Composition for preventing or treating unconventional protein secretion-related diseases

Abstract

The present invention relates to a composition for diagnosing, preventing, or treating diseases related to the regulation of unconventional protein secretion (UPS). The present invention is based on the discovery of a key factor that plays an important role in the activity of unconventional protein secretion, and thus can be effectively used to provide a highly reliable novel target for developing therapeutic agents for related diseases through regulation of the key factor.

Description

Composition for the prevention or treatment of atypical protein transport related diseases

[0001] The present invention relates to a method for diagnosing or treating diseases related to the unconventional protein secretion (UPS) mechanism, specifically cystic fibrosis or infections caused by viruses that utilize the UPS mechanism, by discovering key factors involved in the regulation of the UPS mechanism and regulating the same.

[0002] Cystic fibrosis is a disease caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. When this mutation prevents the protein from folding properly, it is not transported to the cell membrane properly and is degraded in the endoplasmic reticulum (ER), causing the disease to develop.

[0003] Specifically, CFTR is Cl - As a channel protein used for the movement of exocrine fluid, if a mutation causes a malfunction in the protein, secretion failure of the exocrine glands occurs, increasing mucus secretion and causing the mucus to become abnormally sticky, which causes cystic fibrosis. When cystic fibrosis occurs, the secretion and absorption efficiency of the lungs and pancreas decreases, and in the pancreas, sticky mucus blocks the movement of pancreatic fluid, causing digestive disorders. In addition, malabsorption of fat and fat-soluble vitamins occurs, leading to malnutrition, and various symptoms such as sinusitis, nasal polyps, esophagitis, pancreatitis, cirrhosis, rectal prolapse, diabetes, and infertility (especially in men) may occur.

[0004] Meanwhile, in addition to genetic diseases such as cystic fibrosis, the UPS pathway is also known to be utilized by viruses such as SARS-CoV-2 for secretion of spike proteins out of cells. Therefore, if the activity level of the UPS pathway can be controlled, it is expected that infections caused by viruses that utilize the UPS mechanism for self-replication can be prevented or treated.

[0005] Although various factors are expected to be involved in this UPS mechanism, the specific mechanisms or key associated factors remain unknown. Therefore, identifying key factors involved in the UPS pathway, modulating them, could potentially lead to the diagnosis, prevention, or treatment of various diseases associated with the UPS mechanism.

[0006] Accordingly, the inventors of the present invention sought to identify factors that are significantly involved in the regulation of the UPS mechanism and to confirm that controlling these factors can prevent or treat diseases related to the UPS mechanism, specifically cystic fibrosis or SARS-CoV-2 infection.

[0007]

[0008] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0009]

[0010] The present inventors have conducted extensive research to discover essential factors that can regulate the unconventional protein secretion (UPS) mechanism known to be related to the pathogenesis of cystic fibrosis, a disease previously known to be intractable, in order to develop a novel therapeutic composition.

[0011] As a result, it was confirmed that among the proteins belonging to various OSBP families, when the expression of the OSBPL9 protein was inhibited, the atypical protein transport mechanism was reduced, and conversely, when it was overexpressed, the atypical protein transport mechanism was significantly increased. Through this, it was discovered that cystic fibrosis and various diseases related to the atypical protein transport mechanism can be prevented or treated by regulating the expression of OSBPL9, thereby completing the present invention.

[0012] Accordingly, the purpose of the present invention is to provide a composition for preventing or treating diseases related to the regulation of unconventional protein secretion (UPS).

[0013] Another object of the present invention is to provide a composition for diagnosing diseases related to the regulation of non-typical protein transport mechanisms.

[0014] Another object of the present invention is to provide a method for providing information necessary for diagnosing a disease related to the regulation of atypical protein transport mechanisms.

[0015] Another object of the present invention is to provide a method for screening a composition for preventing or treating a disease related to the regulation of atypical protein transport mechanisms.

