Antisense oligonucleotides targeting fungi and uses thereof

Abstract

The present invention relates to: an antisense oligonucleotide (ASO) targeting an FKS1 gene, an FKS2 gene, a CHS3 gene and / or a GWT1 gene of a Candida sp. fungus; an antibacterial composition comprising same; and a pharmaceutical composition for preventing or treating diseases caused by pathogenic fungi. The ASO according to the present invention can be used as a gene therapeutic agent having a fungus-specific antibacterial effect by specifically inhibiting FKS1, FKS2, CHS3 and / or GWT1 genes, and can be a useful gene therapeutic agent that can be used in combination with or replace conventional antifungal agents for antifungal agent-resistant fungi or multidrug-resistant fungi.

Description

Fungal-targeting antisense oligonucleotides and uses thereof

[0001] The present invention relates to an antisense oligonucleotide (ASO) targeting a gene of a fungus of the genus Candida and its use, and more particularly, to an antisense oligonucleotide comprising a specific base sequence that complementarily binds to the FKS1 gene, the FKS2 gene, the CHS3 gene and / or the GWT1 gene, and an antibacterial composition comprising the same, and a pharmaceutical composition for preventing or treating a disease caused by a pathogenic fungus.

[0002]

[0003] Candidiasis is a diverse group of infectious diseases caused by the fungus Candida. Prior to the development of antibiotics, candidiasis was not a major problem. However, with the widespread use of antibiotics, the incidence of all forms of candidiasis has increased dramatically, with Candida now the fourth most common cause of bacteremia or bloodstream infections in hospitalized patients. Bacteremia caused by Candida is called candidemia and is the most common form of invasive candidiasis. The mortality rate from candidemia is 30-50%, which is much higher than that of other fungal infections (Lass-Florl, C et al, Nat Rev Dis Primers, 2024, 10).

[0004] The medical costs associated with Candida infections are significant, with annual medical costs for candidemia exceeding 2.4 trillion won in the United States. This burden of candida has been similarly confirmed in a domestic multicenter study, and Candida has recently been reported as the most common cause of bacteremia in intensive care units (ICUs) in Korea (Amit, C et al., Nanomedicine, 2019, 14, 605-622).

[0005] Candida is a normal part of human flora, commonly found on the skin, digestive tract, and female reproductive organs. When hospitalized patients receive broad-spectrum antibiotics, the number of common bacteria decreases significantly, while the number of Candida increases significantly, increasing the risk of infection. Invasive candidiasis typically begins through damage to the gastrointestinal mucosa or the invasion of Candida into the bloodstream through a catheter. Once candidemia occurs, the infection can easily spread to various organs, causing infections in various areas.

[0006] Since azoles have been primarily used to treat candidiasis, azole-resistant C. glabrata and C. krusei have been considered antifungal-resistant Candida. With the development of organ transplant medicine and anticancer treatments, the number of immunocompromised patients has increased, leading to increased use of antifungal agents, and consequently, the number of antifungal-resistant Candida has increased. Long-term candidemia data from Asan Medical Center in Seoul also show that C. glabrata is more prevalent than the representative strain, C. albicans. Recently, these antifungal-resistant Candida have acquired resistance to echinocandins, which has become a problem, and cases are on the rise.

[0007] For example, Candida auris was first discovered in Japan in 2009, and outbreaks are currently being reported in several countries, including Korea, the United States, and Europe. C. auris is a new multidrug-resistant Candida, with more than 90% of strains resistant to one or more antifungal drugs, and about 30% resistant to two or more antifungal drugs. Some strains are resistant to all antifungal drugs, and are therefore called the superbug of fungi. C. auris can easily spread through the environment and by the hands of medical staff, which can cause outbreaks in hospitals. In addition, some disinfectants are known to have low disinfecting effects against C. auris, which is also a problem in infection control.

[0008] The increasing resistance of C. glabrata to echinocandins, the most effective antifungal agents, and the emergence of a new superbug, multidrug-resistant C. auris, require rapid development of new antifungal agents.

[0009]

[0010] The challenge of developing antifungal drugs lies in the fact that antifungal agents that inhibit fungal metabolic processes in eukaryotic cells, such as those of humans, can also act on human cells, causing toxicity. Drugs targeting protein enzymes found only in some fungi (e.g., chitin synthase) have been developed, but they have shown limited effectiveness against Candida.

[0011] Echinocandin's agonistic enzyme, beta-D-gluan synthase (FKS1, FKS2), is found only in fungi and plays a crucial role in cell wall synthesis for fungal survival, making it an important target for antifungal drug development. While new drugs are being developed, mutations in the FKS1 and FKS2 genes in Candida cause echinocandin resistance, making new antifungal drugs with the same mechanism of action ineffective against this resistant Candida. Furthermore, resistant Candida may be selected shortly after the introduction of these drugs into clinical practice. With the rise in antifungal drug-resistant fungi, there is a pressing need to develop antifungal drugs that are effective against multidrug-resistant fungi.

[0012] Resistance to azoles and echinocandins used in fungi is mainly caused by genetic mutations in target proteins or increased expression of related genes. Therefore, to fundamentally avoid this, new drugs must be developed targeting completely new targets. However, the development of new antifungal agents is difficult due to the similarity of metabolic processes and protein enzymes with those of humans.

[0013]

[0014] Accordingly, the present inventors have made great efforts to develop a therapeutic agent having an antibacterial effect that can be used in combination with or as a substitute for existing antifungal agents, and as a result, they have proposed a new concept of an antifungal agent that selectively inhibits fungal genes using an antisense oligonucleotide (ASO) using gene therapy technology, and selected FKS1, FKS2 (also called GSC1 and GSC2, respectively; beta glucan synthase 1 and 2) and CHS3 (chitin synthase) genes related to cell wall synthesis, and GWT1 (GPI-anchored wall transfer protein 1) genes related to inositol acylation as target genes of the ASO, and confirmed that when delivered to fungi by loading them into a nanocarrier, an effective antifungal effect was exhibited, and thus the present invention was completed.

[0015]

[0016] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0017]

[0018] Summary of the invention

[0019] The purpose of the present invention is to provide an antisense oligonucleotide (ASO) that complementarily binds to a fungal gene.

[0020] Another object of the present invention is to provide an antibacterial composition comprising the antisense oligonucleotide.

[0021] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by a pathogenic fungus, and a method for preventing or treating the disease, comprising the antisense oligonucleotide.

[0022] Another object of the present invention is to provide a use of the antisense oligonucleotide for preventing or treating a disease caused by a pathogenic fungus and a use of the antisense oligonucleotide for preparing a medicament for preventing or treating a disease caused by a pathogenic fungus.

[0023]

[0024] To achieve the above purpose, the present invention provides an antisense oligonucleotide (ASO) that complementarily binds to one or more genes selected from the group consisting of the FKS1 gene, the FKS2 gene, the CHS3 gene, and the GWT1 gene of a fungus of the genus Candida.

