][G][T]TTAGTGGTT[G][G]T (SEQ ID NO: 21) and / or a modified oligonucleotide consisting of a base sequence in which 1 to 3 bases are added, deleted, or substituted in the base sequence of SEQ ID NO: 21 and has ubiquitin ligase RFFL knockdown activity, wherein a base enclosed in [ ] represents a base represented by general formula (I): (wherein Base represents a base), wherein the internucleoside linkages contained in the modified oligonucleotide are, independently of one another, phosphodiester linkages or phosphorothioate linkages and contain 0 to 6 phosphodiester linkages, or a salt thereof. Item 5. A composition for use in treating cystic fibrosis, comprising the modified oligonucleotide or a salt thereof according to any one of Items 1 to 4. Item 6. The composition according to Item 5, wherein the treatment comprises administration of elexacaftor, ivacaftor, or tezacaftor. Item 7. A kit for treating cystic fibrosis, comprising the modified oligonucleotide or a salt thereof according to any one of Items 1 to 4, and elexacaftor, ivacaftor, or tezacaftor. Item 8. The composition according to Item 5 or 6, or the kit according to Item 7, for use in a subject having a mutation in which phenylalanine at position 508 in the CFTR amino acid sequence is deleted. Item 9. A method for treating cystic fibrosis, comprising administering to a subject in need thereof a therapeutically effective amount of the modified oligonucleotide or a salt thereof according to any one of Items 1 to 4. Item 10. The method according to Item 9, further comprising administering to the subject therapeutically effective amounts of elexacaftor, ivacaftor, and tezacaftor. Item 11. The method according to Item 9 or 10, wherein the subject has a mutation in which phenylalanine at position 508 in the CFTR amino acid sequence is deleted. Item 12. The modified oligonucleotide or a salt thereof according to any one of Items 1 to 4, for use as a medicine. Item 13. The modified oligonucleotide or a salt thereof according to any one of Items 1 to 4, for use in the treatment of cystic fibrosis. Item 14. The modified oligonucleotide or a salt thereof according to Item 13, wherein the treatment comprises administration of elexacaftor, ivacaftor, and tezacaftor. Item 15. Item 16. A kit comprising the modified oligonucleotide or a salt thereof according to any one of Items 1 to 4, and elexacaftor, ivacaftor, and tezacaftor, for use in the treatment of cystic fibrosis.Item 17. The kit according to Item 16, which is applied to a subject having a mutation in which phenylalanine at position 508 in the CFTR amino acid sequence is deleted.

[0012] According to the technology of the present disclosure, it is possible to effectively inhibit the function of ubiquitin ligase RFFL and improve the plasma membrane expression of mutant CFTR protein.

[0013] The present inventors have previously reported a small molecule compound that inhibits RFFL (Non-Patent Document 3). In cell line evaluation, this small molecule compound slightly enhanced the efficacy of the CF therapeutic agent Trikafta. However, further investigation by the present inventors revealed that the efficacy of this small molecule compound was not confirmed in evaluation using a primary culture model of CF patient airway epithelium (CF-HBE model), which is the final evaluation system in preclinical trials. Furthermore, although many siRNAs for knocking down RFFL are commercially available, the present inventors' investigations showed that commercially available RFFL siRNAs failed to sufficiently knock down RFFL in the CF-HBE model, nor were they found to enhance the efficacy of the CF therapeutic agent Trikafta (data not shown). As described above, no small molecule compound or siRNA capable of sufficiently inhibiting RFFL function has been found to date, even in the CF-HBE model, which is the final evaluation system in preclinical trials. On the other hand, the technology disclosed herein can exert excellent RFFL function inhibition and amelioration of the plasma membrane expression of mutant CFTR protein in the CF-HBE model.

[0014] The results of Test 1-2 are shown in a heat map. The results of Test 1-3 are shown. The graph shows the relative chemiluminescence intensity of each cell (i.e., the expression level of ΔF508-CFTR-HRP protein at the plasma membrane) with the mock chemiluminescence intensity set to 100%. Tests were performed in n=3, and significance was tested using one-way ANOVA (Dunnett's method). * p<0.05, ** p<0.01, *** p<0.001. The results of Test 1-4 are shown. The graph shows the relative fluorescence intensity of each cell (i.e., relative cell viability) with the mock fluorescence intensity set to 100%. Tests were performed in n=3, and significance was tested using one-way ANOVA (Dunnett's method). * p<0.05, ** p<0.01, *** p<0.001. The results of Test 2-2 are shown in a heat map. The results of Test 2-3 are shown. The test was performed with n=3, and significance was tested by one-way ANOVA (Dunnett's method). * p<0.05, ** p<0.01, **** p<0.0001. The results of Tests 2-4 are shown. The test was performed with n=3. The results of Test 3-2 are shown. The graph shows the relative chemiluminescence intensity of each cell, with the chemiluminescence intensity in NEG#8 set as 100%. The test was performed with n=9, and significance was tested by one-way ANOVA (Dunnett's method). * p<0.05, **** p<0.0001. The results of Test 3-3 are shown. The graph shows the relative fluorescence intensity of each cell, with the fluorescence intensity in NEG#8 set as 100%. The test was performed with n=9. The results of Test 4 are shown. Left: Western blotting results. Ponceau S staining was used as a loading control. Figure right: RFFL protein levels quantified by densitometry. Results are shown as relative values, with the RFFL protein levels in NEG#8 at each time point taken as 100%. Tests were performed on n=6, and significance was tested using Student's t-test. * p<0.05, ** p<0.01, **** p<0.0001. Results of Test 5 are shown. Figure left (ΔF508-CFTR function): CFTR channel activity quantified by short-circuit current measurement is shown, expressed as a relative value, with the measured value in NEG#8 + Trikafta taken as 100%.Right (RFFL mRNA): RFFL mRNA levels quantified after short-circuit current measurement are shown as relative values, with the value measured with NEG#8 + Trikafta set to 100%. Significance tests were performed using a paired t-test. * p<0.05, ** p<0.01. The results of Experiment 6 are shown. The CFTR mutant type is indicated in the upper left corner of each graph. In these experiment results, "NEG" indicates NEG#8, "3074B" indicates ASO37, and "TRI" indicates Trikafta addition. Each result is shown as a relative value, with the value measured with NEG#8 transfection and DMSO addition set to 100%. Experiments were performed on n=5-6, and significance tests were performed using two-way ANOVA (Holm-Sidak multiple comparison). Significant interactions were detected using P. int * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Results of Test 6 (continued) Results of Test 6 (continued) Results of Test 8 (continued) In these test results, "3074B" represents ASO37. Left: Results of TLR9 activity assay. Right: Cell viability. Data are shown as mean ± standard deviation (SD) (n=3).

[0015] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, modified oligonucleotides or salts thereof, as well as compositions and kits containing the modified oligonucleotides or salts thereof. The present disclosure includes all that is disclosed herein and that would be recognized by a person skilled in the art.

[0016] 1. Modified Oligonucleotides of the Present Disclosure The modified oligonucleotides or salts thereof encompassed by the present disclosure have a ubiquitin ligase RFFL knockdown activity, and consist of a base sequence of 21 bases or less that includes any of the base sequences shown in TAGTGGTTG, TAGGCACC, SEQ ID NOs: 2 to 3, and SEQ ID NOs: 6 to 20, and have at least two modified nucleosides within 5 bases from the 5' end and at least one modified nucleoside within 5 bases from the 3' end, wherein the modified nucleosides are each independently represented by one of the general formulas (I) to (III): (wherein Base represents a base). Hereinafter, such modified oligonucleotides encompassed by the present disclosure may be referred to as "modified oligonucleotides of the present disclosure." In this specification, the base symbol T represents thymine or uracil, and C represents cytosine or 5-methylcytosine.

[0017] 1-1. Nucleotide Sequence As described above, the modified oligonucleotide of the present disclosure consists of a nucleotide sequence of 21 nucleotides or less that includes any of the nucleotide sequences shown in SEQ ID NOS: 6 to 20, TAGTGGTTG, TAGGCACC, SEQ ID NOS: 2 to 4, and 6 to 20. In a preferred embodiment of the modified oligonucleotide of the present disclosure, the nucleotide sequence includes any of the nucleotide sequences shown in SEQ ID NOS: 6 to 20, as well as GGTTTAGTGGTTGGT (SEQ ID NOS: 21), CCTGATGGTACTGCT (SEQ ID NOS: 23), AGCCTGATGGTACTG (SEQ ID NOS: 24), GGGTTTAGTGGTTGG (SEQ ID NOS: 25), TAGGCACCATTCAGC (SEQ ID NOS: 26), TTAGTGGTTGATGG (SEQ ID NOS: 27), GGGTTGGAATAGGCC (SEQ ID NOS: 28), AGGGTTGGAATAGGC (SEQ ID NOS: 29), CC The modified oligonucleotide comprises a base sequence of 21 bases or less that contains any of the base sequences set forth in SEQ ID NOs: 9 to 12, 14, 21, and 23 to 27.

