Pharmaceutical composition for preventing or treating Avellino corneal dystrophy using antisense oligonucleotides (ASO)

Abstract

The present invention relates to: a pharmaceutical composition of an antisense oligonucleotide, which induces inhibition of the expression of an R124H mutant of a TGFBI protein for the prevention or treatment of Avellino corneal dystrophy; a use of the composition; or methods of treatment thereof. In particular, the present invention selectively acts on R124H TGFBI mRNA (R124H TGFBI mRNA is a precursor of R124H TGFBI protein causing Avellino corneal dystrophy), thereby inducing inhibition of R124H TGFBI expression while minimizing the effect on WT TGFBI expression, and thus can very effectively prevent or treat Avellino corneal dystrophy.

Description

Technical Field

[0001] The present disclosure relates to an ASO pharmaceutical composition for preventing or treating Avellino corneal dystrophy, wherein the ASO pharmaceutical composition inhibits the expression of R124H mutant TGFBI (pathogenic protein); uses thereof; and methods of treating the same. Background Art

[0002] The cornea is a vital organ in the eye, ensuring vision and maintaining vision. Maintaining a clean cornea is crucial, as a cloudy cornea directly leads to decreased vision and difficulty in daily life. Conditions in which the cornea becomes cloudy due to the accumulation of opaque deposits and vision deteriorates are called corneal dystrophies. Symptoms of these conditions typically worsen with age.

[0003] Among corneal dystrophies, granular corneal dystrophy is a slowly progressive corneal abnormality that usually begins in infancy and gradually causes corneal clouding, leading to severe vision loss after age 60. It is also called granular corneal degeneration, granular corneal dystrophy, or granular corneal dystrophy. There are two types of granular corneal dystrophy. Type 1 granular corneal dystrophy is a very rare form that was first described in Germany by Arthur Groenouw in 1890. Type 2 granular corneal dystrophy (Avellino corneal dystrophy), also a very rare form of corneal dystrophy, was first described by Folberg in 1988 and named after the first four patients who developed the condition, originating from the Avellino region of Italy.

[0004] Granular corneal dystrophy is known to be caused by point mutations in the transforming growth factor-induced (TGFBI) gene located on chromosome 5q31. The R124H mutation (R124HTGFBI), in which arginine at position 124 of the TGFBI protein is replaced with histidine, is one of the most commonly observed mutations and causes type 2 granular corneal dystrophy, also known as Avellino dystrophy.

[0005] TGFBI is a 68kDa extracellular matrix protein composed of 683 amino acids that is primarily involved in cell adhesion, migration, and differentiation. It is primarily expressed in corneal fibroblasts (keratocytes) and corneal epithelial cells, where it interacts with the collagen component of the corneal stroma and plays a role in corneal wound healing and the construction of the extracellular matrix. However, unlike the normal protein, mutant forms of TGFBI do not fold correctly in three dimensions. As a result, they readily aggregate and resist degradation, leading to the accumulation of opaque, insoluble deposits in the corneal stroma or Bowman's membrane, thus causing granular corneal dystrophy.

[0006] Granular corneal dystrophy type 2 (GCD 2), also known as Avellino corneal dystrophy, is an autosomal dominant inherited disorder, and approximately 1 in 870 people in Korea carry the R124H TGFBI mutation. Most patients are heterozygous for this mutation, and the prevalence and severity of symptoms increase with age. Homozygous mutants are extremely rare (approximately 1 in 1,000 or 1 in 10,000, compared to heterozygous mutants) and typically present in childhood with more severe symptoms than heterozygous individuals.

[0007] Current treatments include corneal transplantation and superficial keratectomy to remove the area of ​​the cornea where deposits have formed. While corneal transplantation can potentially maintain a cloudiness-free state for extended periods, it is a highly invasive procedure that requires a donor and carries the risk of recurrence. Keratectomy is less invasive and can help temporarily preserve vision until corneal transplantation, but repeat surgery is necessary to maintain corneal clarity. This repeated treatment often leads to presbyopia due to a decrease in refractive index, and the risk of recurrence and progression is high due to genetic predisposition.

[0008] Various research efforts have been directed toward developing therapeutic agents, but no successful treatment has been achieved to date. For example, KR10-1370659 aims to reduce protein deposition through autophagy induced by rapamycin or melatonin, and KR 10-1394538 attempts to use vitamin D3 to reduce TGFBI protein expression. US2008 / 0267946 attempts to use a TGF-β antibody in patients with Avellino corneal dystrophy to alleviate symptoms exacerbated by TGF-β activation caused by intense light exposure (e.g., ultraviolet light).