[0025] The present invention also provides an antibacterial composition comprising the antisense oligonucleotide.

[0026] The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by a pathogenic fungus, comprising the antisense oligonucleotide.

[0027] The present invention also provides a method for preventing or treating a disease caused by a pathogenic fungus, comprising administering the antisense oligonucleotide.

[0028] The present invention also provides the use of the antisense oligonucleotide for preventing or treating a disease caused by a pathogenic fungus and the use of the antisense oligonucleotide for preparing a medicament for preventing or treating a disease caused by a pathogenic fungus.

[0029]

[0030] Figure 1 is a schematic diagram showing the effect of inhibiting target gene expression using ASO according to the present invention.

[0031] Figure 2 is a diagram showing the conserved sequence common to both FKS1 and FKS2 of Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Candida auris, and the conserved sequence common to CHS3 of Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Candida auris.

[0032] Figure 3 is a graph showing the results of analyzing the effect of inhibiting GSC1 and CHS3 mRNA expression by delivering a gene (ASO) therapeutic agent loaded with Anti-GSC1 ASO candidates 1 and 2 (Figure 3A) or Anti-CHS3 ASO candidates 1 and 2 (Figure 3B) to Candida albicans.

[0033] Figure 4 is a graph showing the results of confirming the antibacterial effect of a gene therapy (ASO) with multiple ASOs targeting growth genes on C. albicans according to the treatment time (Figure 4A) and the amount of ASO loaded (Figure 4B), and is a graph showing the results of comparing the antibacterial effects according to single and multiple ASO loading (Figure 4C).

[0034] Figure 5 is a diagram showing the results of confirming the degree of cell death and morphological changes of Candida albicans by a gene therapy (ASO) containing multiple ASOs (Anti-GSC1 and Anti-CHS3 ASOs) for two genes related to cell wall synthesis, using a confocal microscope (Figure 5A), a scanning electron microscope (Figure 5B), and a transmission electron microscope (Figure 5C).

[0035] Figure 6 is a graph showing candidate gene (ASO) sequences (Figure 6A) for screening gene (ASO) therapeutic agents that can commonly inhibit multiple species of Candida fungi (including the Candida fungi species specified in Figure 2) and their corresponding antibacterial effects against C. albicans (Figure 6B).

[0036] Figure 7 is a diagram showing the results of an analysis of the antibacterial effect of a therapeutic agent loaded with multiple ASOs (Anti-GSC1 ASO and Anti-CHS3 ASO) for two genes related to the synthesis of the cell wall of the fungus against different Candida species (Figure 7A) and the results of an analysis of the antibacterial effect of Anti-GSC2 ASO and Anti-GWT1 ASO (Figure 7B).

[0037] Figure 8 is a graph showing the results of comparing the antibacterial effects against C. albicans between a chemically modified Gapmer form of the gene (Gapmer-ASO) to improve stability in the body and a non-modified general gene (Non-modified ASO; ASO).

[0038] Figure 9 is a diagram showing the results of CFU values ​​(antibacterial effect) in the kidney (Figure 9A) and tissue analysis (H&E, PAS) (Figure 9B) after inoculation with various doses of a gene (ASO) therapeutic agent carrying a multi-target ASO in a mouse model of disseminated Candida infection.

[0039] Figure 10 is a graph comparing the results of CFU values ​​(antibacterial effect) in the kidney after inoculation of a gene (ASO) therapeutic agent carrying the multi-target ASOs selected in Figures 6 and 7 in a mouse model of disseminated Candida infection.

[0040] Figure 11 is a diagram showing the results of tissue analysis (H&E, PAS) (Figure 11A) and a table quantifying the results (Figure 11B) after administering a gene therapy (ASO) containing a multi-target ASO to a mouse model of dermal Candida infection.

[0041] Figure 12 is a graph showing the results of measuring the survival rate for 7 days after administering a gene (ASO) treatment loaded with a multi-target ASO to a mouse model of disseminated candidal infection to evaluate the survival rate improvement effect of the gene (ASO) treatment.

[0042] Figure 13 is a graph showing the results of measuring body weight changes for 7 days after administering a gene (ASO) therapy loaded with a multi-target ASO to a normal mouse model to evaluate the in vivo toxicity of the gene (ASO) therapy.

[0043]

[0044] Detailed description of the invention and preferred embodiments

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0046]

[0047] In one aspect, the present invention relates to an antisense oligonucleotide (ASO) that complementarily binds to one or more genes selected from the group consisting of the FKS1 gene, the FKS2 gene, the CHS3 gene, and the GWT1 gene of a fungus of the genus Candida.

[0048]

[0049] In the present invention, FKS1, also called GSC1, and FKS2, also called GSC2, are subunits of 1,3-beta glucan synthase. CHS3 is chitin synthase III. GWT1 is GPI-anchored wall transfer protein 1.

[0050]

[0051] Antisense oligonucleotide (ASO) technology modulates the transfer of information from genes to proteins by altering the intermediate metabolism of mRNA via single-stranded RNA or DNA. Specifically, by selecting a sufficiently complementary and specific base sequence that hybridizes, the desired suppression of target protein expression is achieved. Because ASOs bind sequence-specifically to the target gene, they do not affect the expression of other genes. Therefore, ASO technology is not only a useful tool for analyzing the in vivo role of specific proteins, but also has potential for use as a gene therapy for specific diseases (KJ Scanlon, et al., FASEBJ. 1995 Oct;9(13):1288-96).

[0052] Antisense DNA binds to the target mRNA to form an RNA / DNA double helix, and this RNA / DNA double helix structure is attacked and degraded by RNase H (RNase H; a type of ribonuclease that specifically degrades mRNA in which RNA / DNA hybrid double helix is ​​formed) existing in the body. Antisense RNA forms an RNA / RNA double helix and is attacked by RNase L to cause degradation of the target mRNA. RNase L is a ribonuclease that preferentially degrades single-stranded RNA around the double-stranded RNA strand (ST Crooke, Annu Rev Pharmacol Toxicol. 1992:32:329-76).

[0053] In this specification, "nucleotide" means a monomer molecule constituting a nucleic acid composed of a combination of a nucleobase, a sugar moiety, and a phosphate group, and the nucleotide may be interpreted as a concept including all unmodified or modified nucleobases, sugar moieties, and / or phosphate groups, such as nucleotide analogs, modified nucleotides, non-natural nucleotides, and non-standard nucleotides.

[0054] In this specification, "nucleoside" means a glycosylamine, which is considered to be a portion of a nucleotide excluding a phosphate group, and means a monomer molecule composed of a nucleobase and a sugar moiety. The nucleoside can be interpreted as a concept that includes all nucleosides in which the nucleobase "G / " or the sugar moiety is modified or not, just like a nucleotide.