[0018] In another embodiment of the modified oligonucleotide of the present disclosure, the base sequence consists of any of the base sequences set forth in SEQ ID NOS: 6 to 38, with one to six bases added, deleted, or substituted, and the modified oligonucleotide has the activity of knocking down ubiquitin ligase RFFL (the ubiquitin ligase RFFL knockdown activity is described below). In a preferred embodiment of the modified oligonucleotide of the present disclosure, the base sequence consists of any of the base sequences set forth in SEQ ID NOS: 9 to 12, 14, 21, and 23 to 27, with one to six bases added, deleted, or substituted, and the modified oligonucleotide has the activity of knocking down ubiquitin ligase RFFL. Here, the number of bases that can be added, deleted, or substituted is preferably one to five, more preferably one to three, and even more preferably one or two. Furthermore, the position of the addition, deletion, or substitution is preferably near the 5' and / or 3' end of the modified oligonucleotide.

[0019] In another preferred embodiment of the modified oligonucleotide of the present disclosure, the base sequence consists of any of the base sequences shown in SEQ ID NOs: 6 to 38. In a more preferred embodiment of the modified oligonucleotide of the present disclosure, the base sequence consists of any of the base sequences shown in SEQ ID NOs: 9 to 12, 14, 21, and 23 to 27.

[0020] As described above, the base length of the modified oligonucleotides of the present disclosure is 21 bases or less, preferably 20 or 19 bases or less, more preferably 18 bases or less, and even more preferably 17 bases or less. The lower limit of the base length of the modified oligonucleotides of the present disclosure is not particularly limited, and may be, for example, 9, 10, 11, 12, 13, 14, or 15 bases or more. The base length of the modified oligonucleotides of the present disclosure may be 9 to 21 bases, preferably 13 to 20 bases, more preferably 14 to 19 bases, even more preferably 15 to 18 bases, and most preferably 15 bases.

[0021] In an embodiment in which the modified oligonucleotide of the present disclosure comprises a base sequence containing any one of TAGTGGTTG, TAGGCACC, the base sequences of SEQ ID NOS: 2 to 4, and 6 to 20, and further comprising an optional additional base, the type of additional base is not particularly limited. Examples of bases that can be added include adenine, cytosine, guanine, thymine, uracil, hypoxanthine, xanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 7-methylguanine, and 5,6-dihydroxyuracil. Of these, the additional base is preferably at least one selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, and 5-methylcytosine.

[0022] In an embodiment in which the modified oligonucleotide of the present disclosure comprises a base sequence comprising any one of TAGTGGTTG, TAGGCACC, SEQ ID NOs: 2 to 4, and SEQ ID NOs: 6 to 20, and further comprising an optional additional base, the additional base may be added to the 5' end, the 3' end, or both the 5' end and the 3' end of any one of the base sequences represented by SEQ ID NOs: 1 to 27.

[0023] 1-2. Modified Nucleosides As described above, the modified oligonucleotide of the present disclosure has at least two modified nucleosides within 5 bases from the 5'-end and at least one modified nucleoside within 5 bases from the 3'-end, wherein the modified nucleosides are each independently represented by the general formulas (I) to (III): (wherein Base represents a base). In the present disclosure, a base constituting a modified nucleoside represented by any of general formulas (I) to (III) may be indicated by enclosing the base in "{}" (braces). It is particularly preferred that the modified nucleoside is a modified nucleoside represented by general formula (I).

[0024] The modified nucleic acid having the structure represented by general formula (I) is provided by Luxna Biotech under the name AmNA (registered trademark), and has the characteristics of improved nuclease resistance, improved base-specific hybridization, and reduced hepatotoxicity compared to the locked amino acid (LNA) described below. In the present disclosure, a base constituting a modified nucleoside represented by general formula (I) may be indicated by enclosing the base in square brackets ("[ ]").

[0025] The modified nucleic acid having the structure represented by general formula (II) is provided by Luxna Biotech under the name scpBNA (registered trademark), and has the characteristics of improved nuclease resistance, improved base-specific hybridization, reduced hepatotoxicity, and improved hydrophobicity compared to LNA. Note that in the present disclosure, the fact that a certain base constitutes a modified nucleoside having the structure represented by general formula (II) may be indicated by enclosing the base in "( )" (parentheses).

[0026] Modified nucleic acids having a structure represented by general formula (III) are commonly known as LNAs, and are characterized by improved nuclease resistance, improved base-specific hybridization, and reduced cytotoxicity compared to natural nucleic acids. In the present disclosure, a base constituting a modified nucleoside represented by general formula (III) may be indicated by enclosing the base in "< >" (angle brackets).

[0027] As described above, the modified oligonucleotide of the present disclosure has at least two modified nucleosides represented by any one of general formulas (I) to (III) within five bases from the 5'-end. The number of modified nucleosides contained within five bases from the 5'-end of the modified oligonucleotide of the present disclosure may be 2 to 5, preferably 2 to 4, and more preferably 2 or 3. Furthermore, it is particularly preferred that the modified oligonucleotide of the present disclosure has all three bases from the 5'-end be modified nucleosides represented by any one of general formulas (I) to (III).

[0028] The modified oligonucleotide of the present disclosure has at least one modified nucleoside represented by any one of general formulas (I) to (III) within 5 bases from the 3'-end. The number of modified nucleosides contained in the modified oligonucleotide of the present disclosure within 5 bases from the 3'-end may be 1 to 5, preferably 2 to 4, and more preferably 2 or 3. Furthermore, the modified oligonucleotide of the present disclosure particularly preferably has 2 or 3 modified nucleosides represented by any one of general formulas (I) to (III) within 3 bases from the 5'-end.

[0029] Although not particularly limited, when the base constituting the modified nucleoside is a base represented by the base symbol C (ie, cytosine or 5-methylcytosine), the base is particularly preferably 5-methylcytosine.

[0030] Although not particularly limited, the modified oligonucleotide of the present disclosure preferably has a modified nucleoside represented by any of general formulas (I) to (III) only in a portion within 5 bases from the 5'-end and / or within 5 bases from the 3'-end, and more preferably has a modified nucleoside represented by any of general formulas (I) to (III) only in a portion within 3 bases from the 5'-end and / or within 3 bases from the 3'-end.

[0031] Nucleosides other than the modified nucleosides represented by any one of general formulas (I) to (III) contained in the modified oligonucleotides of the present disclosure are not particularly limited, but are preferably ribonucleosides or deoxyribonucleosides, and particularly preferably deoxyribonucleosides. Furthermore, the bases constituting the nucleosides other than the modified nucleosides represented by any one of general formulas (I) to (III) are preferably one or more selected from the group consisting of adenine, cytosine, 5-methylcytosine, guanine, thymine, and uracil. A particularly preferred embodiment is one in which the nucleoside other than the modified nucleosides represented by any one of general formulas (I) to (III) is a deoxyribonucleoside, and the bases constituting the deoxyribonucleoside are one or more selected from the group consisting of adenine, cytosine, 5-methylcytosine, guanine, and thymine.

[0032] 1-3. Internucleoside Bonds Although not particularly limited, the internucleoside bonds contained in the modified oligonucleotide of the present disclosure are preferably, independently of one another, phosphodiester bonds or phosphorothioate bonds. A phosphorothioate bond has a structure in which one of the oxygen atoms constituting the phosphodiester bond is replaced with a sulfur atom. In the present disclosure, a phosphodiester bond may be represented by "o" and a phosphorothioate bond may be represented by "s."

[0033] Although not particularly limited, the number of phosphodiester bonds contained in the modified oligonucleotide of the present disclosure is preferably 0 to 6. The upper or lower limit of the range may be 0, 1, 2, 3, 4, 5, or 6. The number of phosphodiester bonds contained in the modified oligonucleotide of the present disclosure is more preferably 0 to 4, even more preferably 1 to 4, and particularly preferably 2 or 3.

[0034] It is generally known that by substituting phosphodiester bond for phosphorothioate bond for internucleoside bond contained in oligonucleotide, the nuclease resistance of the oligonucleotide can be improved.On the other hand, it is generally known that by substituting phosphodiester bond for phosphorothioate bond for internucleoside bond contained in oligonucleotide, the biological toxicity of the oligonucleotide, such as hepatotoxicity, can be enhanced.That is, from the viewpoint of improving nuclease resistance, more phosphorothioate bond is considered to be preferable, but from the viewpoint of reducing biological toxicity, fewer phosphorothioate bond is considered to be preferable.

[0035] Furthermore, as described below, the modified oligonucleotides of the present disclosure contain a sequence complementary to a portion of the mRNA for the ubiquitin ligase RFFL, and are presumed to exhibit excellent effects of inhibiting RFFL function and improving the plasma membrane expression of mutant CFTR protein by forming base pairs with the mRNA. Therefore, if the modified oligonucleotides of the present disclosure are degraded by nucleases, the effects of inhibiting RFFL function and improving the plasma membrane expression of mutant CFTR protein may be reduced.