[0009] However, these attempts have not yet led to successful drug development, and their mechanism of action is inherently limited, as it is not directly related to the pathogenic R124H TGFBI. In particular, once the R124H TGFBI protein aggregates in the cornea, it is difficult to dissolve or remove with drugs. Even surgery can trigger new aggregation. Therefore, a more effective strategy would be to prevent pathogenic protein aggregation at an early stage by specifically inhibiting R124H TGFBI.

[0010] Antisense oligonucleotides (ASOs) are single-stranded DNA-based oligonucleotides that specifically bind to mRNA with a complementary sequence, thereby suppressing the expression of the target protein by recruiting an RNase called RNase H (which degrades mRNA) or by blocking translation into protein. The present disclosure aims to provide a method for fundamentally treating or preventing Avellino corneal dystrophy induced by R124H TGFBI using an ASO that specifically inhibits the expression of R124H TGFBI, the pathogenic protein of Avellino corneal dystrophy. Summary of the Invention Technical Purpose

[0011] The present disclosure aims to provide a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, which uses ASO to inhibit R124H TGFBI expression; its use; and its method of treatment.

[0012] In addition, another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, which uses ASO to selectively inhibit R124H mutant TGFBI while minimizing the effect on the expression of normal TGFBI (wild-type TGFBI); its use; and its treatment method. Technical Solution

[0013] To achieve the above objectives, the present disclosure provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising an antisense oligonucleotide having a nucleotide sequence that inhibits the expression of R124H mutant TGFBI.

[0014] Furthermore, the present disclosure provides use of an antisense oligonucleotide having a nucleotide sequence that inhibits the expression of TGFBI having an R124H mutation as an agent for preventing or treating Avellino corneal dystrophy.

[0015] Furthermore, the present disclosure provides a method for preventing or treating Avellino corneal dystrophy, comprising administering an antisense oligonucleotide having a nucleotide sequence that inhibits expression of R124H mutant TGFBI. Beneficial effects

[0016] The development of gene therapy is essential for the effective treatment of Avellino corneal dystrophy, a genetic disease with a high unmet medical need. In particular, once the pathogenic R124H TGFBI protein accumulates in the cornea, it is difficult to dissolve or remove it with drugs, and even surgery may trigger new aggregations. Therefore, to block the pathogenic mechanism, the present disclosure provides a technical advantage by using R124H TGFBI-specific antisense oligonucleotides that act at the mRNA stage before the R124H TGFBI protein is produced. By inhibiting the expression of R124H TGFBI, the causative protein of Avellino corneal dystrophy, the disease can be effectively prevented or treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A diagram showing the encoding mRNA sequence and the corresponding amino acid sequence at the position including amino acid 124 of normal WT TGFBI protein and mutant R124HTGFBI protein is shown.

[0018] Figure 2 A schematic diagram shows an assay method for obtaining ASOs that selectively inhibit R124H TGFBI, using a reporter gene system that indicates the expression level of WT TGFBI protein or the expression level of R124H TGFBI protein.

[0019] FIG3 shows the results of detecting the expression levels of WT TGFBI protein and R124H TGFBI protein after transfection with ASO at the indicated concentrations.

[0020] Figure 4 Shown are the results of detecting the expression levels of WT TGFBI protein and R124H TGFBI protein after treatment with ASO in the absence of a transfection agent (gymnotic delivery).

[0021] Figure 5 Shown are the results of detecting the expression levels of WTTGFBI protein and R124H TGFBI protein after treatment with various concentrations of ASO in the absence of a transfection agent (naked delivery). DETAILED DESCRIPTION

[0022] Hereinafter, preferred embodiments of the present disclosure will be described. However, the embodiments of the present disclosure may be modified into various other forms, and the scope of the present disclosure is not limited to the following embodiments.

[0023] The present disclosure aims to prevent or treat Avellino corneal dystrophy by inhibiting the production of TGFBI protein (the causative protein of the disease) using DNA-based antisense oligonucleotides (ASOs), and more preferably, to prevent or treat Avellino corneal dystrophy by selectively inhibiting the production of R124H mutant TGFBI protein in the TGFBI protein.