[0055] As used herein, "oligonucleotide" means an oligonucleotide or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their analogs, and the oligonucleotide generally includes an oligonucleotide composed of a covalent bond between a nucleobase, a sugar, and a nucleoside (backbone) that exist in a living body, as well as a modified or substituted oligonucleotide composed of a nucleotide analogue, a modified nucleotide, a non-natural nucleotide, or a non-standard nucleotide that acts similarly thereto. Such modified or substituted oligonucleotides have properties such as enhanced cellular uptake, enhanced nucleic acid target affinity, and increased stability in the presence of a nuclease, compared to unmodified or unsubstituted oligonucleotides.

[0056] In this specification, "antisense oligonucleotide (ASO)" is interpreted to include an oligonucleotide capable of hybridizing with a target nucleic acid sequence by hydrogen bonding. Antisense oligonucleotides include, but are not limited to, oligonucleotides, oligonucleotide analogs, oligonucleotide mimetics, siRNA, single-stranded siRNA (ss siRNA), short hairpin RNA (shRNA), microRNA mimics, ribozymes, external guide sequence oligonucleotides, and other oligonucleotides that hybridize with a target nucleic acid sequence to regulate its expression, and the antisense oligonucleotides are interpreted as a concept including single-stranded and double-stranded oligonucleotides.

[0057]

[0058] In one embodiment of the present invention, the antisense oligonucleotide has a nucleic acid base sequence that includes a reverse complement of a target portion of a target nucleic acid sequence to be targeted when described in the 5' to 3' direction. Preferably, the antisense oligonucleotide can complementarily bind to a nucleic acid base sequence of an FKS1 gene, an FKS2 gene, a CHS3 gene, and / or a GWT1 gene. The FKS1 gene, an FKS2 gene, a CHS3 gene, and / or a GWT1 gene are nucleic acids targeted by the antisense oligonucleotide, and may be selected from mRNA and pre-mRNA including introns, exons, and untranslated regions.

[0059] In the present invention, the antisense oligonucleotide may be characterized by having a base sequence that is at least 70%, at least 80%, at least 90%, or completely complementary to any sequence of the nucleic acid base sequence of the FKS1 gene, the FKS2 gene, the CHS3 gene, and / or the GWT1 gene, but is not limited thereto.

[0060] In the present invention, the antisense oligonucleotide that complementarily binds to the FKS1 gene may be characterized by including a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 8, and the antisense oligonucleotide that complementarily binds to the FKS2 gene may be characterized by including a base sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10, but is not limited thereto.

[0061] In the present invention, the antisense oligonucleotide that complementarily binds to the CHS3 gene may be characterized by including a base sequence represented by any one of SEQ ID NO: 11 to SEQ ID NO: 13, and the antisense oligonucleotide that complementarily binds to the GWT1 gene may be characterized by including a base sequence represented by SEQ ID NO: 14, but is not limited thereto.

[0062] In the present invention, it is preferable that the antisense oligonucleotide complementarily binding to the FKS1 gene comprises a base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 5, and the antisense oligonucleotide complementarily binding to the CHS3 gene comprises a base sequence represented by SEQ ID NO: 11, but is not limited thereto.

[0063]

[0064] The above antisense oligonucleotide selects one or more target sites in the nucleic acid base sequence of the FKS1 gene, the FKS2 gene, the CHS3 gene, and / or the GWT1 gene, and selects an oligonucleotide sufficiently complementary to the target site so as to hybridize sufficiently specifically with the target site, thereby obtaining the desired effect on regulating the expression of FKS1, FKS2, CHS3, and / or GWT1.

[0065] As used herein, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleic acid bases that pair by forming hydrogen bonds.

[0066] As used herein, the term “hybridizable” or “complementary” or “substantially complementary” means that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that can non-covalently bind to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid), i.e., form, “anneal,” or “hybridize” pairs of adenine (A) with thymine (T), adenine (A) with uracil (U), and guanine (G) with cytosine (C), under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength.

[0067] In one embodiment of the present invention, the hybridization occurs between the antisense oligonucleotide disclosed herein and a nucleic acid sequence of the FKS1 gene, FKS2 gene, CHS3 gene, and / or GWT1 gene. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleic acid bases of the nucleic acid molecules.

[0068] Hybridization can occur under a variety of conditions. Stringent conditions depend on the sequence and the nature and composition of the nucleic acid molecule being hybridized. Methods for determining whether a sequence can specifically hybridize with a target nucleic acid are well known in the art.

[0069]

[0070] As used herein, the term "complementary" refers to the property of two nucleotides to be able to precisely pair. For example, if two different nucleic acids or oligonucleotides have base sequences written in the 5' to 3' direction, and a certain portion of the base sequence of one nucleic acid or oligonucleotide is aligned in the opposite direction, the two nucleic acids or oligonucleotides are said to be complementary if they non-covalently bind to a certain portion of the other nucleic acid or oligonucleotide, i.e., form a pairing of adenine (A) and thymine (T), a pairing of adenine (A) and uracil (U), and a pairing of guanine (G) and cytosine (C).

[0071] Accordingly, the terms "specifically hybridizable" and "complementary" can be interpreted as terms used to indicate a sufficient degree of complementarity or precise pairing between an oligonucleotide and a DNA or RNA target to enable stable, specific binding. It is known in the art that the sequence of an antisense oligonucleotide need not be 100% complementary to the sequence of the target nucleic acid with which it is specifically hybridized.

[0072] The above antisense oligonucleotide is interpreted as having a degree of complementarity sufficient to prevent non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desirable, i.e., under physiological conditions in the case of in vivo analysis or treatment, or under assay performance conditions in the case of in vitro analysis.

[0073] That is, if the antisense oligonucleotide can specifically hybridize with the target nucleic acid, non-complementary nucleic acid bases between the antisense oligonucleotide and the target nucleic acid can be tolerated. Furthermore, the antisense oligonucleotide can hybridize with one or more nucleic acid portions (e.g., a loop structure, a mismatch, or a hairpin structure) such that the intervening or adjacent portions are not involved in hybridization.

[0074] According to one specific example, the antisense oligonucleotide of the present invention or the modified oligonucleotide constituting the antisense oligonucleotide may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to a nucleic acid sequence of the FKS1 gene, the FKS2 gene, the CHS3 gene, and / or the GWT1 gene. The percent complementarity of the antisense oligonucleotide with the target nucleic acid can be determined by a conventional method known in the art.

[0075] As used herein, “fully complementary” means that each nucleic acid base of the antisense oligonucleotide can form precise base pairing with the corresponding nucleic acid base of the target nucleic acid.

[0076] In one embodiment of the present invention, the non-complementary nucleic acid base may be located at the 5' end or the 3' end of the antisense oligonucleotide. Alternatively, the non-complementary nucleic acid base or bases may be located within the antisense oligonucleotide. When two or more non-complementary nucleic acid bases are present, they may be adjacent (i.e., linked) or non-adjacent. In one embodiment of the present invention, the non-complementary nucleic acid base may be located in the wing portion of a gapmer antisense oligonucleotide.