[0016] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0017]

[0018] According to one aspect of the present invention, the present invention provides a composition for preventing or treating a disease related to regulation of unconventional protein secretion (UPS), which comprises as an active ingredient an agent for regulating the expression of the OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

[0019] The present inventors have conducted extensive research to discover essential factors that can regulate the unconventional protein secretion (UPS) mechanism, known to be related to the pathogenesis of cystic fibrosis, in order to develop a novel therapeutic composition for the disease. As a result, among the various OSBP family proteins, we confirmed that inhibiting the expression of the OSBPL9 protein in particular decreased the unconventional protein secretion mechanism, whereas conversely, overexpressing it significantly increased the unconventional protein secretion mechanism. Through this, we discovered that regulating the expression of OSBPL9 can prevent or treat cystic fibrosis, as well as various diseases related to the unconventional protein secretion mechanism, thereby completing the present invention.

[0020] In this specification, the term “OSBPL9 (Oxysterol binding protein-like 9)” refers to a protein encoded by the OSBPL9 gene in humans, and can be used interchangeably with ORP-9, ORP9, etc. The protein belongs to the oxysterol-binding protein (OSBP) family, which includes various intracellular lipid receptor proteins.

[0021] The term “unconventional protein secretion (UPS)”, as used herein, may also be referred to as “unconventional protein transport mechanism” or “ER / Golgi-independent protein secretion mechanism”, and refers to a mechanism by which proteins are transported to the cell membrane or extracellular matrix without passing through the endoplasmic reticulum (ER) or Golgi apparatus.

[0022] As used herein, the term “disease related to regulation of non-canonical protein transport mechanisms” refers to a disease caused by regulation (increase or decrease) of non-canonical protein transport mechanisms. Specifically, diseases related to regulation of non-canonical protein transport mechanisms as used herein include: ① neurodegenerative diseases including Alzheimer’s (AD) and Parkinson’s disease (PD) that are related to the production of misfolded proteins and their secretion by the UPS; ② immune regulation-related diseases that are related to the secretion of inflammasomes including immune-related factors such as interleukins through the UPS pathway; ③ diseases such as cancer or metastatic cancer that are known to be related to protein secretion and exosome production by the UPS pathway; ④ fibrosis, specifically cystic fibrosis; And ⑤ It includes all types of diseases that can be prevented or treated by modulating atypical protein transport mechanisms, including but not limited to the diseases mentioned above, such as infections caused by viruses that utilize the UPS pathway for survival, specifically SARS-CoV-2.

[0023] Accordingly, in the case of diseases corresponding to ①, ②, ③ and ⑤ above, prevention or treatment can be achieved by suppressing the UPS pathway by suppressing the expression of the OSBLP9 protein according to the present invention; and in the case of diseases corresponding to ④ above, prevention or treatment can be achieved by activating the UPS pathway by promoting the expression of the OSBLP9 protein according to the present invention.

[0024]

[0025] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0026] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. When the composition of the present invention is administered to a subject, the expression of the OSBPL9 protein or the gene encoding it is regulated, thereby suppressing the development of symptoms caused by a disease related to the regulation of a non-canonical protein transport mechanism, eliminating them, or alleviating them. Therefore, the composition of the present invention may be a composition for treating these diseases on its own, or may be administered together with other pharmacological ingredients and used as a therapeutic adjuvant for the diseases. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “therapeutic adjuvant” or “therapeutic adjuvant.”

[0027] As used herein, the term “expression modulating agent” means an activator that increases the expression of the OSBPL9 protein or the gene encoding it, or an inhibitor that decreases the expression thereof.

[0028]

[0029] According to a specific embodiment of the present invention, the expression regulating agent is an inhibitor or activator of the OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

[0030] The term “inhibitor” as used herein means a substance that causes a decrease in the activity or expression of a target gene, not only when the activity or expression of the target gene becomes undetectable or exists at an insignificant level, but also when the activity or expression of the target gene is decreased to such an extent that the biological function of the target gene is significantly reduced.