[0036] Because modified nucleosides represented by any of general formulas (I) to (III) are themselves highly resistant to nucleases, high nuclease resistance can be maintained even when the internucleoside linkage containing the modified nucleoside is a phosphodiester linkage. Therefore, from the viewpoint of effectively inhibiting RFFL function and improving the plasma membrane expression of mutant CFTR protein while reducing biotoxicity, the modified oligonucleotide of the present disclosure preferably has an internucleoside linkage containing a modified nucleoside represented by any of general formulas (I) to (III) that is a phosphodiester linkage. In this embodiment, it is more preferable that the internucleoside linkage not containing the modified nucleoside is a phosphorothioate linkage.

[0037] As described above, the modified oligonucleotide of the present disclosure has at least two modified nucleosides within five bases from the 5'-end and at least one modified nucleoside within five bases from the 3'-end. Therefore, it is preferable that the modified oligonucleotide of the present disclosure contains phosphodiester bonds only near the 5'-end and / or 3'-end and does not contain phosphodiester bonds in other portions. It is particularly preferable that the modified oligonucleotide of the present disclosure contains phosphodiester bonds only near the 5'-end and / or 3'-end and that all internucleoside bonds other than phosphodiester bonds are phosphorothioate bonds.

[0038] Although not particularly limited, the position of the phosphodiester bond contained in the modified oligonucleotide of the present disclosure is preferably only present within five internucleoside bonds from the 5' end and / or within five internucleoside bonds from the 3' end of the internucleoside bond contained in the modified oligonucleotide, and is not present in any other portion, and more preferably only present within three internucleoside bonds from the 5' end and / or within three internucleoside bonds from the 3' end, and is not present in any other portion.

[0039] More specifically, the internucleoside linkages contained in the modified oligonucleotides of the present disclosure are preferably, for example, ssoss...sssss, ssoss...ssoss, sooss...ssoos, sssss...ssoso, sosss...ssooo, or sosss...ssoso, where "..." indicates that an s follows, with "o" indicating a phosphodiester bond and "s" indicating a phosphorothioate bond. Also, sssss…sssso, sssss…sssos, sssss…ssoss, sssss…sssoo, sssss…ssoos, sssss…ssooo, ossss…sssss, ossss…sssso, ossss…sssos, ossss…ssoss, ossss…sssoo, ossss…ssoos, ossss…ssoso, sosss…sssss, sos ss…sssso, sosss…sssos, sosss…ssoss, sosss…sssoo, sosss…ssoos, sosss…ssooo, ssoss…sssso, ssoss… sssos, ssoss…sssoo, ssoss…ssoos, ssoss…ssoso, ssoss…ssooo, oosss…sssss, oosss…sssso, oosss…sss os, oosss…ssoss, oosss…sssoo, oosss…ssoos, oosss…ssoso, oosss…ssooo, sooss…sssss, sooss…sssso, sooss…sssos, sooss…ssoss, sooss…sssoo, sooss…ssoso, sooss…ssooo, ososs…sssss, ososs…sssso, oso It may be ss...sssos, ososs...ssoss, ososs...sssoo, ososs...ssoos, ososs...ssoso, oooss...ssooo, oooss...sssss, oooss...sssso, oooss...sssos, oooss...ssoss, oooss...sssoo, oooss...ssoos, oooss...ssoso, or oooss...ssooo.

[0040] 1-4. Specific embodiments As preferred embodiments of the modified oligonucleotides of the present disclosure, {G}{C}{C}GGGTCACTC{T}{C}T, {A}{G}{T}CCTTTCTGA{T}{C}C, {C}{C}{G}TTTTGGTCT{T}{C}G, {T}{T}{G}GGTACAGGA{G}{G}A, {G}{C}{G}TTTAGGGTT{T}{G}C, {A}{A}{A}GGCTGTGGT{T}{C}A, {A}{T}{T}CCCCCAACT{C}{C}T, {A}{C}{C}ATTCAGCCT{T}{G}C, {A}{A}{A}GGGGTTTAGTG{G}{T}T, {T}{T}{T}CTGAGCCATAGT{C}{C}{A}, {G}{A}{C}TTAACAAACAGG{C}{A}{C}, {C}{T}{G}ACCTACATAGAT{T}{T}{C}, {G}{C}{C}TTGCTTCAGTAT{T}{A}{G}, {G}{T}{C}AGTTCTCACCTT{A}{T}{G}, {G}{T}{C}CCAATCCTTCCA{G}{C}{T}, {G}{G}{T}TTAGTGGTT{G}{G}T, {G}{G}{T}TTAGTGGTT{G}{G}{T}, {C}{C}{T}GATGGTACT{G}{C}T, {A}{G}{C}CTGATGGTA{C}{T}G, {G}{G}{G}TTTAGTGGT{T}{G}G, {T}{A}{G}GCACCATTC{A}{G}C, {T}{T}{A}GTGGTTGGTGA{T}{G}G, {G}{G}{G}TTGGAATAG{G}{C}C, {A}{G}{G}GTTGGAATA{G}{G}C, {C}{C}{T}TGTATAGCC{G}{G}G, {T}{C}{C}TTGTATAGC{C}{G}G, {G}{C}{C}TGATGGTAC{T}{G}C, {T}{A}{G}TGGTTGGTG{A}{T}G, {G}{T}{T}TAGTGGTTGGT{G}{A}T, {G}{G}{G}TTTAGTGGTTG{G}{T}G, {T}{T}{T}GAATAGGCACCA{T}{T}{C}, {T}{G}{T}TTTTGAATAGGC{A}{C}{C}, and{T}{T}{T}AGTGGTTGGTGA{T}{G}{G}. Note that a base enclosed in "{}" (braces) indicates that the base constitutes a modified nucleoside represented by any one of the general formulae (I) to (III). Although not particularly limited, in this embodiment, a nucleoside other than a modified nucleoside represented by any one of the general formulae (I) to (III) is preferably a deoxyribonucleoside. Furthermore, in this embodiment, the base represented by C constituting a modified nucleoside represented by any one of the general formulae (I) to (III) is preferably 5-methylcytosine. Furthermore, in this embodiment, the base represented by T constituting a nucleoside other than a modified nucleoside represented by any one of the general formulae (I) to (III) is preferably thymine.

[0041] Thanks for watching the snowflakes. G#C(C)GGGTCACTC(T(C)T、 (A(C)(T)CCTTTCTGA(T(C)C、 CTTCAGGATTTTGGTCT(T) C6G、 TT(TC)G GGTACAGGA G0G0A、 G0C0GTTTAGGGTTT00G0C、 000000 GGCTGTGGT10C0A、 A0T0T CCCCCAACT+C0CT、 ATTCAGCCT+T0G0C、 AAGGGTTTAGTGG0G0T0T CTGAGCCATAGTG0C0A3 GA0AACCTTAAACAAACAGG C0AAC6, CCTACTG ACCTACATAGAT!T6C0 GTGCTTCAGTAT1T6A6G6、 GT#C6AGTTCTCACCTT(A6G0、 G#T6CCACAATCCTTCCA(G)C6T6、 G#G6T6TTAGTGGTT+G6G6T. TTAGTGGTT, G6G(T)、 G6G6(T)TTAGTGGTT,G6G(T)、 C#C(T)GATGGTACT(G(C)T、 (A(C)(C)CTGATGGTA(C(T)G、 G0G0G0TTTAGTGGT1T0G6G,T0A0G0GCACCATTC:A0G0C. (T8T0A GTGGTTGGTGA1TGG 、 GTGGGG TTGGAATAGG0C6C、 A0G0G0GTTGGAATA0G0G0C、,C000000 TGTATAGCCG0G0G0 TTGTTAGC+C0G0G+G000000 TGATGGTAC+T0G0C、 T#AGTGGTTGGTG#A#TG 、,G#T#T#TAGTGGTTGGT G0AT、 GTTAGTGGTTG G0TG, T0T0TGAATAGGCACCAT0C0 TTG0T0TTTTGAATAGGC1A0C6C3[T][T][T]AGTGGTTGGTGA[T][G]<G>. A base enclosed in "[ ]" (brackets) indicates that the base constitutes a modified nucleoside represented by the general formula (I) above. A base enclosed in "( )" (parentheses) indicates that the base constitutes a modified nucleoside represented by the general formula (II) above. A base enclosed in "< >" (angle brackets) indicates that the base constitutes a modified nucleoside represented by the general formula (III) above. Although not particularly limited, in this embodiment, a nucleoside other than a modified nucleoside represented by any of the general formulae (I) to (III) above is preferably a deoxyribonucleoside. Furthermore, in this embodiment, a base represented by C that constitutes a modified nucleoside represented by any of the general formulae (I) to (III) above is preferably 5-methylcytosine. Furthermore, in this embodiment, the base represented by T constituting the nucleoside other than the modified nucleoside represented by any one of the above general formulae (I) to (III) is preferably thymine.