[0077] In one embodiment of the present invention, the antisense oligonucleotide of the present invention may include those complementary to a nucleic acid base sequence portion of the FKS1 gene, the FKS2 gene, the CHS3 gene, and / or the GWT1 gene. As used herein, "portion" means a predetermined number of nucleic acid bases that are adjacent (i.e., bound) within a region or portion of a target nucleic acid. The portion may also mean a predetermined number of adjacent nucleic acid bases of the antisense oligonucleotide. In one embodiment, the antisense oligonucleotide may be complementary to a portion of at least 8 nucleic acid bases of the target portion, may be complementary to a portion of at least 12 nucleic acid bases, or may be complementary to a portion of at least 15 nucleic acid bases.

[0078]

[0079] In the present invention, the antisense oligonucleotide may be characterized by including a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 14, and including one or more chemical modifications, but is not limited thereto.

[0080] In the present invention, the modified oligonucleotide may comprise one or more modifications selected from one or more modified internucleoside linkers, one or more modified nucleosides comprising a modified sugar moiety, and one or more modified nucleosides comprising a modified nucleobase.

[0081]

[0082] Modification of sugar moieties

[0083] In one embodiment of the present invention, the modified nucleoside may be a modified nucleoside comprising a non-bicyclic modified sugar moiety and / or a bicyclic or tricyclic sugar moiety, and / or a sugar moiety modified with a sugar surrogate or sugar mimetic.

[0084] In the present invention, the modified nucleoside is, for example, 2'-O-alkyl such as 2'-O-methyl, 2'-O-alkoxy such as 2'-O-methoxy, 2'-O-alkoxyalkyl such as 2'-O-methoxyethyl, 2'-amino, 2'-allyl, 2'-fluoro, 2'-arabino-fluoro, 2'-ON-substituted acetamide such as 2'-OCH2C(=O)-NHCH3(NMA), 2'-O-benzyl and 2'-O-methyl-4-pyridine, 4'-O-methyl, 5'-methyl, It may be a sugar moiety having one or more substituents selected from the group consisting of 5'-vinyl and 5'-methoxy, but is not limited thereto.

[0085] The antisense oligonucleotide according to the present invention may optionally comprise one or more modified nucleosides having substituted or modified sugar moieties. Modification of the sugar moieties imparts nuclease stability, binding affinity, or other advantageous biological properties to the antisense oligonucleotide. The (pento)furanosyl sugar ring of the natural nucleoside may be modified in a variety of ways, including, but not limited to, addition of a substituent (particularly at the 2' position); bridging of two different ring atoms to form a bicyclic nucleic acid (BNA); and substitution of an atom or group such as -S-, -N(R)-, or -C(R1)(R2) at the ring oxygen at the 4' position. Modified sugar moieties include, but are not limited to, substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH2 (2'-OMe) or 2'-O(CH2)2-OCH3 (2'-O-methoxyethyl or 2'-MOE) substituents; and bicyclic modified sugars (BNAs) having 4'-(CH2)nO-2' (n=1 or n=2) bridges. Methods for preparing such modified sugars are known in the art. The base portion of a nucleoside comprising a modified sugar moiety can be maintained to hybridize with a target nucleic acid.

[0086] In the present invention, the modified nucleoside comprises one of F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; O-alkyl-O-alkyl; O-alkyl-O-alkyl-N(dialkyl); or O-alkyl-carboxylamide at the 2' position (wherein alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10Alkenyl and alkynyl). O[(CH2)nO]m CH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, O(CH2)n0(CH2)nN[(CH2)mCH3]2, O(CH2)nC(=O)-NHCH3 and O(CH2)n0N[(CH2)mCH3]2 are particularly preferred (wherein n and m are 0 to about 10).

[0087] Preferably, the modification may comprise a 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE, i.e., an alkoxyalkoxy group) (Martin et al., HeIv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group, and the modification may comprise a 2'-dimethylaminooxyethoxy (i.e., a (CH2)2ON(CH3)2 group, also known as 2'-DMAOE), and a 2'-dimethylaminoethoxyethoxy [i.e., a 2'-O(CH2)2O(CH2)2--N(CH3)2 group, also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE].

[0088] The bicyclic or tricyclic sugar moiety may be selected from the group consisting of, but is not limited to, locked nucleic acid (LNA), constrained ethyl bicyclic nucleic acid (cEt), 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), and tricyclo-DNA.

[0089] In one embodiment, the modified nucleoside may comprise a sugar substitute having a six-membered ring or an acyclic moiety. The sugar substitute may be selected from the group consisting of, but is not limited to, a morpholino ring such as a phosphorodiamidate morpholino oligomer (PMO), a cyclohexenyl ring, a cyclohexyl ring, and a tetrahydropyranyl ring such as a hexitol, anitol, mannitol, or a fluorohexitol. Various other bicyclic and tricyclic sugar substituted ring systems that can be used to modify the nucleosides introduced into the antisense oligonucleotides according to the present invention are known in the art. The activity of these ring systems can be enhanced through various substitution processes.

[0090] Additionally, the above-described sugar substitute may be, but is not limited to, a non-cyclic moiety such as, for example, an unlocked nucleic acid (UNA) or a peptide nucleic acid (PNA).

[0091] Peptide nucleic acid (PNA) is a type of nucleic acid analogue in which nucleobases are linked by peptide bonds rather than phosphate bonds. It has peptide bonds instead of phosphodiester bonds, and has nucleobases such as adenine, thymine, guanine, and cytosine, so it can specifically hybridize with nucleic acids. PNA is not found in nature, but is artificially synthesized by chemical methods, and can form double-stranded nucleic acids with complementary base sequences through hybridization. In addition, PNA is not only chemically stable because it is electrically neutral, but it also has the characteristic of being biologically stable because it is not degraded by nucleases or proteases. Although the N-aminoethylglycine backbone is the most widely used PNA, PNAs with modified backbones can also be used, as is known in the art (PE Nielsen and M. Egholm "An Introduction to PNA" in PE Nielsen (Ed.) "Peptide Nucleic Acids: Protocols and Applications" 2nd Ed. Page 9 (Horizon Bioscience, 2004)).

[0092] Unlocked nucleic acid (UNA) is a modified nucleoside that lacks the C2'-C3' linkage of ribose. Due to its open-chain structure, its steric configuration is not restricted, allowing for the control of oligonucleotide flexibility. The inclusion of UNA in an antisense oligonucleotide is known to lower the Tm value by approximately 5°C to 10°C and reduce off-target binding.

[0093]

[0094] Nucleobase modification

[0095] In the present invention, the modified nucleoside is pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2-deoxyuridine, N-ethylpiperidine 7'-EAA triazol modified adenine, N-ethylpiperidine 6'-triazol modified adenine, 6'-phenylpyrrolocytosine, 2',4'-difluorotoluylribonuleoside and It may be characterized by being a modified nucleoside comprising one or more modified nucleobases selected from the group consisting of 5'-nitroindole, but is not limited thereto.