[0031] Inhibitors of target genes include, but are not limited to, shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) antisense oligonucleotide, or CRISPR system containing guide RNA (gRNA) that recognizes the target gene, which suppresses the expression of the gene whose sequence is already known in the art at the gene level, antibodies or aptamers that suppress at the protein level, as well as compounds, peptides, and natural products that suppress the activity thereof, and all gene and protein level suppression means known in the art can be used.

[0032] The term “shRNA (small hairpin RNA)” as used herein refers to an RNA sequence that forms a tight hairpin structure to suppress the expression of a target gene through RNA interference, which is a single-stranded structure consisting of 50-70 nucleotides that forms a stem-loop structure in vivo. Typically, a long RNA of 19-29 nucleotides complementarily forms a double-stranded stem by base pairing on both sides of a loop region of 5-10 nucleotides, and is transduced into cells through a vector containing a U6 promoter to ensure constant expression, and is usually passed on to daughter cells to ensure heritable suppression of the expression of the target gene.

[0033] The term “siRNA” in this specification refers to a short double-stranded RNA that can induce RNAi (RNA interference) by cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and either blunt-ended or cohesive-ended is possible as long as it can suppress the expression of the target gene through the RNAi effect. The cohesive-ended structure can be either a 3-terminal protruding structure or a 5-terminal protruding structure.

[0034] In this specification, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is an oligonucleotide that is not expressed in cells and has a short stem-loop structure and suppresses target gene expression through complementary binding to the mRNA of the target gene.

[0035] As used herein, the term "ribozyme" refers to an RNA molecule that functions like an enzyme, recognizing a specific base sequence in RNA and cleaving it. A ribozyme consists of a region that specifically binds to a complementary base sequence of a target mRNA strand and a region that cleaves the target RNA.

[0036] As used herein, the term "PNA (Peptide Nucleic Acid)" refers to a molecule that possesses properties of both nucleic acids and proteins and can complementarily bind to DNA or RNA. PNA is not found in nature and is artificially synthesized through chemical methods. It forms a double strand through hybridization with a natural nucleic acid of complementary base sequence, thereby regulating the expression of target genes.

[0037] As used herein, the term “antisense oligonucleotide” refers to a nucleic acid molecule that is a nucleotide sequence complementary to a sequence of a specific mRNA and binds to the complementary sequence in the target mRNA, thereby inhibiting its translation into protein, translocation into the cytoplasm, maturation, or any other essential activity for its overall biological function. Antisense oligonucleotides can be modified at one or more base, sugar, or backbone positions to enhance their potency (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatoms, heterocyclic sugar sulphonates, etc.

[0038] As used herein, the term "guide RNA (gRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized region. A representative example of such gene editing systems is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0039] According to the present invention, the expression inhibitor of the present invention may be an antibody or an antigen-binding fragment thereof that specifically binds to the protein and inhibits its activity, or an aptamer that specifically binds to the protein. The antibody that specifically recognizes the target protein is a polyclonal or monoclonal antibody, and is particularly preferably a monoclonal antibody.

[0040] The antibodies of the present invention can be produced by methods commonly practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816,567), or the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production are described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984.

[0041] As used herein, the term “antigen binding fragment” means a portion of a polypeptide in the overall structure of an immunoglobulin capable of binding an antigen, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0042] In this specification, the term “specifically binding” has the same meaning as “specifically recognizing” and means that an antigen and an antibody (or fragment thereof) specifically interact through an immunological reaction.

[0043] The term “aptamer” as used herein refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. The general contents of aptamers are described in detail in Hoppe-Seyler F, Butz K “Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R . “An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24):14272-7(1998).