[0042] Further preferred embodiments of the modified oligonucleotide of the present disclosure include the modified oligonucleotides shown in the table below. Although not particularly limited, in this embodiment, the nucleoside other than the modified nucleoside represented by any one of the general formulae (I) to (III) above is preferably a deoxyribonucleoside. Furthermore, in this embodiment, the base represented by C constituting the modified nucleoside represented by any one of the general formulae (I) to (III) above is preferably 5-methylcytosine. Furthermore, in this embodiment, the base represented by T constituting the nucleoside other than the modified nucleoside represented by any one of the general formulae (I) to (III) above is preferably thymine.

[0043]

[0044]

[0045] Particularly preferred embodiments of the modified oligonucleotide of the present disclosure include the modified oligonucleotides shown in the table below. Although not particularly limited, in this embodiment, the nucleoside other than the modified nucleoside represented by the general formula (I) is preferably a deoxyribonucleoside. Furthermore, in this embodiment, the base represented by C constituting the modified nucleoside represented by the general formula (I) is preferably 5-methylcytosine. Furthermore, in this embodiment, the base represented by T constituting the nucleoside other than the modified nucleoside represented by the general formula (I) is preferably thymine.

[0046]

[0047] A most preferred embodiment of the modified oligonucleotide of the present disclosure is a modified oligonucleotide having a base sequence and modified nucleoside positions of [G][G][T]TTAGTGGTT[G][G]T (SEQ ID NO: 21), with an internucleoside linkage of ssosssssssssss. Although not particularly limited, in this embodiment, nucleosides other than the modified nucleoside represented by general formula (I) are preferably deoxyribonucleosides. Furthermore, in this embodiment, the base represented by C constituting the modified nucleoside represented by general formula (I) is preferably 5-methylcytosine. Furthermore, in this embodiment, the base represented by T constituting the nucleoside other than the modified nucleoside represented by general formula (I) is preferably thymine. This embodiment is expected to exhibit particularly excellent RFFL knockdown activity and amelioration of mutant CFTR protein plasma membrane expression. Furthermore, this embodiment is expected to exhibit particularly excellent safety (e.g., low immunostimulation, low cytotoxicity, etc.).

[0048] 1-5. Salts The modified oligonucleotides of the present disclosure may optionally form pharmaceutically acceptable salts. In the present disclosure, the term "modified oligonucleotides of the present disclosure" is used to encompass not only the modified oligonucleotides themselves but also their pharmaceutically acceptable salts, unless otherwise specified.

[0049] Specific salts include, for example, alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts such as ammonium salt; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetrahydrofuran ... Examples of the amine salt include organic salts such as methylammonium salts and tris(hydroxymethyl)aminomethane salts; inorganic acid salts such as hydrohalogenated salts such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide, nitrate, perchlorate, sulfate and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate, arylsulfonates such as benzenesulfonate and p-toluenesulfonate, organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate and aspartate.

[0050] The modified oligonucleotides of the present disclosure may be in the form of a solvate. Examples of the solvate include solvates with water, methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, dimethoxyethane, and the like.

[0051] 1-7. The modified oligonucleotide of the present disclosure may be one type selected from the above-described modified oligonucleotides, or two or more types may be used in combination.

[0052] 2. Method for Producing Modified Oligonucleotides of the Present Disclosure The modified oligonucleotides of the present disclosure can be produced by conventionally known methods or methods that can be easily conceived by those skilled in the art from conventionally known methods. For example, the modified nucleoside represented by the above general formula (I) can be produced according to the description in Patent Document 1. The modified nucleoside represented by the above general formula (II) can be produced according to the description in Patent Document 2. In addition, methods for producing the modified nucleoside represented by the above general formula (III), methods for introducing a phosphodiester bond, and methods for introducing a phosphorothioate bond, etc., are widely known in the technical field.

[0053] Additionally, modified oligonucleotides of the present disclosure are also available through publicly available modified oligonucleotide synthesis services, such as those offered by GeneDesign.

[0054] 3. Effects of Modified Oligonucleotides of the Present Disclosure 3-1. Effect of Knocking Down Ubiquitin Ligase RFFL As shown in the Examples below, modified oligonucleotides of the present disclosure have the effect of knocking down ubiquitin ligase RFFL. Specifically, they have at least one of the effects of reducing RFFL mRNA levels and reducing the expression level of RFFL protein. Whether the modified oligonucleotides of the present disclosure have these effects can be confirmed by conventionally known methods or methods that can be easily derived from conventionally known methods. For example, the effect of reducing RFFL mRNA levels can be confirmed by extracting mRNA from cells or tissues to which the modified oligonucleotides of the present disclosure have been applied and performing quantitative reverse transcription PCR (qRT-PCR) using the mRNA as a template. Furthermore, for example, the effect of reducing the expression level of RFFL protein can be confirmed by purifying protein from cells or tissues to which the modified oligonucleotides of the present disclosure have been applied and performing Western blotting.

[0055] 3-2. Effect of Improving Plasma Membrane Expression of Mutant CFTR Protein As shown in the Examples below, the modified oligonucleotides of the present disclosure can have the effect of improving the plasma membrane expression of mutant CFTR protein. As mentioned above, CFTR is a channel protein, and therefore, its expression at the plasma membrane is necessary for CFTR to perform its function. The effect of the modified oligonucleotides of the present disclosure to improve the plasma membrane expression of mutant CFTR protein can be confirmed by conventionally known methods or methods that can be easily derived from conventionally known methods. For example, the plasma membrane expression of mutant CFTR protein can be evaluated by applying the modified oligonucleotides of the present disclosure to cells stably expressing a fusion protein of mutant CFTR and HRP (horseradish peroxidase), adding an HRP substrate, and detecting and quantifying the chemiluminescence generated by HRP enzymatic activity.

[0056] In this evaluation system, if the mutant CFTR-HRP protein is expressed intracellularly rather than on the plasma membrane, the HRP substrate present outside the cell will hardly come into contact with HRP, and therefore little chemiluminescence will be generated. On the other hand, if the mutant CFTR-HRP protein is expressed on the plasma membrane, the HRP substrate will be able to come into contact with HRP on the plasma membrane, and therefore the amount of chemiluminescence will be increased. In other words, in this evaluation system, an increase in the amount of chemiluminescence compared to the control will be evaluated as an improvement in the plasma membrane expression of the mutant CFTR protein.

[0057] Furthermore, the ability of the modified oligonucleotides of the present disclosure to improve plasma membrane expression of mutant CFTR protein may be confirmed, for example, by applying the modified oligonucleotides of the present disclosure to cells stably expressing a fusion protein of mutant CFTR and a HiBiT tag, and then quantifying the HiBiT-tagged protein expressed on the plasma membrane using a known reagent such as the Nano Glo HiBiT Extracellular Detection System (Promega).

[0058] 3-3. CFTR Channel Activity Improving Effect As shown in the Examples below, the modified oligonucleotides of the present disclosure can also enhance CFTR channel activity. This effect can be confirmed, for example, by measuring short-circuit current.

[0059] 3-4. Enhancement of the efficacy of CFTR modulating drugs As shown in the examples below, when the modified oligonucleotides of the present disclosure are used in combination with a CFTR modulating drug, such as the CF therapeutic agent Trikafta, they can exhibit a more potent effect on improving the plasma membrane expression of mutant CFTR protein than when the CFTR modulating drug is used alone. That is, the modified oligonucleotides of the present disclosure can enhance the efficacy of the CFTR modulating drug. This effect can be confirmed by comparing the efficacy of the combined use of the modified oligonucleotides of the present disclosure with the efficacy of the CFTR modulating drug when used alone.

[0060] 3-5. Other Properties As shown in the Examples below, the modified oligonucleotides of the present disclosure can exert a sustained RFFL knockdown effect over a long period of time. Furthermore, since the effect of improving plasma membrane expression of mutant CFTR protein and the effect of enhancing the efficacy of CFTR modulators are presumed to be due to RFFL knockdown, it is reasonably presumed that the modified oligonucleotides of the present disclosure can also exert these effects over a long period of time. Furthermore, as shown in the Examples below, the modified oligonucleotides of the present disclosure can have low cytotoxicity. Cytotoxicity can be confirmed by conventionally known methods. Furthermore, as shown in the Examples below, the modified oligonucleotides of the present disclosure can have low immunostimulatory properties. Immunostimulatory properties can be confirmed by conventionally known methods (e.g., TLR9 activity assays, etc.).

[0061] 4. Uses As described above, the modified oligonucleotides of the present disclosure have the effect of knocking down the ubiquitin ligase RFFL and / or improving the plasma membrane expression of mutant CFTR protein. Because CFTR gene mutations are the cause of cystic fibrosis (CF), improving the plasma membrane expression of CFTR contributes to the fundamental treatment of CF. Furthermore, the present inventors have previously found that knocking down the ubiquitin ligase RFFL can improve the plasma membrane expression of CFTR (Non-Patent Document 1). Therefore, the technology of the present disclosure is preferably used in the treatment of cystic fibrosis.