[0096] Unmodified or natural nucleic acid bases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0097] The modified nucleoside may also include nucleobase modifications or substitutions. Nucleobase modifications or substitutions are structurally distinct, but are functionally interchangeable with naturally occurring or synthetic unmodified nucleobases. Naturally occurring nucleobases and modified nucleobases can participate in hydrogen bonding. These nucleobase modifications impart nuclease stability, binding affinity, or other favorable biological properties to the antisense oligonucleotide. For example, certain nucleobase substitutions, such as 5-methylcytosine substitutions, are known to increase nucleic acid duplex stability by 0.6-1.2°C, and thus may be particularly useful in enhancing the binding affinity of antisense oligonucleotides to target nucleic acids.

[0098] For example, the modified nucleobases include 5'-hydroxymethyl cytosine, xanthine, hypoxanthine, 2'-aminoadenine, 6'-methyl and other alkyl derivatives of adenine and guanine, 2'-propyl and other alkyl derivatives of adenine and guanine, 2'-thiouracil, 2'-thiothymine and 2'-thiocytosine, 5'-halouracil and cytosine, 5'-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6'-azo uracil, cytosine and thymine, 5'-uracil (pseudo-uracil), 4'-thiouracil, 8'-halo, 8'-amino, 8'-thiol, 8'-thioalkyl, 8'-hydroxy and others. 8'-substituted adenines and guanines, 5'-halo (especially 5'-bromo), 5'-trifluoromethyl and other 5'-substituted uracils and cytosines, 7'-methylguanine and 7'-methyladenine, 2'-F-adenine, 2'-amino-adenine, 8'-azaguanine and 8'-azaadenine, 7'-deazaguanine and 7'-deazaadenine and 3'-deazaguanine and 3-deazaadenine, tricyclic pyrimidines such as phenoxazine cytidine (lH-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps include, but are not limited to, substituted phenoxazine cytidines such as 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).

[0099] The modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B. ed., CRC Press, 1993.

[0100]

[0101] Modified internucleoside linker

[0102] In the present invention, in the antisense oligonucleotide, the modified internucleoside linking group may be characterized by being at least one modified internucleoside linking group selected from the group consisting of phosphorothioate, phosphorodithioate, phosphotriester, phosphoramidate, mesyl phosphoramidate, methylphosphonate, methoxypropyl-phosphonate, and boranophosphate.

[0103] As is well known in the art, a nucleoside is a combination of a nucleobase and a sugar moiety. A nucleotide further comprises a phosphate group covalently bonded to the sugar moiety of the nucleoside. In nucleotides comprising a pentofuranosyl sugar, the phosphate group can be bonded to the 2', 3', or 5' hydroxyl group of the linked sugar. In the formation of an oligonucleotide, the phosphate groups covalently bond to adjacent nucleosides to form a linear polymer compound. In turn, each end of the linear polymer structure also bonds to form a circular structure, although an open linear structure is generally preferred. In the oligonucleotide structure, the phosphate groups typically form the internucleoside backbone of the oligonucleotide, and the naturally occurring bonds and backbones of RNA and DNA are 3' to 5' phosphodiester bonds. An antisense oligonucleotide according to one embodiment may comprise one or more modified internucleoside linkages in addition to naturally occurring internucleoside linkages, which are often selected over antisense oligonucleotides comprising naturally occurring internucleoside linkages due to their properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0104] A specific example of a preferred antisense oligonucleotide that can be used in the present invention is an oligonucleotide comprising a modified backbone or unnatural internucleoside linkage. As defined above, oligonucleotides with a modified backbone include nucleotides containing a phosphorus atom in the backbone and nucleotides that do not contain a phosphorus atom in the backbone. Furthermore, as used in the art, a modified oligonucleotide that does not contain a phosphorus atom in the internucleoside backbone is also construed as an oligonucleotide in the present specification.

[0105] In an antisense oligonucleotide according to one embodiment, the modified linkage between nucleosides may include linkages between nucleosides that contain phosphate as well as linkages between nucleosides that do not contain phosphate. The linkages between representative phosphate-containing nucleosides may be phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, methyl and other alkyl phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates having their normal 3'-5' linkage, 2'-5' linkage analogs, mesyl phosphoramidate, thionophosphoramidate, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphate and boranophosphate and compounds having opposite polarity in which one or more internucleotide linkages are 3'-3', 5'-5' or 2'-2' linkages, It is not limited to this.

[0106] In the present invention, the modified oligonucleotide comprises a gap segment composed of linked deoxynucleosides, a 5' wing segment composed of linked nucleosides, and a 3' wing segment composed of linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and the nucleoside of each wing segment may include, but is not limited to, a modified sugar moiety or sugar substitute.

[0107] In one embodiment of the present invention, the modified oligonucleotide may be characterized by, but is not limited to, comprising a gap segment consisting of 8 to 10 linked deoxynucleosides; a 5' wing segment consisting of 3 to 5 linked nucleosides; and a 3' wing segment consisting of 3 to 5 linked nucleosides, wherein each nucleoside of each wing segment comprises a modified sugar moiety.

[0108] In a gapmer, an internal region containing a number of nucleotides that support RNase H cleavage is located between an external region containing a number of nucleosides that are chemically different from the nucleosides of the internal region. For an antisense oligonucleotide having a gapmer motif, the gap segment may support the cleavage of the target nucleic acid, while the wing segment may comprise a modified oligonucleotide containing modified nucleosides to enhance stability, affinity, and exonuclease resistance.

[0109] Optionally, the gap segment may also comprise a modified oligonucleotide. The modified oligonucleotide may comprise one or more modifications selected from one or more modified internucleoside linkers, one or more modified nucleosides comprising a modified sugar moiety, and one or more modified nucleosides comprising a modified nucleobase, each of which is as described above.

[0110]

[0111] Formulation

[0112] To facilitate the use of oligonucleotides, a variety of formulations have been developed to deliver oligonucleotides to a subject or cellular environment, for example, by minimizing degradation, facilitating delivery and / or uptake, or providing other beneficial properties to the oligonucleotide in the formulation.

[0113] In the present invention, the antisense oligonucleotide for reducing the expression of the FKS1 gene, the FKS2 gene, the CHS3 gene, and / or the GWT1 gene can be suitably formulated so that, when administered to a subject either systemically or into the immediate environment of the target cell, a sufficient portion of the oligonucleotide enters the cell to reduce the expression of the FKS1 gene, the FKS2 gene, the CHS3 gene, and / or the GWT1 gene. In one embodiment, the antisense oligonucleotide can be formulated in the form of a buffer solution, for example, a phosphate-buffered saline solution, a liposome, a micelle structure, or a capsid. It can also be formulated in water or an aqueous solution (for example, pH-adjusted water) or a basic buffered aqueous solution (for example, PBS).

[0114] In the present invention, the introduction of oligonucleotides into cells can be facilitated by using a formulation of oligonucleotides containing cationic lipids. For example, cationic lipids such as lipofectamine, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc.), or FuGene 6 (Roche), all of which can be used according to the manufacturer's protocol. Such formulations can include lipid nanoparticles.