[0044] The term “activator” in this specification refers to an effective ingredient that enhances the expression level or activity of OSBPL9 (Oxysterol binding protein-like 9) protein, and includes, but is not limited to, nucleic acid molecules, peptides, proteins, compounds, and natural products that enhance the expression of OSBPL9, a protein whose sequence and structure are already known in the art, at the gene or protein level, or enhance its inherent biological activity. Accordingly, “OSBPL9 protein activator” is used with the same meaning as “OSBPL9 protein agonist.”

[0045] In the present specification, “method for increasing the expression level of OSBPL9 protein at the gene level” includes, but is not limited to, in vivo delivery of mRNA of a target gene, and any gene and protein level activation means known in the art may be used.

[0046]

[0047] According to a specific embodiment of the present invention, the disease related to regulation of the unconventional protein secretion (UPS) mechanism is fibrosis, and in this case, the expression regulator is an expression activator.

[0048] Fibrosis, a disease that can be prevented or treated with the preventive or therapeutic composition of the present invention, specifically cystic fibrosis, is a disease that occurs when a mutation occurs in the CFTR gene, causing an abnormality in protein folding, resulting in the protein not being properly transported to the cell membrane but being degraded in the endoplasmic reticulum (ER). Accordingly, when the non-canonical protein transport mechanism (UPS) is activated by activating the activity or expression of the OSBPL9 protein according to the present invention to transport the CFTR protein with an abnormality in folding to the cell membrane, even if the activity is lower than that of the normal protein, it can function as a channel to a certain extent, making it possible to prevent or treat cystic fibrosis.

[0049]

[0050] According to a specific embodiment of the present invention, the disease related to regulation of the unconventional protein secretion (UPS) mechanism is an infection of a virus that utilizes the unconventional protein secretion (UPS), and in this case, the expression regulating agent is an expression inhibitor.

[0051] As used herein, the term “viruses utilizing unconventional protein secretion (UPS)” refers to virus species that secrete viral virulence factors outside the host cell through an unconventional protein secretion mechanism during the replication process after infection in the host cell. Specifically, these viruses include, but are not limited to, VP22 of Herpes simplex virus type I, VP40 of the Ebola virus, human immunodeficiency virus-1 (HIV-1), and SARS-CoV-2 virus.

[0052] “Infection by a virus using an unconventional protein secretion (UPS)”, which is a disease that can be prevented or treated with the preventive or therapeutic composition of the present invention, means an infection by a virus that uses the UPS pathway in a host cell for virus replication. Accordingly, when the unconventional protein secretion (UPS) is inhibited by inhibiting the activity or expression of the OSBPL9 protein according to the present invention, replication of a virus using an unconventional protein secretion (UPS) and propagation through it can be prevented, thereby treating the infection by the virus.

[0053]

[0054] According to a specific embodiment of the present invention, the fibrosis is cystic fibrosis.

[0055] In this specification, the term "cystic fibrosis (CF)" can be used interchangeably with cystic fibrosis or mucoviscidosis, and refers to a type of autosomal recessive inherited disease in which the transport of chloride ions and sodium through epithelial tissue is impaired, resulting in thick, sticky mucous membranes. Cystic fibrosis primarily causes problems in the lungs, but can also affect the pancreas, liver, and intestines. The excessive mucus produced by CF itself reduces the secretion and absorption efficiency of the lungs and pancreas. In the pancreas, the sticky mucus blocks the movement of pancreatic juice, causing digestive problems and malabsorption of fats and fat-soluble vitamins. This can even lead to nutritional deficiencies. In addition, cystic fibrosis can cause sinusitis, nasal polyps, esophagitis, pancreatitis, cirrhosis, rectal prolapse, diabetes, and infertility (especially in men). Additionally, symptoms may appear in which excessive mucus inhibits the movement of pathogens, increasing the likelihood of bacterial overgrowth and infection at the contact site.

[0056] According to a specific embodiment of the present invention, the cystic fibrosis is caused by a mutation selected from the group consisting of ΔF508-CFTR, N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR and V520F-CFTR.