[0062] Furthermore, as described above, the modified oligonucleotides of the present disclosure may have the effect of enhancing the efficacy of CFTR modulating drugs. Therefore, the technology of the present disclosure is preferably used to enhance the efficacy of CFTR modulating drugs in combination therapy with CFTR modulating drugs. A specific example of the CFTR modulating drug is Trikafta (registered trademark), a triple combination drug consisting of elexacaftor, ivacaftor, and tezacaftor. Given the effect of knocking down the ubiquitin ligase RFFL, which induces degradation of mutant CFTR proteins, and improving plasma membrane expression of mutant CFTR proteins, it is reasonably expected that the technology of the present disclosure will also enhance the efficacy of CFTR modulating drugs other than Trikafta.

[0063] The technology of the present disclosure can be applied to, for example, humans and non-human mammals (e.g., rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.), with humans being preferred.

[0064] The technology of the present disclosure can be applied to subjects suffering from CR, particularly subjects with a mutation in which phenylalanine at position 508 in the CFTR amino acid sequence is deleted (ΔF508-CFTR). The technology of the present disclosure can also be effective for subjects with mutations other than ΔF508 (rare CFTR). Specific rare CFTR mutations include S492F, R560S, A561E, L1077P, G85E, W1282X, R347P, T70(Q1411X), and N1303K. In other words, subjects with these mutations are also preferred subjects for the technology of the present disclosure. Furthermore, subjects with CFTR mutations classified as class 6 in the CFTR classification, such as T70(Q1411X), are also preferred subjects for the technology of the present disclosure.

[0065] Furthermore, the ubiquitin ligase RFFL is involved in the ubiquitination-mediated degradation of the CFTR protein, and this degradation also occurs in the presence of Trikafta. Because the technology of the present disclosure can knock down RFFL and improve the plasma membrane expression of mutant CFTR protein, it can be preferably applied to subjects in whom the desired therapeutic effect has not been achieved with existing CFTR therapeutic agents such as Trikafta (elexacaftor, ivacaftor, and tezacaftor).

[0066] The method of application of the technology of the present disclosure is not particularly limited as long as the desired effect is achieved. For example, the modified oligonucleotide of the present disclosure or a composition containing the same may be made into a liquid or powder form and inhaled by a subject through the mouth and / or nose. Alternatively, the modified oligonucleotide of the present disclosure or a composition containing the same may be applied to the affected area, or may be administered orally, or may be administered by injection into or around the lesion, or may be administered by injection or infusion intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, or the like, or may be administered by perfusion via a catheter.

[0067] The frequency of application of the technology of the present disclosure is not particularly limited. For example, it may be applied once or multiple times a day, once or multiple times a week, once or multiple times a month, or once or multiple times a year. The application period of the technology of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. Furthermore, the dose of the modified oligonucleotide of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. The application frequency, application period, and dose can be adjusted appropriately by those skilled in the art depending on the condition of the subject, the course of treatment, etc.

[0068] The technology of the present disclosure may be optionally combined with other pharmaceutical compositions and / or treatment methods, etc., that are administered to a subject with CF. When the technology of the present disclosure is combined with other pharmaceutical compositions and / or treatment methods, etc., they may be administered to a subject simultaneously, or separately at any time. Furthermore, when the technology of the present disclosure is used in combination with an existing CF treatment, such as Trikafta, the technology of the present disclosure may be administered before, after, during, or simultaneously with the administration of the CF treatment.

[0069] In the technology of the present disclosure, the modified oligonucleotides of the present disclosure may be mixed with pharmaceutically acceptable bases, carriers, excipients, diluents, solubilizers, emulsifiers, preservatives, pH adjusters, adjuvants, chelating agents, etc., and these components may be used alone or in combination of two or more. That is, the present disclosure also encompasses compositions containing the modified oligonucleotides of the present disclosure. Such compositions may be referred to as "compositions of the present disclosure." The matters described with respect to the modified oligonucleotides of the present disclosure are incorporated by reference as appropriate into the compositions of the present disclosure, and the matters described with respect to the compositions of the present disclosure are incorporated by reference as appropriate into the modified oligonucleotides of the present disclosure.

[0070] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. The preservatives may be used alone or in combination of two or more.

[0071] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphate, acetic acid, nitric acid, and chemically possible salts thereof, as well as sodium hydroxide, potassium hydroxide, etc. pH adjusters can be used alone or in combination of two or more types so that the pH of a composition containing the modified oligonucleotide of the present disclosure is in the range of 4 to 8, preferably 5 to 7.

[0072] The method for preparing a composition containing the modified oligonucleotide of the present disclosure is not particularly limited as long as the effects of the present disclosure are achieved. For example, the composition can be prepared according to a method known in the art. More specifically, the composition can be prepared by mixing the modified oligonucleotide of the present disclosure and other components with sterile distilled water.

[0073] The present disclosure also encompasses a kit for treating cystic fibrosis, comprising a modified oligonucleotide of the present disclosure and elexacaftor, ivacaftor, or tezacaftor. The kit for treating cystic fibrosis may be referred to as the "kit of the present disclosure." The matters described with respect to the modified oligonucleotide of the present disclosure and the composition of the present disclosure are incorporated by reference into the kit of the present disclosure, as appropriate, and the matters described with respect to the kit of the present disclosure are incorporated by reference into the modified oligonucleotide of the present disclosure and the composition of the present disclosure, as appropriate.

[0074] The timing of administration of the modified oligonucleotides of the present disclosure, as well as elexacaftor, ivacaftor, and tezacaftor contained in the kit of the present disclosure, is not particularly limited as long as the desired effect is achieved. For example, all components may be administered simultaneously, or each component may be administered at a different time, or two or three components may be administered simultaneously with the remaining components administered at different times. When not all components are administered simultaneously, the interval between each administration is not limited as long as the desired effect is achieved, and the order of administration is also not limited. Furthermore, administration of any component may be repeated until the desired effect is achieved. Furthermore, the administration routes of each component may be the same or different.

[0075] 5. Possible Mechanism: While not wishing to be bound by any theory, it is believed that the modified oligonucleotides disclosed herein exert their function on CFTR mutants via the following mechanism: Because the modified oligonucleotides disclosed herein have a base sequence complementary to a portion of RFFL mRNA, they form base pairs with RFFL mRNA. As a result, RFFL mRNA is cleaved by a nuclease, reducing the level of RFFL mRNA and the amount of its translation product, RFFL protein (RFFL knockdown). The reduction in RFFL protein suppresses the ubiquitination of mutant CFTR protein, which in turn suppresses the degradation of mutant CFTR protein that occurs following ubiquitination. As a result, the mutant CFTR protein that remains undegraded is translocated to the plasma membrane (improving plasma membrane expression of mutant CFTR protein).

[0076] The inventors have previously confirmed that siRNAs that effectively knocked down RFFL in CFBE cells failed to sufficiently knock down RFFL in the CF-HBE model, the final evaluation system in preclinical trials (data not shown). As demonstrated in the Examples below, the modified oligonucleotides of the present disclosure can also knock down RFFL and improve mutant CFTR function in the CF-HBE model. While the reason for this is unclear, it is possible that the introduction of modified nucleosides with specific structures and / or the selection of base sequences contributed to the effects in the CF-HBE model. More specifically, the introduction of modified nucleosides with specific structures and / or the selection of base sequences may have improved nuclease resistance compared to conventional siRNAs and / or base-specific hybridization to RFFL mRNA, resulting in RFFL knockdown and mutant CFTR function improvement in the CF-HBE model.

[0077] Furthermore, because siRNA has low resistance to nucleases, when siRNA is introduced into cells, it is usually necessary to protect the siRNA from nucleases by, for example, encapsulating it in liposomes (liposomalization). However, liposomes are known to be cytotoxic. The administration of the modified oligonucleotides of the present disclosure does not require the use of such liposomes, which can be said to be another advantage of the modified oligonucleotides of the present disclosure. In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure encompasses any combination of the constituent features described herein.

[0078] Furthermore, the various characteristics (numerical values, properties, structures, functions, etc.) described in each embodiment of the present disclosure may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0079] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0080] 1.1-1. Design of Modified Oligonucleotides The present inventors first attempted to design modified oligonucleotides capable of effectively knocking down RFFL. Specifically, 28 base sequences complementary to human RFFL mRNA were selected using a proprietary algorithm that took into account factors such as toxicity risk, GC content, ease of dimer formation, and risk of off-target effects. The three bases from the 5' end and the second and third bases from the 3' end of the selected base sequence were modified nucleosides represented by the general formula (I) above, and the remaining bases were deoxyribonucleosides. Furthermore, all internucleoside linkages were phosphorothioate. The human RFFL mRNA sequence (SEQ ID NO: 39) is registered in the NCBI database RefSeq (https: / / www.ncbi.nlm.nih.gov / refseq / ) under accession number NM_001017368.2.

[0081] 1-2. Quantification of RFFL knockdown effect The modified oligonucleotides designed as described in 1-1 were obtained using GeneDesign's modified oligonucleotide synthesis service. The RFFL knockdown effect of the modified oligonucleotides was quantified using the following method.