[0115] Additionally, the formulation may include an excipient. The excipient may include a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to a cell, tissue, organ, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013). The excipient imparts improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient to the composition. Additionally, the excipient may be a buffer (e.g., sodium citrate, sodium phosphate, tris(II) base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).

[0116] In one embodiment, the oligonucleotide may be lyophilized to extend its shelf life, and then prepared into a solution prior to use. Accordingly, excipients in compositions comprising the oligonucleotide according to the present invention may include a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrrolidone) or a disintegration temperature modifier (e.g., dextran, ficoll, or gelatin).

[0117] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers may include normal saline, bacteriostatic water, Cremophor EL.TM. (BASF), or phosphate-buffered saline (PBS). The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol), and sodium chloride in the composition. Sterile injectable solutions may be prepared by incorporating the oligonucleotide in the required amount in a selected solvent, along with one or a combination of ingredients enumerated above, as required, followed by sterile filtering. The pharmaceutical composition may contain at least about 0.1% of a therapeutic agent (e.g., an antisense oligonucleotide for reducing the expression of WFDC2) or more, but the percentage of active ingredient is preferably from about 1% to about 80% by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be taken into account in the preparation of the formulation.

[0118]

[0119] From another aspect, the present invention relates to an antibacterial composition comprising the above antisense oligonucleotide.

[0120] In the present invention, the antibacterial composition may be characterized by having an antibacterial effect against fungi of the genus Candida.

[0121] In the present invention, the Candida fungus may be characterized by being at least one selected from the group consisting of C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. auris, C. krusei, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. kefyr, C. famata, C. rugosa, and C. lipolytica, but is not limited thereto.

[0122] In the present invention, the term “antibacterial” means an activity that inhibits the growth or proliferation of fungi including microorganisms.

[0123] The antibacterial composition of the present invention can be used as a single agent, and can be manufactured and used as a composite agent by additionally including a composition known to have a recognized antibacterial effect.

[0124] According to one embodiment of the present invention, an antibacterial agent or antibacterial food composition comprising the antibacterial composition is provided.

[0125]

[0126] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a disease caused by a pathogenic fungus, comprising the antisense oligonucleotide.

[0127] In another aspect, the present invention relates to a method for preventing or treating a disease caused by a pathogenic fungus, comprising administering the antisense oligonucleotide to a subject.

[0128] In another aspect, the present invention relates to the use of the antisense oligonucleotide for preventing or treating diseases caused by pathogenic fungi.

[0129] In another aspect, the present invention relates to the use of the antisense oligonucleotide for the manufacture of a medicament for the prevention or treatment of diseases caused by pathogenic fungi.

[0130] In the present invention, the disease caused by the pathogenic fungus may be characterized by being selected from the group consisting of candidal dermatitis, candidal granuloma, candidal stomatitis, candidal vaginitis, candidal balanitis, candidal urethritis, candidal enteritis, candidal meningitis, candidal endocarditis, candidal sepsis, candidal onychomycosis, and neonatal candidiasis, but is not limited thereto.

[0131] In this specification, the term “prevention” means any act of inhibiting or delaying the progression of a disease caused by a pathogenic fungus by administering the composition of the present invention, and “treatment” means inhibiting the development of a disease caused by a pathogenic fungus, alleviating or eliminating symptoms.

[0132] The pharmaceutical composition according to the present invention may contain a pharmaceutically effective amount of the antisense oligonucleotide alone, or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, improve, and treat the target disease.

[0133] The above “pharmaceutically acceptable” means physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal upset or dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition may further include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.

[0134] The term "carrier" is defined as a compound that facilitates the introduction of a nucleic acid structure into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of living organisms.

[0135] The term "diluent" is defined as a compound that stabilizes the biologically active form of the target compound and is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the field. A commonly used buffer solution is phosphate-buffered saline, as it mimics the salt content of human body fluids. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely alter the biological activity of a compound.

[0136] A pharmaceutical composition comprising an antisense oligonucleotide according to the present invention may be administered to a patient as such or as a pharmaceutical composition mixed with other active ingredients or with a suitable carrier or excipient, such as in combination therapy.

[0137] The pharmaceutical composition may further comprise, in addition to the above ingredients, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0138] The pharmaceutical composition according to the present invention may be formulated as an injectable formulation, such as an aqueous solution, suspension, or emulsion, or as a lyophilized formulation, but is not limited thereto. Furthermore, the composition may be formulated as desired for each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Sciences.

[0139] The term "administration" of the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

[0140] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A physician of ordinary skill can easily determine and prescribe an amount effective to achieve the intended purpose of treating or preventing GSC. More specifically, a therapeutically effective amount means an amount effective to prolong the survival of a subject to be treated, or to prevent, alleviate, or relieve the symptoms of a disease. Determination of a therapeutically effective amount is within the capabilities of a person of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0141] In the present invention, “subject” means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering an antisense oligonucleotide according to the present invention, and preferably means a human.

[0142] The pharmaceutical composition according to the present invention can be used in combination with conventional therapeutic agents. This means that the antisense oligonucleotide according to the present invention and the pharmaceutical composition comprising it can be administered simultaneously with, or sequentially or in reverse order with, conventional therapeutic agents such as antifungal agents. These agents can be administered in combination in an appropriate effective amount within the scope of those skilled in the art.

[0143]

[0144] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0145]

[0146] Example 1: Selection of ASO targets in antifungal drug-resistant fungi (Candida)

[0147] Among existing antifungal drug targets, ERG11 (lanosterol 14α-demethylase) is a target of azole antifungals. However, since azole antifungals are fungistatic drugs, this gene was deemed unlikely to be a potent ASO target and was therefore excluded from the candidate gene list.

[0148] FKS1 (GSC1) and FKS2 (beta-D-glucan synthase) are targets of echinocandin antifungals. Because they exhibit fungicidal effects when inhibited, they were considered potent ASO targets and were therefore selected as candidate ASOs. We searched for conserved sequences common to both FKS1 and FKS2 from five representative Candida species: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Candida auris. Through this, we constructed an FKS1 (GSC1) ASO that would most effectively act with the corresponding base sequence (Fig. 2).

[0149] Chitin synthase III (CHS3) is crucial for fungal survival and is known as a target for the development of new antifungal agents. We explored conserved sequences common to the five Candida species mentioned above and developed a CHS3 ASO that would most effectively target the corresponding sequence (Fig. 2).

[0150] Additionally, we produced an ASO for the GWT1 (GPI-anchored wall transfer protein 1) gene, which is known to be required for inositol acylation of GPI (glycosylphosphatidylinositol) anchors in yeast.

[0151] Based on a study showing that deletion of FKS1 and CHS3 in Candida results in lethality (Tong et al., Science 2004; 303: 808), we selected these two genes as targets and produced 19-24 mer ASOs based on these deletions. We confirmed that the selected ASOs and the human chromosome had identical base sequences, and since only 10-12 mer of the ASOs matched, we expected no off-target effects.