[0057] Cystic fibrosis is mainly caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein, which lead to a folding defect in the CFTR. These cystic fibrosis-causing mutations include, but are not limited to, ΔF508-CFTR, N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR, and V520F-CFTR, and these mutations cause a folding defect in CFTR and lead to Cl- Any mutation that causes impaired function as a channel protein may be included.

[0058] According to a specific embodiment of the present invention, the virus is SARS-CoV-2.

[0059] The term “SARS-CoV-2” in this specification refers to the virus that causes coronavirus disease 2019 (COVID-19), which is also contagious to humans, and may be referred to as the 2019 novel coronavirus (2019-nCoV) or coronavirus-19 (Covid 19). SARS-CoV-2 is classified into four classes by the US government’s SARS-CoV-2 Interagency Group (SIG): Variant Being Monitored (VBM); Variant of interest (VOI); Variant of concern (VOC); and Variant of High Consequence (VOHC). VBM includes alpha, beta, gamma, epsilon, eta, iota, kappa, mu, and zeta, and VOC includes delta and omicron. The virus that is the target of the prevention or treatment of the present invention is composed of all mutations described above, but is not limited thereto and includes all types of mutations that will occur in the future.

[0060]

[0061] According to another aspect of the present invention, the present invention provides a composition for diagnosing a disease related to regulation of unconventional protein secretion (UPS), which comprises as an active ingredient an agent for measuring the expression level of OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

[0062] In this specification, the meaning of “OSBLP9 protein” and “diseases related to regulation of unconventional protein secretion (UPS)” and examples of various diseases included therein have already been described above, so description thereof is omitted to avoid excessive duplication.

[0063] The term “diagnosis” as used herein includes determining the susceptibility of an individual to a particular disease, determining whether a particular disease has developed in a current individual, and determining the prognosis of an individual suffering from a particular disease.

[0064] The term “diagnostic composition” as used herein means an integrated mixture or device including a means for measuring the expression level of the OSBPL9 protein or a gene encoding it to determine whether or not a disease related to the regulation of unconventional protein secretion (UPS) in a subject has occurred or to predict the possibility of occurrence, and may also be expressed as a “diagnostic kit.”

[0065] When the disease related to the regulation of the above-mentioned unconventional protein secretion (UPS) mechanism is fibrosis, specifically cystic fibrosis, a decrease in the expression level of the OSBLP9 protein or the gene encoding it is judged to indicate the onset of the disease.

[0066] In the case where the disease related to the regulation of the above-mentioned unconventional protein secretion (UPS) mechanism is an infection by a virus that uses the unconventional protein secretion (UPS), specifically an infection by SARS-CoV-2, an increase in the expression level of the OSBLP9 protein or the gene encoding it is judged to indicate the onset of the disease.

[0067]

[0068] According to another aspect of the present invention, the present invention provides a method for providing information necessary for diagnosing a disease related to regulation of unconventional protein secretion (UPS), comprising the step of measuring the expression level of OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same in a biological sample isolated from an individual.

[0069] In this specification, the meaning of “OSBLP9 protein” and “diseases related to regulation of unconventional protein secretion (UPS)” and examples of various diseases included therein, as well as specific diseases that can be diagnosed based on an increase or decrease in the expression level of the OSBLP9 protein or the gene encoding it, have already been described above, and therefore, description thereof is omitted to avoid excessive duplication.

[0070] As used herein, the term “subject” refers to a subject that provides a sample for measuring the expression level of the OSBPL9 protein or the gene encoding it, and is ultimately the subject of analysis for the onset of a disease related to the regulation of unconventional protein secretion (UPS). The subject includes, without limitation, a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, a monkey, a chimpanzee, a baboon, or a rhesus macaque, and is specifically a human. Since the composition of the present invention provides information for predicting not only the current onset of a disease but also the risk of developing a disease in the future, the subject of the present invention may be a patient suffering from a disease related to the regulation of unconventional protein secretion (UPS) or may be a subject that has not yet developed a disease related to the regulation of unconventional protein secretion (UPS).