[0082] CFBE41o- cells (hereafter simply referred to as "CFBE cells" or "CFBE") were transfected with 20 nM of modified oligonucleotides using Lipofectamine RNAiMax (Thermo Fisher Scientific). CFBE cells are a CF human bronchial epithelial cell line derived from a CF patient homozygous for the ΔF508-CFTR mutation. Transfection was performed according to the recommended Lipofectamine RNAiMax protocol. After culturing the cells for 24 hours, mRNA was collected. Endogenous RFFL mRNA was quantified by quantitative RT-PCR using the mRNA. The results are shown in Figure 1. This study confirmed that the modified oligonucleotides listed in the table below have excellent RFFL knockdown effects in CFBE cells.

[0083]

[0084] In this example, all bases that do not constitute a modified nucleoside represented by any of general formulas (I) to (III) constitute a deoxyribonucleoside. Furthermore, when the base that constitutes a modified nucleoside represented by any of general formulas (I) to (III) is a base represented by C, the C represents 5-methylcytosine. On the other hand, when the base that constitutes a deoxyribonucleoside is a base represented by C, the C represents cytosine. In this example, all Ts are thymine.

[0085] 1-3. Measurement of ΔF508-CFTR plasma membrane expression levels by HRP assay. CFBE cells stably expressing ΔF508-CFTR-HRP were transfected with the modified oligonucleotides shown in 1-2 to have RFFL knockdown effects. The expression levels of ΔF508-CFTR-HRP protein at the plasma membrane were measured.

[0086] Specifically, CFBE cells stably expressing ΔF508-CFTR-HRP were seeded in 96-well plates and transfected with 20 nM of modified oligonucleotides using Lipofectamine RNAiMax (Thermo Fisher Scientific). Transfection was performed according to the recommended Lipofectamine RNAiMax protocol. After transfection, the cells were cultured at 37°C for 3 days, followed by 26°C for 2 days to promote plasma membrane expression of ΔF508-CFTR-HRP protein. After incubation at 37°C for 1 hour, HRP substrate (Super Signal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was added to each well, and the chemiluminescence intensity of the HRP substrate was measured using a plate reader (Varioskan, Thermo Fisher Scientific). As can be seen from this test system, the higher the measured chemiluminescence intensity, the greater the plasma membrane expression of ΔF508-CFTR-HRP protein. The results are shown in Figure 2. Figure 2 shows the relative chemiluminescence intensity (i.e., the plasma membrane expression level of ΔF508-CFTR-HRP protein) of each cell, relative to the chemiluminescence intensity of mock cells transfected in the same manner but without modified oligonucleotides (mock). Experiments were performed in triplicate, and significance was tested by one-way ANOVA (Dunnett's method). * p<0.05, ** p<0.01, *** p<0.001.

[0087] As shown in Figure 2, the expression level of ΔF508-CFTR-HRP protein at the plasma membrane was increased in CFBE cells transfected with the modified oligonucleotides compared to mock-transfected cells. These results suggest that the modified oligonucleotides tested in this study have the effect of improving the plasma membrane expression of mutant CFTR protein.

[0088] 1-4. Evaluation of alamarBlue Cytotoxicity. Next, we evaluated the cytotoxicity of the modified oligonucleotides tested in 1-3. Specifically, after measuring the chemiluminescence intensity in 1-3, the cells on the 96-well plate were washed with PBS(-) and incubated in medium containing alamarBlue cell viability detection reagent (Thermo Fisher Scientific) for 2 hours at 37°C. The fluorescence intensity of alamarBlue was then quantified using a plate reader (Varioskan, Thermo Fisher Scientific). The alamarBlue cell viability detection reagent contains resazurin as its active ingredient. When added to cells, the reagent turns red and emits strong fluorescence due to the reducing environment of live cells. Therefore, the more live cells present in the system, the stronger the fluorescence detected. Each experiment was performed with n = 3, and significance was tested using one-way ANOVA (Dunnett's method). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0089] The results are shown in Figure 3. Figure 3 shows the relative fluorescence intensity (i.e., relative cell viability) of each cell, with the mock fluorescence intensity set to 100%. As shown in Figure 3, all cells maintained a cell viability of 50% or more. In particular, ASO20 and ASO21 maintained a cell viability comparable to that of the mock. These results suggest that the modified oligonucleotides tested in this study all had relatively low cytotoxicity, with ASO20 and ASO21 having particularly low cytotoxicity.

[0090] 2.2-1. Design of Modified Oligonucleotides Based on the results of Study 1, the inventors attempted to modify ASO20 to obtain modified oligonucleotides with superior activity. As described above, the modified oligonucleotides evaluated in Study 1 have a nucleotide sequence complementary to a portion of human RFFL mRNA. The inventors believed that hybridization of modified oligonucleotides near the sequence complementary to ASO20 in RFFL mRNA would be important for RFFL knockdown and improved plasma membrane expression of mutant CFTR protein. Therefore, modified oligonucleotides were designed that have a nucleotide sequence complementary to a sequence several bases away from the sequence complementary to ASO20 in RFFL mRNA. Furthermore, to further reduce cytotoxicity, modified oligonucleotides were designed in which some of the phosphorothioate linkages in ASO20 were replaced with phosphodiester linkages (ASO36-38 shown in the table below).

[0091] 2-2. Quantification of RFFL knockdown effect The modified oligonucleotides designed as described in 2-1 were obtained using GeneDesign's modified oligonucleotide synthesis service. The RFFL knockdown effect of the modified oligonucleotides was quantified using the method described in 1-2. The results are shown in Figure 4. This test confirmed that the modified oligonucleotides shown in the table below have excellent RFFL knockdown effect.

[0092]

[0093] 2-3. Measurement of ΔF508-CFTR Plasma Membrane Expression Levels by HRP Assay. As in 1-3, CFBE cells stably expressing ΔF508-CFTR-HRP were transfected with each modified oligonucleotide, and the expression levels of ΔF508-CFTR-HRP protein at the plasma membrane were measured. The results are shown in Figure 5. Each experiment was performed in triplicate, and significance was tested using one-way ANOVA (Dunnett's method). * p<0.05, ** p<0.01, **** p<0.0001. As shown in Figure 5, CFBE cells transfected with the modified oligonucleotides listed in the table in 2-2 showed increased plasma membrane expression of ΔF508-CFTR-HRP protein compared to mock. These results suggest that the modified oligonucleotides evaluated in this study have the effect of improving plasma membrane expression of mutant CFTR protein in CFBE cells.

[0094] 2-4. Evaluation of Cytotoxicity Using AlamarBlue As in 1-4, the inventors evaluated the cytotoxicity of the modified oligonucleotides tested in 2-3. The results are shown in Figure 6. The test was performed in triplicate. As shown in Figure 6, all cells maintained high cell viability. These results suggest that all modified oligonucleotides tested in this study have low cytotoxicity.

[0095] 3.3-1. Design of modified oligonucleotides Using a proprietary algorithm updated from 1-1, the present inventors designed new modified oligonucleotides targeting human RFFL mRNA. We also designed modified oligonucleotides (ASOs 36-38) by changing the number and position of phosphodiester bonds or the type of modification of the modified nucleoside (ASOs 70-72, shown in the table below).

[0096] 3-2. Measurement of ΔF508-CFTR plasma membrane expression in combination with Trikafta. As in 1-3, CFBE cells stably expressing ΔF508-CFTR-HRP were transfected with each modified oligonucleotide, and the expression level of ΔF508-CFTR-HRP protein at the plasma membrane was measured. However, unlike 1-3, where plasma membrane expression of ΔF508-CFTR-HRP protein was promoted by incubation at low temperature (26°C), in this study, plasma membrane expression of ΔF508-CFTR-HRP protein was promoted by incubation at 37°C for 2 days with the CF therapeutic agent Trikafta (3 μM VX-661 (tezacaftor), 1 μM VX-445 (elexacaftor), and 1 μM VX-770 (ivacaftor)). In addition, cells treated with the negative control modified oligonucleotide (NEG#8) were used for comparison in the following experiments. NEG#8 is a modified oligonucleotide that has been previously confirmed to have no effect on RFFL and CFTR expression and to have low cytotoxicity. The structure of NEG#8 is shown in the table below. The results are shown in Figure 7. The test was performed on n=9, and significance was tested using one-way ANOVA (Dunnett's method). * p<0.05, **** p<0.0001.

[0097]

[0098] 3-3. Evaluation of cytotoxicity using alamarBlue As in 1-4, the cytotoxicity of the modified oligonucleotides tested in 3-2 was evaluated. The results are shown in Figure 8. The test was performed with n=9.

[0099] 3-4. The results shown in Figure 7 demonstrate that the modified oligonucleotides shown in the table below, when used in combination with the CF therapeutic agent Trikafta, have a superior effect on improving the plasma membrane expression of mutant CFTR protein compared to Trikafta alone (note that ASO20 shown in the table below is the same as that shown in Table 1-2, and ASO37 is the same as that shown in Table 2-2). These modified oligonucleotides were therefore suggested to have the effect of enhancing the efficacy of the CF therapeutic agent Trikafta. Furthermore, as shown in Figure 8, these modified oligonucleotides had low cytotoxicity.