[0152]

[0153] Example 2: Screening of gene therapy candidates in cultured fungi.

[0154] Based on the results of in vitro studies, we aimed to develop single-target ASOs or two or three multi-targeted ASOs. To minimize theoretical predictions and off-target effects, we used a simulation program (RNA fold) to predict the secondary structure of the target gene's mRNA based on its free energy among several potential candidates. We then selected a group of candidates with the highest potential for effectiveness and screened them.

[0155] To analyze the quantitative expression inhibition effect of target gene mRNA, cultured Candida strains were treated with gene (ASO) therapeutic agents, and target gene expression was quantitatively measured using real-time polymerase chain reaction (quantitative real-time PCR, qPCR).

[0156] Anti-GSC1 ASO, Anti-GSC2 ASO, Anti-CHS3 ASO, and Anti-GWT1 ASO candidates were used as ASOs loaded into gene therapy. The conditions for the optimal expression inhibition effect were optimized by treating C. albicans with the gene therapy at various concentrations (10–50 nM) and over time.

[0157] Anti-GSC1 ASO, Anti-GSC2 ASO, Anti-CHS3 ASO, and Anti-GWT1 ASO were loaded onto nanocarriers manufactured using chitosan instead of PEI as the outermost polymer material according to the method described in [Marcel Janis Beha, et al., Materials Science & Engineering C 126 (2021) 112167], and treated with Candida for 2 hours.

[0158] As a result, it was confirmed that the expression level of GSC1 was reduced. In particular, among anti-GSC1 ASO candidates 1 and 2, candidate 1 (GSC1_1: sequence number 2) showed a 53.5% decrease in the GSC1 mRNA expression level, and candidate 2 (GSC1_2: sequence number 3) showed a 38.9% decrease (Fig. 3A).

[0159] Similarly, when anti-CHS3 ASO was loaded onto ASO nanocarriers using the same method and treated with Candida for 2 hours, the expression level of CHS3 was significantly suppressed. Additionally, among the candidates for anti-CHS3 ASO, candidate 1 (CHS3_1: SEQ ID NO: 12) showed a 75.6% decrease in CHS3 mRNA expression, and candidate 2 (CHS3_1: SEQ ID NO: 13) showed a 71.2% decrease (Fig. 3B).

[0160] In summary, we identified the optimal ASO for the selected target gene, and its sequence is as follows:

[0161]

[0162] Example 3: Analysis of target gene inhibition and antibacterial effects of fungal-targeted gene therapy.

[0163] C. albicans was cultured and treated with nanocarriers loaded with multiple anti-GSC1 ASOs and anti-CHS3 ASOs. Bacterial growth was measured using the WST-8 method, and the antifungal effect was found to increase with increasing treatment time of the gene (ASO) therapeutic up to 24 hours (Fig. 4A).

[0164] Furthermore, when the treatment time of the gene (ASO) therapy was fixed at 24 hours and the cells were re-cultured after replacing the medium with antibiotics, the antibacterial effect was confirmed to be maintained for up to 24 hours. In particular, as the amount of ASO multiply loaded into the gene (ASO) therapy increased, a greater antibacterial effect was observed (Fig. 4B).

[0165] Additionally, the results of comparing the antibacterial effects of single and multiple ASO loadings verified that multiple ASO loadings exhibited sufficient antibacterial effects even without an antibacterial agent (Fig. 4C).

[0166]

[0167] Next, to qualitatively confirm the antibacterial effect of the developed gene (ASO) therapeutic, microscopic analysis (Live / Dead PI assay), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were performed. Microscopic analysis (SEM and TEM) allowed for the visual observation of cell death effects, as well as the morphological and morphological changes of Candida due to the antifungal effect.

[0168] The antibacterial effect of the gene therapy (ASO) loaded with multiple ASOs was confirmed using the Live / Dead PI assay. Compared to the control group, the PI fluorescence signal, which is a signal of cell death, was found in almost all cells loaded with the gene therapy (ASO). In particular, in the case of fungi, hyphae, which indicate the degree of virulence, were observed in a form that was almost absent, indicating that the gene therapy (ASO) reduced the virulence of the fungi and appropriately performed the antibacterial action (Fig. 5A).

[0169] When the morphology of the fungus was observed through a scanning electron microscope (SEM), the fungus was observed to have almost no hyphae, similar to the results of the Live / Dead PI assay, and in particular, many spores were observed to have shapes that appeared to be crushed or burst, confirming that an appropriate antibacterial effect occurred (Fig. 5B).

[0170] Similarly, in transmission electron microscopy (TEM) analysis, most strains were observed to have destroyed cell morphology due to cell death, and in particular, cell wall synthesis was inhibited by ASO, resulting in loss of cell wall or a thinning that was more than 7 times thinner than the control group (Fig. 5C).

[0171] Consequently, multi-target gene therapy (ASO) has been demonstrated to exhibit effective antibacterial activity.

[0172]

[0173] Example 4: Analysis of general antibacterial effect against fungi

[0174] Next, to determine whether the developed gene (ASO) therapeutic agent is effective not only against the standard strain C. albicans but also against various Candida strains, several ASO candidates were selected by gene (Fig. 6A), and the most promising gene (ASO) therapeutic agent was selected by treating C. albicans (Fig. 6B).

[0175] The antibacterial effect of a gene (ASO) therapeutic agent containing multiple anti-GSC1 ASO (F.48: SEQ ID NO: 3) and anti-CHS3 ASO (C.57: SEQ ID NO: 13) selected for treatment of various clinical strains of various species, including C. tropicalis, C. parapsilosis, C. glabrata, C. auris, and C. krusei, was evaluated through a cell growth test (WST-8 assay). As a result, although there were differences depending on the Candida species, the antibacterial effect ranged from a minimum of 74.8% to a maximum of 95.7%, and it was confirmed that the antibacterial effect was 85% or higher in most strains (Fig. 7A).

[0176] In addition, the combined antifungal (micafungin) antibacterial effect of Anti-GSC2 ASO and Anti-GWT1 ASO was confirmed, and the antibacterial effect was up to 90.8% for GSC2 and up to 57.4% for GWT1 (Fig. 7B).

[0177] Additionally, to enhance in vivo stability, a chemically modified Gapmer gene (Gapmer-ASO) and an unmodified gene (Non-modified ASO; ASO) were treated with C. albicans and then administered concurrently with an antifungal agent (micafungin) to compare their antibacterial effects. As a result, the Gapmer-ASO treatment showed superior antibacterial activity in most candidate groups, with some candidate groups exhibiting antibacterial effects of up to 84.4% (Fig. 8).

[0178]

[0179] Example 5: Analysis of therapeutic effects in an animal model of fungal infection

[0180] The efficacy of a multi-target gene therapy (ASO) containing anti-GSC1 ASO and anti-CHS3 ASO, which were confirmed to be effective in in vitro experiments, was evaluated in an animal model of infection.