[0071] According to another aspect of the present invention, the present invention provides a method for preventing or treating a disease related to regulation of unconventional protein secretion (UPS), comprising the step of administering to a subject an agent for regulating the expression of OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

[0072] The agent for regulating the expression of the OSBPL9 (Oxysterol binding protein-like 9) protein or the gene encoding it used in the present invention and the disease related to the regulation of the unconventional protein secretion (UPS) mechanism to be prevented or treated using the agent have already been described above, so description thereof is omitted to avoid excessive duplication.

[0073]

[0074] According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating a disease related to the regulation of unconventional protein secretion (UPS), comprising the following steps:

[0075] (a) a step of contacting a test substance with a biological sample containing OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same;

[0076] (b) a step of measuring the expression level of the protein or the gene in the biological sample;

[0077] Diseases related to the regulation of the above unconventional protein secretion (UPS) mechanism include fibrosis or infection by a virus that utilizes the unconventional protein secretion (UPS).

[0078] If the activity or expression level of the protein or gene in the biological sample increases, the test substance is determined to be a composition for preventing or treating fibrosis.

[0079] If the activity or expression level of the protein or gene in the biological sample decreases, the test substance is determined to be a composition for preventing or treating infection by a virus that uses an unconventional protein secretion (UPS) mechanism.

[0080] In this specification, the meaning of “OSBLP9 protein” and “diseases related to regulation of unconventional protein secretion (UPS)” and examples of various diseases included therein have already been described above, so description thereof is omitted to avoid excessive duplication.

[0081] The term "biological sample" in the present invention refers to any sample obtained from a mammal, including a human, that contains cells expressing the aforementioned genes, including, but not limited to, tissues, organs, cells, or cell cultures. More specifically, the biological sample may be a tissue, cell, or culture thereof.

[0082] The term "test substance" used in referring to the screening method of the present invention refers to an unknown substance used in screening to examine whether the substance, added to a sample containing cells expressing the genes of the present invention, affects the activity or expression level of these genes. The test substance includes, but is not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of the gene in a biological sample treated with the test substance can be performed using various expression level and activity measurement methods known in the art.

[0083]

[0084] The features and advantages of the present invention are summarized as follows:

[0085] (a) The present invention provides a composition for diagnosing, preventing or treating a disease related to the regulation of unconventional protein secretion (UPS).

[0086] (b) The present invention can be usefully utilized to provide a highly reliable new target for the development of therapeutic agents for related diseases by discovering a key factor that plays an important role in the activity of a non-typical protein transport mechanism and regulating the same.

[0087]

[0088] Figure 1 depicts the members and structure of the OSBP gene family. Only OSBP family members with FFAT and PH domains were screened. OSBPL10 and OSBPL11 possess putative FFAT domains.

[0089] Figure 2 depicts the results of an experiment conducted to identify genes involved in atypical membrane transport within the OSBP gene family. Figure 2a depicts the results of immunoblotting comparing the amount of CFTR in the membrane after inducing atypical membrane transport with ΔF508-CFTR overexpression in a situation where each gene was knocked down with siRNA, and Figure 2b depicts the results quantified and represented as a bar graph.

[0090] Figure 3 is a diagram illustrating the results of an experiment confirming the degree of atypical membrane transport of CFTR in a situation where OSBPL1, OSBPL7, and OSBPL9 were overexpressed. Figure 3a is a diagram illustrating the results of immunoblotting, and Figure 3b is a diagram illustrating the quantitative results as a bar graph.