[0100]

[0101] On the other hand, as shown in Figure 7, for example, ASO23, ASO73, and ASO74 shown in the table below did not show any potentiating effect on the efficacy of Trikafta.

[0102]

[0103] Based on these results, the inventors considered ASO37 to be a promising candidate for a novel CF treatment. Specifically, ASO37 was evaluated to have particularly excellent RFFL knockdown activity, improved plasma membrane expression of mutant CFTR protein, and enhanced efficacy of Trikafta, while also exhibiting low cytotoxicity. Therefore, we decided to further evaluate the function of ASO37.

[0104] 4. Evaluation of RFFL knockdown durability. CFBE cells stably expressing ΔF508CFTR-3HA were transfected with 20 nM ASO37 using Lipofectamine RNAi Max Transfection Reagent (Thermo Fisher Scientific). Six, 10, and 14 days after transfection, cell lysates were prepared using RIPA buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton-X100, 0.5% sodiumdeoxycholate, 5 μg / ml pepstatin, 5 μg / ml leupeptin, 1 mM PMSF), and RFFL expression was quantified by Western blotting. Anti-RFFL antibody (HPA019492, Sigma-Aldrich) was used for Western blotting. RFFL protein levels were quantified by densitometry, and relative values ​​were calculated, with the RFFL protein level in NEG#8 at each time point set at 100%. The results are shown in Figure 9. The test was performed with n=6, and significance was tested by student t-test. * p<0.05, ** p<0.01, **** p<0.0001.

[0105] As shown in Figure 9, ASO37 maintained a significant RFFL knockdown effect even 14 days after transfection, suggesting that ASO37 may have a long-lasting efficacy in the treatment of CF.

[0106] 5. Evaluation of CFTR Channel Function Using the CF-HBE Model In the previously described experiments 1–4, CFBE cells were used to evaluate modified oligonucleotides. However, we previously confirmed that siRNAs that effectively knocked down RFFL in CFBE cells failed to sufficiently knock down RFFL in the CF-HBE model, the final evaluation system for preclinical studies (data not shown). Therefore, in this study, we evaluated the function of ASO37 using the CF-HBE model. Specifically, we used a CF-HBE model prepared from primary cultured airway epithelial cells (CF-HBE, The University of North Carolina at Chapel Hill, MLI Tissue Procurement and Cell Culture Core Facility) derived from three CF patients with homozygous ΔF508-CFTR mutations (KKD008N, KKD019N, KKD012K).

[0107] 5-1. Preparation of the CF-HBE Model. First, CF-HBE cells were seeded onto Snapwell filter inserts (0.4 μm pore size, Polyester, Corning, catalog no. 3801) pre-coated with human placental collagen type VI (Sigma, C5533-5MG) and cultured at the liquid-liquid interface until confluent. They were then cultured at the air-liquid interface for 28 days using air-liquid interface differentiation medium to differentiate into airway epithelial cells. The medium was changed three times weekly, and the apical side was washed once weekly with sterile PBS (Thermo Fisher Scientific) at 37°C and 5% CO2 for 10 minutes. The differentiated CF-HBE cells on the Snapwell filter inserts (CF-HBE model) were then used for transfection and evaluation, as described below.

[0108] The apical mucus layer was removed 4 days before transfection. Specifically, 250 μL of PBS containing 3 mM dithiothreitol (DTT, FUJI FILM Wako Pure Chemical) was added to the apical surface, incubated for 30 minutes, and then the solution on the apical surface was aspirated to remove the mucus layer. The apical mucus layer was also removed in the same manner the day before transfection.

[0109] 5-2. Transfection Transfection was performed using the free uptake method using a hypotonic solution (PBS) with appropriate modifications to the method described in Non-Patent Document 4. Unlike the lipofection-based transfection performed in Experiments 1 to 4, this method does not use a transfection reagent. Specifically, the apical surface of the CF-HBE model prepared as described above was first transfected with 40 μM of modified oligonucleotides (ASO37 or NEG#8) in 250 μL of hypotonic solution (50% phosphate-buffered saline (PBS, FUJI FILM Wako Pure Chemical)) for 1 hour. After 1 hour, the solution was removed, and the model was again transfected with 250 μL of DPBS containing 40 μM of modified oligonucleotides (ASO37 or NEG#8). Four days after the second transfection, the Cl of CFTR was detected. - Channel function was evaluated. At this point, the transfection solution was completely absorbed by the apical surface.

[0110] 5-3. Short circuit current measurement Cl -Channel function was assessed by the short-circuit current assay described below. For some samples, Trikafta (18 μM VX-661, 3 μM VX-445, and 1 μM VX-770) was applied to the basolateral side 2 days before short-circuit current measurements. For short-circuit current measurements, Snapwell inserts containing CF-HBEs were placed in Ussing chambers (U-2500, Warner Instruments) filled with Krebs-bicarbonate Ringer (KBR) buffer (115 mM NaCl, 19.8 mM NaHCO3, 5.2 mM KHCO3, 2.4 mM Na2HPO4, 0.4 mM NaH2PO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 5 mM glucose, pH 7.4) preheated to 37°C. The KBR buffer was adjusted to pH 7.4 in advance by continuous bubbling with a mixed gas (95% O2, 5% CO2). - To create a concentration gradient, Cl was added to the apical chamber. - Cl replaced with gluconic acid - Free KRB buffer (115 mM D-gluconatesodium salt, 19.8 mM NaHCO3, 5.2 mM KHCO3, 2.4 mM Na2HPO4, 0.4 mM NaH2PO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 5 mM glucose, pH 7.4) was added.

[0111] Short-circuit current measurements were performed at 37°C using a short-circuit current measurement device (CEZ-9100, Nihon Kohden) and a Power Lab 2 / 26 system (AD Instruments). First, 100 μM amiloride (TCI) was added apically to inhibit the epithelial sodium channel (ENaC), and short-circuit current was measured for approximately 3–4 min until the effect plateaued. Next, 10 μM forskolin (FUJI FILM Wako Chemicals) was added apically to activate CFTR, and short-circuit current was measured for approximately 4–5 min until the effect plateaued. Subsequently, CFTR inhibitor-172 (20 μM, Selleck Chemicals) was added apically, and short-circuit current was measured for approximately 4–5 min until the effect plateaued. The current inhibited by CFTR inhibitor-172 treatment was quantified as CFTR channel activity. After short-circuit current measurements, mRNA was collected from CF-HBEs, and RFFL mRNA levels were quantified by quantitative RT-PCR.

[0112] 5-4. Results The results are shown in Figure 10. Significance tests were performed using paired t-tests. * p<0.05, ** p<0.01. In Figure 10, NEG#8 + Trikafta represents a CF-HBE model transfected with NEG#8, then Trikafta was administered, and short-circuit current measurements were then performed. ASO37 + Trikafta represents a CF-HBE model transfected with ASO37, then Trikafta was administered, and short-circuit current measurements were then performed. The control represents an evaluation using a CF-HBE model that was neither transfected nor treated with Trikafta (i.e., untreated). As mentioned above, NEG#8 is a modified oligonucleotide that has been previously confirmed to have no effect on RFFL and CFTR expression and to have low cytotoxicity. Therefore, the results of NEG#8 + Trikafta in Figure 10 can be interpreted as representing the effect of Trikafta alone.

[0113] The left panel (ΔF508-CFTR function) shows CFTR channel activity quantified by short-circuit current measurement, expressed relative to the value measured with NEG#8 + Trikafta (100%). The right panel (RFFL mRNA) shows RFFL mRNA levels quantified after short-circuit current measurement, expressed relative to the value measured with NEG#8 + Trikafta (100%). As shown on the left side of Figure 10, NEG#8 + Trikafta significantly enhanced CFTR channel activity compared with the control. This result is consistent with previous findings that Trikafta promotes translocation of ΔF508-CFTR protein to the plasma membrane and further enhances CFTR channel activity. On the other hand, ASO37 + Trikafta demonstrated significantly higher CFTR channel activity than NEG#8 + Trikafta. These results suggest that ASO37 enhances the efficacy of Trikafta.

[0114] The results of RFFL mRNA levels shown on the right side of Figure 10 demonstrate that ASO37 also exerts an RFFL mRNA knockdown effect in the CF-HBE model. Furthermore, the RFFL mRNA levels in NEG#8 + Trikafta and the control were similar, confirming again that NEG#8 does not affect RFFL expression.

[0115] 5-5. Summary As described above, the present inventors have previously confirmed that siRNAs that effectively knocked down RFFL in CFBE cells failed to sufficiently knock down RFFL in the CF-HBE model, the final evaluation system in preclinical trials. Through this study, the present inventors found that the modified oligonucleotides of the present disclosure can effectively knock down RFFL even in the CF-HBE model, where conventional techniques were unable to sufficiently knock down RFFL. Furthermore, the present inventors have also found that the modified oligonucleotides of the present disclosure can further enhance the efficacy of the CF therapeutic agent Trikafta.