[0024] Avellino corneal dystrophy (granular corneal dystrophy type 2), the disease to be treated in this article, is a dominantly inherited disorder caused by the R124H mutation in the TGFBI protein. The resulting mutant TGFBI protein is misfolded and unstable, leading to the formation of granular deposits in the corneal stroma, which gradually reduces corneal transparency and ultimately causes vision loss.

[0025] The transforming growth factor beta-induced (TGFBI) protein associated with Avellino corneal dystrophy is produced from an mRNA with 2,052 nucleotide units and 684 codons and is ultimately translated into a protein consisting of 683 amino acids. Avellino corneal dystrophy is caused by a point mutation from CGC to CAC induced at codon 124 in the TGFBI coding sequence (CDS). Figure 1 ), and it is a serious hereditary disease with no current cure, and even surgery is prone to recurrence, gradually leading to vision loss.

[0026] Furthermore, depending on the allele, most patients with Avellino corneal dystrophy are heterozygous mutants expressing both normal and mutant proteins, while patients with Avellino corneal dystrophy who are homozygous mutants expressing only the mutant protein are very rare, occurring in approximately 1 in 1,000 or 1 in 10,000. Therefore, in order to develop therapeutic agents, it is necessary to inhibit the entire TGFBI, but additional functions are required to minimize the effects on wild-type TGFBI and selectively inhibit the R124H mutant TGFBI.

[0027] Therefore, the inventors of the present disclosure believe that preventing the production of R124H mutant TGFBI protein will be an effective therapeutic strategy for treating Avellino corneal dystrophy, and based on this insight, completed the present disclosure by identifying a novel ASO nucleotide sequence that inhibits the expression of R124H TGFBI protein.

[0028] Furthermore, since normal WT TGFBI protein plays a role in maintaining cell adhesion and differentiation of tissues in vivo, it is desirable to maintain normal WT TGFBI expression even when the expression of mutant TGFBI is inhibited. Therefore, in the present disclosure, ASO nucleotide sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression were additionally identified.

[0029] Therefore, the present disclosure provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising a single-stranded antisense oligonucleotide having a nucleotide sequence that inhibits the expression of transforming growth factor β-induced (TGFBI) mRNA with an R124H mutation.

[0030] Since Avellino corneal dystrophy is caused by a point mutation from CGC (the mRNA codon for arginine (R) at position 124 of the normal TGFBI protein) to CAC, the present disclosure may include antisense oligonucleotides having the nucleotide sequences shown in Tables 1 to 4, which directly inhibit mRNA, thereby preventing protein translation of R124H TGFBI mRNA.

[0031] Specifically, when comprising an 18-mer antisense oligonucleotide, the present disclosure may be one or more types selected from the following: 9 nucleotide sequences comprising modified deoxyribonucleic acid (DNA) linked by phosphorothioate (PS) bonds (SEQ ID NO: 1 to SEQ ID NO: 9); and 9 nucleotide sequences of unmodified DNA linked by phosphorothioate (PS) bonds (SEQ ID NO: 10 to SEQ ID NO: 18), as shown in Table 1.

[0032] When a 19-mer antisense oligonucleotide is included, it can be one or more types selected from the following: a 10-nucleotide sequence comprising a modified DNA linked by a PS bond (SEQ ID NO: 19 to SEQ ID NO: 28); and a 10-nucleotide sequence comprising an unmodified DNA linked by a PS bond (SEQ ID NO: 29 to SEQ ID NO: 38), as shown in Table 2.

[0033] When a 20-mer antisense oligonucleotide is included, it can be one or more types selected from the following: 11 nucleotide sequences comprising modified DNA linked by PS bonds (SEQ ID NO: 39 to SEQ ID NO: 49); and 11 nucleotide sequences comprising unmodified DNA linked by PS bonds (SEQ ID NO: 50 to SEQ ID NO: 60), as shown in Table 3.

[0034] Furthermore, the present disclosure may be one or more types selected from the nucleotide sequences shown in Table 4 (SEQ ID NO: 61 to SEQ ID NO: 67), which have one or more mismatches in addition to the R124HTGFBI complementary sequence.

[0035] ASO sequences are essentially based on deoxyribonucleic acid (DNA), and when modifications are applied, they are represented as follows.