[0181] To determine the dosage of gene therapy (ASO) containing multiple anti-GSC1 ASO and anti-CHS3 ASO in animal experiments, fungus (C. albicans) was inoculated (IV) into the tail microvessels of 6-week-old ICR mice, and 1 hour later, gene therapy (ASO) containing various doses of ASO was administered intraperitoneally (IP), and the CFU value in the kidney was measured 24 hours later.

[0182] Compared to the control group (NT (non-target) ASO), when the developed gene (ASO) therapeutic agent (F.57 (SEQ ID NO: 5) and C.Ca (SEQ ID NO: 11)) was administered, it was confirmed that colonies did not grow at all selected doses, and a significant therapeutic effect was confirmed in the infected animal model (Fig. 9A).

[0183] In addition, the results of H&E and PAS tissue analysis also showed that when gene (ASO) therapeutics (F.57 (SEQ ID NO: 5) and C.Ca (SEQ ID NO: 11)) were administered, fungi were significantly reduced, and almost no inflammatory cells were found, verifying that the therapeutic efficacy was as great as that of existing antifungal agents (Fig. 9B).

[0184] In particular, when the therapeutic agent loaded with the multi-target ASO selected in FIGS. 6 and 7 was administered to an infected animal model, no colonies grew at all in the kidney (FIG. 10), confirming a strong antibacterial effect.

[0185] Additionally, 6-week-old ICR mice were inoculated with fungus (C. albicans) into the dermis (ID), and 1 hour later, the gene (ASO) therapy was administered (SC), and the CFU value in the kidney was measured 24 hours later. The results of H&E and PAS tissue analysis showed that the fungus was significantly reduced in the case of administration of the developed gene (ASO) therapy compared to the control group, and almost no inflammatory cells were found, verifying that the treatment efficacy was great not only for systemic infection but also for dermal (local) infection (Fig. 11).

[0186] Finally, to evaluate the survival improvement effect of gene therapy (ASO) in an infected animal model, the treatment (F.57 (SEQ ID NO: 5) and C.Ca (SEQ ID NO: 11)) loaded with multi-target ASOs was administered intraperitoneally (IP) and the survival rate was measured for 7 days. As a result, it was confirmed that the treatment showed a survival improvement effect comparable to that of existing antifungal agents (Fig. 12).

[0187]

[0188] Example 6: Toxicity Analysis of Gene (ASO) Therapeutics

[0189] To evaluate the side effects and toxicity of gene (ASO) therapy, a treatment (F.57 (SEQ ID NO: 5) and C.Ca (SEQ ID NO: 11)) loaded with multi-target ASO was administered to a normal mouse model. After measuring the change in body weight for 7 days, it was confirmed that the mice were in a healthy state without any significant difference from the control group (Fig. 13).

[0190]

[0191] The ASO according to the present invention can be used as a gene therapy agent having a fungal-specific antibacterial effect by specifically inhibiting one or more genes selected from the group consisting of FKS1, FKS2, CHS3, and GWT1 genes, and can be a useful gene therapy agent that can be used in combination with or as a substitute for existing antifungal agents against antifungal drug-resistant fungi or multidrug-resistant fungi.

[0192]

[0193] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0194]

[0195] Electronic file attached.

Claims

1. An antisense oligonucleotide (ASO) that complementarily binds to one or more genes selected from the group consisting of the FKS1 gene, FKS2 gene, CHS3 gene, and GWT1 gene of fungi of the genus Candida.

2. In the first paragraph, an antisense oligonucleotide that complementarily binds to the FKS1 gene is characterized in that it includes a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO:

8.

3. In the first paragraph, an antisense oligonucleotide that complementarily binds to the FKS2 gene is characterized in that it includes a base sequence represented by SEQ ID NO: 9 or SEQ ID NO:

10.

4. In the first paragraph, an antisense oligonucleotide that complementarily binds to the CHS3 gene is characterized in that it includes a base sequence represented by any one of SEQ ID NO: 11 to SEQ ID NO:

13.

5. In the first paragraph, an antisense oligonucleotide that complementarily binds to the GWT1 gene is characterized in that it includes a base sequence represented by SEQ ID NO:

14.

6. An antisense oligonucleotide according to claim 1, characterized in that the oligonucleotide comprises at least one chemical modification selected from the group consisting of a modified nucleoside linker, a modified nucleoside comprising a modified sugar moiety, and a modified nucleoside comprising a modified nucleobase.

7. An antisense oligonucleotide characterized in that the modified nucleoside comprises a sugar moiety having one or more substituents selected from the group consisting of 2'-O-methyl, 2'-O-methoxy, 2'-O-methoxyethyl, 2'-amino, 2'-allyl, 2'-fluoro, 2'-arabino-fluoro, 2'-OCH2C(=O)-NHCH3(NMA), 2'-O-benzyl, 2'-O-methyl-4-pyridine, 4'-O-methyl, 5'-methyl, 5'-vinyl, and 5'-methoxy.

8. An antisense oligonucleotide according to claim 6, wherein the modified internucleoside linking group is at least one modified internucleoside linking group selected from the group consisting of phosphorothioate, phosphorodithioate, phosphotriester, phosphoramidate, mesyl phosphoramidate, methylphosphonate, methoxypropyl-phosphonate, and boranophosphate.

9. In the first paragraph, the oligonucleotide comprises a gap segment composed of linked deoxynucleosides, a 5' wing segment composed of linked nucleosides, and a 3' wing segment composed of linked nucleosides. An antisense oligonucleotide characterized in that the gap segment is located between the 5' wing segment and the 3' wing segment, and the nucleoside of each wing segment comprises a modified sugar moiety or sugar surrogate.

10. An antisense oligonucleotide according to claim 9, wherein the oligonucleotide comprises a gap segment consisting of 8 to 10 linked deoxynucleosides; a 5' wing segment consisting of 3 to 7 linked nucleosides; and a 3' wing segment consisting of 3 to 7 linked nucleosides, wherein each nucleoside of each wing segment comprises a modified sugar moiety.

11. An antibacterial composition comprising an antisense oligonucleotide according to any one of claims 1 to 10.

12. An antibacterial composition according to claim 11, characterized in that it has an antibacterial effect against fungi of the genus Candida.

13. An antibacterial composition according to claim 12, wherein the Candida fungus is at least one selected from the group consisting of C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. auris, C. krusei, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. kefyr, C. famata, C. rugosa, and C. lipolytica.

14. A pharmaceutical composition for preventing or treating a disease caused by a pathogenic fungus, comprising an antisense oligonucleotide according to any one of claims 1 to 10.

15. A pharmaceutical composition according to claim 14, characterized in that the disease caused by the pathogenic fungus is selected from the group consisting of candidal dermatitis, candidal granuloma, candidal stomatitis, candidal vaginitis, candidal balanitis, candidal urethritis, candidal enteritis, candidal meningitis, candidal endocarditis, candidal sepsis, candidal onychomycosis, and neonatal candidiasis.

Images