[0116] 6. Examination of the Effect of Modified Oligonucleotides on Improving Plasma Membrane Expression of Rare CFTR Mutants In the above-described Tests 1 to 5, the effect on the ΔF508-CFTR mutant was evaluated. However, there are many other CFTR genetic mutations that cause CF besides the ΔF508 mutation (although, as already mentioned, approximately 90% of CF patients have the ΔF508 mutation). Therefore, the inventors investigated whether the modified oligonucleotides disclosed herein are also effective against CFTR mutants other than the ΔF508-CFTR mutant (rare CFTR mutants).

[0117] Specifically, the airway epithelial cell line BEAS-2B stably expressing fusion proteins of each rare CFTR variant with an extracellular HiBiT tag (rare CFTR-HiBiT(Ex)) was seeded in a 96-well plate. The cells were transfected with 20 nM NEG#8 or ASO37 using Lipofectamine RNAiMax (Thermo Fisher Scientific). Transfection was performed according to the Lipofectamine RNAiMax recommended protocol. The medium was replaced the day after transfection and cultured at 37°C for 2 days. The medium was then replaced with dimethyl sulfoxide (DMSO) or the CF treatment Trikafta (3 μM VX-661 (tezacaftor), 1 μM VX-445 (elexacaftor), and 1 μM VX-770 (ivacaftor)), and the cells were cultured for an additional 2 days at 37°C.

[0118] Two days later, the plasma membrane expression of rare CFTR-HiBiT was measured using the Nano Glo HiBiT Extracellular system (Promega) with a Luminoskan and VarioskanFlash microplate reader (Thermo Fisher Scientific). The results are shown in Figures 11 to 13. Tests were performed on n = 5 to 6, and significance was tested by two-way ANOVA (Holm-Sidak multiple comparison). Significant interactions were detected by P int* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Each result is shown as a relative value, with the measurement value for NEG#8 transfection and DMSO addition set at 100%. The CFTR mutant type is indicated in the upper left of each graph. In these test results, "NEG" represents NEG#8, "3074B" represents ASO37, and "TRI" represents Trikafta addition.

[0119] For all rare CFTR mutants tested, ASO37 transfection with Trikafta (ASO37 + Trikafta) significantly increased CFTR plasma membrane expression compared with NEG#8 transfection with Trikafta (NEG#8 + Trikafta). In particular, for the R560S mutant (serine at position 560 is replaced by arginine) shown in Figure 11, Trikafta alone did not significantly improve CFTR plasma membrane expression (compared with NEG#8 + DMSO and NEG#8 + Trikafta). However, the combination of Trikafta with ASO37 significantly improved CFTR plasma membrane expression. Furthermore, for the R347P, T70(Q1411X), and N1303K mutants shown in Figure 13, ASO37 alone without Trikafta significantly improved CFTR plasma membrane expression.

[0120] 7. The effect of modified oligonucleotides encompassed by the present disclosure on improving CFTR plasma membrane expression (percentage of CFTR expression in the control) evaluated in Sub-Studies 1 to 3 is summarized in the table below. That is, the data shown in the table below corresponds to the data shown in Figures 2, 5, and 7. Note that "control" here refers to mock in Tests 1 and 2, and NEG#8 in Test 3.

[0121]

[0122]

[0123] 8. TLR9 activity assay and cell viability assessment: To evaluate whether ASO37 induces TLR9 (toll-like receptor 9)-mediated immune responses, a TLR9 activity assay was performed using HEK-Blue hTLR9 cells. Furthermore, cell viability was assessed to confirm the cytotoxicity of ASO37.

[0124] Specifically, 5 × 10 HEK-Blue hTLR9 cells (Invivogen) were cultured at 4 °C for 1 min. 4 Cells were seeded at a density of 100 cells / well into 96-well plates (Corning) and cultured in DMEM medium supplemented with HEK-Blue Detection (Invivogen). Three wells of cells per condition were immediately stimulated with ASO37, the positive control SY-ODN18 (an 18-nucleotide phosphorothioate oligodeoxynucleotide known to have TLR9-stimulating activity; see Non-Patent Document 5), or the negative control water for 18 hours. SEAP activity in the medium, an indicator of NF-κB activity induced by TLR9 activation, was then determined for each well by measuring absorbance at 650 nm using an ARVO X3 microplate reader (PerkinElmer). The obtained TLR9 activity values ​​(n = 3 wells) were normalized based on the negative control value, and the results were calculated as the mean and standard deviation (SD).

[0125] Cell viability was assessed by WST-8 assay (Cell Counting Kit-8, Dojindo Laboratories) according to the manufacturer's protocol. The obtained data (n = 3 wells) were normalized to the negative control value, as were the TLR9 activity values, and the results were calculated as the mean and standard deviation (SD).

[0126] The results are shown in Figure 14. In Figure 14, "3074B" represents ASO37. The positive control, SY-ODN18, increased TLR9 activity in a concentration-dependent manner. In contrast, ASO37 did not increase TLR9 activity at any concentration (Figure 14, left). Furthermore, regardless of the concentration of ASO37 added, cell viability remained nearly 100%, and no cytotoxicity was observed (Figure 14, right).

[0127] These results demonstrate that ASO37 does not have TLR9-mediated immunostimulatory activity and is not cytotoxic, suggesting its safety.

Claims

1. A modified oligonucleotide or a salt thereof, wherein the modified oligonucleotide has a ubiquitin ligase RFFL knockdown activity and is selected from the group consisting of TAGTGGTTG, GGGTTGGAATAGGC (SEQ ID NO: 2), CCTTGTATAGCCGG (SEQ ID NO: 3), CCTGATGGTACTG (SEQ ID NO: 4), TAGGCACC, GCCGGGTCACTCTCT (SEQ ID NO: 6), AGTCCTTTCTGATCC (SEQ ID NO: 7), CCGTTTTGGTCTTCG (SEQ ID NO: 8), TTGGGTACAGGAGGA (SEQ ID NO: 9), GCGTTTAGGGTTTGC (SEQ ID NO: 10), AAAGGCTGTGGTTCA (SEQ ID NO: 11), ATTCCCCCAACTCCT (SEQ ID NO: 12), ACCATTCAGCCTTGC (SEQ ID NO: 13), AAAGGGGTTTAGTGGTT (SEQ ID NO: 14), TTTCTGAGCCATAGTCCA (SEQ ID NO: 15), GACTTAACAAACAGGCAC (SEQ ID NO: 16), CTGACCTACATAGATTTC (SEQ ID NO: 17), GCCTTGCTTCAGTATTAG (SEQ ID NO: 18), The nucleic acid sequence of the present invention is 21 bases or less in length and contains GTCAGTTCTCACCTTATG (SEQ ID NO: 19) or GTCCCAATCCTTCCAGCT (SEQ ID NO: 20), and has at least two modified nucleosides within five bases from the 5'-end and at least one modified nucleoside within five bases from the 3'-end, wherein the modified nucleosides are each independently represented by one of the general formulas (I) to (III): (wherein Base represents a base) or a salt thereof.

2. The modified oligonucleotide or a salt thereof according to claim 1, wherein the internucleoside bonds contained in the modified oligonucleotide are, independently of each other, phosphodiester bonds or phosphorothioate bonds.

3. The modified oligonucleotide or salt thereof according to claim 1 or 2, wherein the modified nucleoside is a modified nucleoside represented by general formula (I).

4. A modified oligonucleotide or a salt thereof, wherein the modified oligonucleotide is a modified oligonucleotide consisting of [G][G][T]TTAGTGGTT[G][G]T (SEQ ID NO: 21), and / or a modified oligonucleotide consisting of a base sequence in which 1 to 3 bases are added, deleted, or substituted in the base sequence of SEQ ID NO: 21, and has the activity of knocking down ubiquitin ligase RFFL, wherein the bases enclosed in [ ] are those represented by general formula (I): (wherein Base represents a base), wherein the internucleoside bonds contained in the modified oligonucleotide are independently phosphodiester bonds or phosphorothioate bonds and contain 0 to 6 phosphodiester bonds, or a salt thereof.

5. A composition for use in treating cystic fibrosis, comprising the modified oligonucleotide or salt thereof according to claim 1, 2, or 4.

6. The composition of claim 5, wherein the treatment comprises administration of elexacaftor, ivacaftor, and tezacaftor.

7. A kit for treating cystic fibrosis, comprising the modified oligonucleotide or salt thereof according to claim 1, 2, or 4, and elexacaftor, ivacaftor, or tezacaftor.

8. The composition described in claim 5 for use in a subject having a mutation in which phenylalanine at position 508 in the CFTR amino acid sequence is deleted.

9. The composition described in claim 6 for use in a subject having a mutation in which phenylalanine at position 508 in the CFTR amino acid sequence is deleted.

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