[0036] *=2'-O-methoxyethyl (2'-MOE)

[0037] m=5-methyl

[0038] - = phosphorothioate linkage (PS linkage)

[0039] Table 1 18-mer antisense oligonucleotide sequence

[0040] Table 2 19-mer antisense oligonucleotide sequence

[0041] Table 3 20-mer antisense oligonucleotide sequence

[0042] Table 4 Modified ASO sequences with mismatched sequences

[0043] In particular, the present disclosure provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences in the nucleotide sequence of Table 1 below: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, wherein the one or more types of antisense oligonucleotides inhibit R124H TGFBI expression.

[0044] Preferably, the present disclosure provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 as nucleotide sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression; and more preferably, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 45.

[0045] In one aspect, the present disclosure provides a use as a medicament for preventing or treating Avellino corneal dystrophy, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences as a nucleotide sequence that inhibits the expression of R124H TGFBI mRNA: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45.

[0046] Preferably, the present disclosure provides a use as an agent for preventing or treating Avellino corneal dystrophy, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences as nucleotide sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45; and more preferably, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 45.

[0047] In another aspect, the present disclosure provides a method for preventing or treating Avellino corneal dystrophy, comprising administering an antisense oligonucleotide having a nucleotide sequence that inhibits expression of R124H TGFBI. In particular, the present disclosure provides a method for preventing or treating Avellino corneal dystrophy, wherein the nucleotide sequence comprises one or more types of antisense oligonucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45.

[0048] Preferably, the present disclosure provides a method for preventing or treating Avellino corneal dystrophy, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 as nucleotide sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression; and more preferably, comprising one or more types of antisense oligonucleotides selected from the group consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 45.

[0049] Furthermore, while the present disclosure is structurally based on 18-mers to 20-mers, even if the nucleotide sequences of SEQ ID NOs: 1 to 67 are used and one to six nucleic acids or nucleic acid derivatives are additionally formed at their 5' and 3' ends, it does not depart from the scope of the present disclosure. Even if the nucleotide sequences of SEQ ID NOs: 1 to 67 are used and a biodegradable or non-biodegradable linker is additionally attached to their 5' or 3' ends, or another compound is additionally formed without attachment, it does not depart from the scope of the present disclosure. The present disclosure encompasses the use of the nucleotide sequences of SEQ ID NOs: 1 to 67 and further encompasses embodiments in which a biodegradable or non-biodegradable linker is attached to the sugar moiety or nucleobase of an internal nucleotide, or to an internucleotide linkage, or in which other compounds are formed with such a linker, which does not depart from the scope of the present disclosure.

[0050] Furthermore, the present disclosure provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, wherein the antisense oligonucleotide comprises one or more chemical modifications in the sugar moiety or nucleobase of the ribonucleic acid, or an internucleotide linkage of the ribonucleic acid.

[0051] That is, the antisense oligonucleotides according to the present disclosure may be modified in various ways that can be conceived by those skilled in the art, but as long as the nucleotide sequence itself remains unchanged, such modifications also fall within the scope of the present disclosure. Here, the extent to which the nucleotide sequence itself remains unchanged means that even if a portion of the 5'- or 3'-terminus of the nucleotide sequence or an internal portion thereof is added, substituted, or deleted, approximately 90% or more of the nucleotide sequence is retained.

[0052] As an example of modification, even if the binding site of the nucleic acid has a modification (e.g., phosphorothioate, phosphoramidothioate, phosphonate, or methylphosphonate), as long as the nucleotide sequence is unchanged, it does not depart from the scope of the present disclosure. In addition, even if a chemical modification (e.g., 2'-O-methoxyethyl (2'-MOE), 2'-fluoro, 2'-O-methyl (2'-OMe), locked nucleic acid (LNA), peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), N3'-P5' phosphoramidate, 2'-deoxy-2'-fluoro-β-D-arabinoic acid analog (FANA), cyclohexene nucleic acid (CeNA), tricyclic DNA (tcDNA), 2-thiothymidine, 3'-fluorohexanol nucleic acid (FHNA), ethylene bridged nucleic acid (ENA), or (S)-constrained ethyl (cEt) modification) is introduced into the nucleic acid structure, as long as the nucleotide sequence remains unchanged, it does not depart from the scope of the present disclosure. 5-Methylpyrimidine can be applied to the base portion as needed, and even if additional fluorinated modifications are made, it does not depart from the scope of the present disclosure.