[0091] Figure 4 is a diagram showing the results of measuring the current flow of CFTR under the conditions of ΔF508-CFTR / ΔF508-CFTR+ORP9, where CFTR reaches the cell membrane and Cl -When it acts as a channel, the current flow increased. After opening the pores of CFTR with Forskolin+IBMX and measuring the current flow, we treated it with a CFTR inhibitor (CFTRinh-172) to confirm whether the current was caused by CFTR. Figure 4a shows the results confirming that CFTR current was not observed in ΔF508-CFTR overexpressing cells, and Figure 4b shows the results confirming that a CFTR current of approximately 2500 pA was measured in ΔF508-CFTR + ORP9 overexpressing cells.

[0092] Figure 5 is a drawing showing the results of measuring the current flow of CFTR when VX-809 was treated or ORP9 was overexpressed under conditions inducing ΔF508-CFTR expression. When ORP9 was treated under conditions inducing ΔF508-CFTR expression (Figure 5a), a current amount that was as much as 20 times higher was observed compared to when VX-809 was treated (Figure 5b), confirming the significantly superior cystic fibrosis treatment effect of ORP9 compared to conventional therapeutic agents.

[0093] Figure 6 shows the results of Western blot analysis to confirm the degree of atypical membrane transport of CFTR by quantifying CFTR translocated to the membrane when ORP9 was overexpressed for several mutations causing cystic fibrosis, namely N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR, and V520F-CFTR. The measurement results showed that cystic fibrosis was effectively treated when ORP9 of the present invention was overexpressed not only for ΔF508-CFTR but also for various other mutations causing cystic fibrosis.

[0094] Figure 7 is a diagram illustrating the results of measuring the current flow of CFTR through patch clamp to more closely examine the therapeutic effect of ORP9 on cystic fibrosis under conditions of inducing N1303K-CFTR expression among various additional mutations. While no CFTR current was observed in cells induced to express N1303K-CFTR (7a), a CFTR current of approximately 250 pA was measured in N1303K-CFTR + ORP9 overexpressing cells, confirming a significant recovery in the current amount (Fig. 7b).

[0095] Figure 8 is a bar graph quantifying the current flow when ORP9 is treated compared to when conventional treatments such as VX-809, VX-445 + VX-661, or PI4K2α are treated in cells expressing ΔF508-CFTR. It can be confirmed that the treatment effect of ORP9 overexpression is significantly superior to that of conventional treatments for cystic fibrosis.

[0096]

[0097] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0098]

[0099] Example

[0100] Experimental and analytical methods

[0101] Plasmid cloning and siRNA transfection

[0102] Plasmids expressing pCMV-ΔF508-CFTR were prepared according to a previously reported method. The pCMV3-OSBP, pCMV3-OSBPL1A, pCMV3-OSBPL7, and pCMV3-OSBPL9 plasmids were purchased commercially and cloned into the pCMV3 vector (Sinobiological #HG20205-UT, #MG5A7386-CM, #HG23120-U, #HG21379-CM). The pcDNA3-ARF1-Q71L-HA plasmid was commercially custom synthesized and cloned into the pcDNA3 vector.

[0103] To generate additional mutants known to cause cystic fibrosis, plasmids expressing N1303K-CFTR, ΔI507-CFTR, R560T-CFTR, R1066C-CFTR, V520F-CFTR, and A561E-CFTR were all generated by Site Directed Mutagenesis of the WT-CFTR plasmid (Biofact #LP116-500).

[0104] ON-TARGETplus human OSBP-specific, OSBP2-specific, OSBPL1A-specific, OSBPL3-specific, OSBPL6-specific, OSBPL7-specific, OSBPL9-specific, OSBPL10-specific, OSBPL11-specific and control scrambled siRNAs were purchased commercially (SMARTpool siRNAs: OSBP, gene ID 5007; OSBP2, gene ID 23762; OSBPL1A, gene ID 114876; OSBPL3, gene ID 26031; OSBPL6, gene ID 114880; OSPBL7, gene ID 114881; OSBPL9, gene ID 114883; OSBPL10, gene ID 114884; OSBPL11, gene ID 114885, Lafayette, CO, USA).