[0053] The pharmaceutical composition according to the present disclosure may be provided in one or more formulations selected from the group consisting of gel, emulsion, injection, and aerosol according to conventional methods, but is not limited thereto.

[0054] In another embodiment of the present disclosure, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers and diluents, antioxidants, buffers, bacteriostats, dispersants and surfactants commonly used in the manufacture of pharmaceutical compositions.

[0055] Specifically, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, polyvinyl pyrrolidone and water can be used as vehicles and diluents, and in addition, non-aqueous solvents may include propylene glycol, polyethylene glycol and vegetable oils such as olive oil, ethyl oleate and synthetic oil.

[0056] The above-mentioned pharmaceutical compositions can be administered to the subject in a conventional manner by intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, intraocular or intradermal routes.

[0057] The preferred dosage of antisense oligonucleotide may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the administration route and cycle, and may be appropriately selected by those skilled in the art, and the scope of the present disclosure is not limited thereto.

[0058] In the present disclosure, a "subject" may be a mammal including a human, but is not limited to these examples.

[0059] Example

[0060] Hereinafter, the present disclosure will be described in more detail by way of examples. These examples are only intended to illustrate the present disclosure in more detail, and according to the gist of the present disclosure, those skilled in the art will understand that the scope of the present disclosure is not limited by these examples.

[0061] Example 1-Example 67: Preparation of antisense oligonucleotides targeting R124H TGFBI mRNA

[0062] Antisense oligonucleotides of each SEQ ID NO were prepared using an oligonucleotide synthesizer. Synthesis was performed using DNA-based modifications according to the sequence annotations in Table 5.

[0063] <Applied Modifications>

[0064] *=2'-O-methoxyethyl (2'-MOE)

[0065] m=5-methyl

[0066] - = phosphorothioate linkage (PS linkage)

[0067] Table 5

[0068] Experimental Example 1: Establishment of a reporter gene system to evaluate the inhibition of human WT TGFBI and human R124H TGFBI expression Control Capacity

[0069] To obtain an ASO that selectively acts on R124H TGFBI compared to WT TGFBI, a system was constructed in which a reporter gene was expressed when WT TGFBI and R124H TGFBI proteins were expressed separately. Figure 1 As shown, human WT TGFBICDS or human R124HTGFBI CDS was inserted into the psiCHECK-2 vector (Promega), and the reporter gene Renilla luciferase was designed to be expressed when the protein of human WT TGFBI or human R124H TGFBI was expressed. In the vector, a control reporter gene, firefly luciferase gene, was inserted, which was designed to be expressed when the plasmid was transfected to normalize the transfection degree of the plasmid, thereby enabling accurate and reproducible results to be obtained regarding the effect on the expression of the target gene WT TGFBI or R124H TGFBI.

[0070] Experimental Example 2: Evaluation of the inhibitory ability of human WT TGFBI and human R124H TGFBI expression (by ASO transfection) Assessment of staining)

[0071] The plasmids in Experimental Example 1 (psi-CHECK2_WT TGFBI and psi-CHECK2_R124H TGFBI) were transfected into HEK-293 cells, which have a very low endogenous TGFBI expression level, to evaluate the example. 4 The density of cells / well was seeded in 96-well plates and cultured overnight at 37°C in DMEM (Thermo Fisher Scientific) culture medium containing 10% FBS in the presence of 5% CO. Psi-CHECK2_WT TGFBI plasmid or psi-CHECK2_R124H TGFBI plasmid was transfected into different wells of 96-well plates with 20 ng per well using lipofectamine 2000 (Thermo Fisher Scientific), and after 6 hours, the ASOs of the embodiment were transfected using lipofectamine RNAiMAX (Thermo Fisher Scientific) to give 0 nM, 25 nM, 50 nM, and 100 nM per well. 24 hours after ASO treatment, the fluorescence generated by Renilla luciferase and firefly luciferase expression was measured using a microplate reader (GloMax, Promega) by a Dual-Glo luciferase assay system (Promega). The fluorescence value of Renilla luciferase measured in each well was divided by the fluorescence value of Firefly luciferase for correction, and then the value of the control group not treated with ASO was set to 1 to express the value of the ASO-treated group relatively, as shown in FIG3 .

[0072] As a result, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more at a concentration of 25 nM were Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, Example 22, Example 23, Example 24, Example 43, Example 44 and Example 45, and their nucleotide sequences corresponded to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In addition, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more at a concentration of 25 nM and failed to inhibit the expression of WT TGFBI by 50% or more at the same concentration were Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, Example 22, Example 23, Example 24, Example 43, Example 44 and Example 45, and their nucleotide sequences correspond to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In addition, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more at a concentration of 25 nM, while failing to inhibit the expression of WT TGFBI by 50% or more at 50 nM are Examples 2, 3, 4, 5, 6, 7, 8, and 45, and their nucleotide sequences correspond to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, and 45, respectively. In addition, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more at a concentration of 25 nM, while failing to inhibit the expression of WT TGFBI by 50% or more at 100 nM are Examples 2, 3, 4, 5, 6, and 7, and their nucleotide sequences correspond to SEQ ID NO: 2, 3, 4, 5, 6, and 7, respectively.

[0073] In addition to the above sequences, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more at a treatment concentration of 50 nM are Examples 1 and Example 14, and these examples did not inhibit the expression of WT TGFBI by 50% or more at the same concentration, and their nucleotide sequences correspond to SEQ ID NO: 1 and SEQ ID NO: 14, respectively.

[0074] In addition to the above sequences, the ASO that inhibited R124H human TGFBI expression by 50% or more at a treatment concentration of 100 nM is Example 17, and the example did not inhibit WT TGFBI expression by 50% or more at the same concentration, and its nucleotide sequence corresponds to SEQ ID NO: 17.

[0075] In addition, regardless of the treatment concentration, the ASOs in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, Example 14, Example 22, Example 23, Example 24, Example 38, Example 43, Example 44, Example 45, Example 61, Example 63, Example 66 and Example 67 exhibited an inhibition rate against R124H human TGFBI expression that was at least 20% higher than that against WT human TGFBI, and their nucleotide sequences correspond to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

[0076] In addition, regardless of the treatment concentration, the ASOs in Examples 2, 3, 4, 5, 38, 45, 61, 66, and 67 exhibited an inhibition rate against R124H human TGFBI expression that was at least 30% higher than that against WT human TGFBI, and their nucleotide sequences correspond to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 38, SEQ ID NO: 45, SEQ ID NO: 61, SEQ ID NO: 66, and SEQ ID NO: 67, respectively.

[0077] Furthermore, as a result of experiments on ASOs 9, 10, 13, 16, 32, 33, 35, 38, etc., it was found that ASOs 9, 10, 13, and 16 with sequence numbers of 18-mer or shorter, or ASOs 32, 33, 35, and 38 with sequence numbers of 19-mer or longer, had an inhibitory effect on the R124H mutant TGFBI of less than 50%, and had almost no difference in inhibitory effect on the wild-type TGFBI and the R124H mutant TGFBI, or conversely, the wild-type TGFBI was more inhibited ( FIG. 3 ).

[0078] Experimental Example 3: Evaluation of the inhibitory ability of human WT TGFBI and human R124H TGFBI expression (by naked delivery of ASO) Evaluation sent)

[0079] The extent of expression inhibition of WT TGFBI and R124H TGFBI was additionally assessed by naked ASO delivery to cells without transfection reagents or other delivery aids. HEK-293 cells were plated at 1.7 × 10 4Cells were seeded at a density of 10 cells / well in a 96-well plate and cultured overnight in DMEM medium containing 10% FBS at 37°C in the presence of 5% CO2. 20 ng of psi-CHECK2_WTTGFBI plasmid or psi-CHECK2_R124H TGFBI plasmid was transfected into different wells of the 96-well plate using lipofectamine 2000 (Thermo Fisher Scientific). Six hours later, the ASOs of Examples 3, 4, 5, 22, 23, 24, 43, 44, and 45 were diluted in culture medium to 0 nM and 200 nM per well, respectively. 24 hours after ASO treatment, the fluorescence generated by the expression of Renilla luciferase and firefly luciferase was measured using a microplate reader (GloMax, Promega) and the Dual-Glo Luciferase Assay System (Promega). The fluorescence value of Renilla luciferase measured in each well was divided by the fluorescence value of Firefly luciferase for correction, and then the value of the control group not treated with ASO was set to 1, and the value of the ASO-treated group was expressed relative to that of the control group. Figure 4 shown.

[0080] As a result, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more were Example 3, Example 4, Example 5, Example 22, Example 23, Example 24, Example 43, Example 44, and Example 45, each of which had a nucleotide sequence comprising the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45. Furthermore, the ASOs that inhibited the expression of R124H human TGFBI by 50% or more but failed to inhibit the expression of WT TGFBI by 50% or more were Example 3, Example 4, Example 5, Example 22, Example 23, and Example 45, each of which had a nucleotide sequence comprising the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 45.

[0081] In addition, the ASOs whose expression inhibition rate for R124H human TGFBI reaches or exceeds 20% of that for WT human TGFBI are Examples 3, 4, 5, 22, 23, 24, 44 and 45, and their nucleotide sequences contain the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 44 and SEQ ID NO: 45.

[0082] In addition, the ASOs whose expression inhibition rate for R124H human TGFBI reaches or exceeds 30% of that for WT human TGFBI are Examples 3, Example 4, Example 5, Example 22, Example 23 and Example 45, and their nucleotide sequences contain the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 45.

[0083] In addition, for the same experimental procedure, as an example, Example 5 and Example 45 were treated at various concentrations ranging from 0.78 nM to 100 nM, and the IC for inhibiting 50% of WT TGFBI or R124H TGFBI expression was obtained using GraphPad Prism 9.5.1. 50 .

[0084] Results, such as Figure 5 As shown in the figure, in the whole treatment concentration range, the expression inhibition rate of R124HTGFBI in Example 5 and Example 45 was higher than that in WT TGFBI. 50 The IC of R124H TGFBI in Example 5 was obtained. 50 was 10.56 nM, and the IC for WT TGFBI 50 The IC value for R124H TGFBI in Example 45 was obtained as 5.71 nM. 50 was 5.71 nM, and the IC for WT TGFBI 50 When the selectivity of R124H TGFBI relative to WT TGFBI is expressed as the IC 50 Divided by the IC of R124H TGFBI 50 When the obtained values ​​(WT / Mut ratio) were compared, Example 5 exhibited excellent selectivity with a WT / Mut ratio of approximately 91.7, and Example 45 with a WT / Mut ratio of approximately 19.1.

[0085] Table 6 IC 50 and R124H TGFBI selectivity (WT / Mut ratio)

[0086] In the comprehensive analysis of the above test results, one or more antisense oligonucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 were analyzed as ASOs. At the overall concentrations tested in Experimental Example 2, they inhibited the expression of R124H human TGFBI by 50% or more, and may be useful for inhibiting the expression of R124H TGFBI.

[0087] Preferably, one or more antisense oligonucleotides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 were analyzed as ASOs that inhibited the expression of R124H human TGFBI by 50% or more at a minimum concentration of 25 nM in Experimental Example 2, and failed to inhibit the expression of WT TGFBI by 50% or more at the same concentration, which may be useful in selectively inhibiting the expression of R124H TGFBI while minimizing the effect on the expression of WT TGFBI.

[0088] More preferably, upon analysis, regarding the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 45, which as ASO inhibited the expression of R124H human TGFBI by 50% or more at the lowest concentration of 25 nM in Experimental Example 2, and failed to inhibit the expression of WT TGFBI by 50% or more at a higher concentration of 50 nM, and regarding the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 45, which as ASO inhibited the expression of R124H human TGFBI by 50% or more by naked delivery (200 nM) in Experimental Example 3 without delivery, and failed to inhibit the expression of WT TGFBI by 50% or more, are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 45. One or more types of antisense oligonucleotides from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 45 may be useful in selectively inhibiting R124H TGFBI expression while minimizing effects on WTTGFBI expression.

[0089] Although the specific parts of the present disclosure have been described in detail above, it is clear to those skilled in the art that these detailed descriptions are only preferred exemplary embodiments and the scope of the present disclosure is not limited thereto. In other words, the essential scope of the present disclosure is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising a single-stranded antisense oligonucleotide having a nucleotide sequence that inhibits the expression of transforming growth factor β-induced (TGFBI) with an R124H mutation, wherein: The antisense oligonucleotide is selected from one or more oligonucleotides in the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO:

45.

2. The pharmaceutical composition according to claim 1, wherein The antisense oligonucleotide comprises one or more oligonucleotides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO:

45.

3. The pharmaceutical composition according to claim 1, wherein The antisense oligonucleotide comprises one or more oligonucleotides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:

45.

4. The pharmaceutical composition according to claim 1, wherein The antisense oligonucleotides contain one or more chemical modifications in the sugar moiety or the nucleobase structure of the ribonucleic acid, or in the internucleotide linkages.

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