[0105] Plasmid transcription was performed using Lipofectamine LTX reagent (Invitrogen #15338500, Waltham, MA, USA), and siRNA was transcribed using Lipofectamine RNAimax transcription material (Invitrogen #13778100). Both transcriptions were performed according to the manufacturer's protocol.

[0106]

[0107] Chemical reagents and antibodies

[0108] The following antibodies were purchased commercially: anti-CFTR (Millipore #05-583, Billerica, MA, USA; Alomone Labs #ACL006, Jerusalem, Israel), anti-HA (Cell Signaling Technology #2367, Danvers, MA, USA), anti-Myc (Cell Signaling Technology #2276), anti-DYK (Cell Signaling Technology #2368), and anti-Aldolase A (Santa Cruz #sc-390733, Dallas, TX, USA).

[0109]

[0110] Surface biotinylation and immunoblotting

[0111] HEK293 cells were grown on 6-well plates washed with poly-D-lysine. Forty-eight hours after transfection, cells were washed three times with ice-cold PBS. Cell surface proteins were biotinylated with Sulfo-NHS-SS-Biotin (Thermo Pierce #21331, Waltham, MA, USA) in cold PBS for 30 min on ice in the dark. After the reaction, the cells were quenched with 1% bovine serum albumin (BSA) in PBS. The BSA solution was then washed with ice-cold PBS. Cells were lysed in staining solution [20 mM Tris (pH 7.4), 150 mM NaCl, 1% (v:v) NP40, 0.5% (v:v) sodium deoxycholate, 10% glycerol, 1 mM EDTA, and protease inhibitor mixture (Roche #04693159001)] and centrifuged at 13,200 rpm for 20 min at 4°C. After centrifugation, only the supernatant was collected, and the protein concentration was measured using the Bradford assay (Bio-RAD #5000006, Hercules, CA, USA). Next, 400 μg of protein from each sample was incubated with 300 μL 5% streptavidin agarose (Thermo Pierce #20349) at 4°C overnight. Biotinylated proteins bound to agarose were eluted with 2X SDS sample solution containing 0.02 g / ml DL-dithioiothreitol (DTT, Sigma-Aldrich #43815) at 37°C for 30 min. The whole cell lysate and eluted biotinylated samples were then loaded onto SDS-polyacrylamide gels (SDS-PAGE, KOMA BIOTECH #KG50105; Seoul, Korea), separated by gel electrophoresis, and transferred to nitrocellulose membranes (Amersham #10600004, Chicago, IL, USA).The membrane was incubated in a blocking solution containing 5% skimmed milk in TBS-T for 30 minutes and blotted with the appropriate primary and HRP-conjugated secondary antibodies. The blots were observed using enhanced chemiluminescence detection reagent (ECL, Amersham #RPN2134).

[0112]

[0113] Electrophysiology: Whole-cell recordings

[0114] CFTR channel activity was measured in HEK 293T cells using the whole-cell clamp technique. Cells were transferred to a bath mounted on the stage of an inverted microscope (Ti-2, Nikon), and whole-cell patches were made by membrane rupture after Giga Ohm sealing. Bath solution was infused at a rate of 5 mL / min. Voltage and current recordings were performed at room temperature (22–25°C). Patch pipettes with a resistance of 2–4 MΩ were connected to the head stage of a patch clamp amplifier (Axopatch-200B, Molecular Devices, Sunnyvale, CA, USA). The bath solution contained (in mM): 140 N-methyl-D-glucamine chloride (NMDG-Cl), 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, and 10 sucrose (pH adjusted to 7.4 with NMDG). The pipette solution contained (in mM): 140 N-methyl-D-glucamine chloride (NMDG-Cl), 5 EGTA, 1 MgCl2, 3 MgATP, and 10 HEPES (pH adjusted to 7.2 with NMDG). To record CFTR currents, voltage ramps from -100 to +100 mV were delivered every 10 s from a holding voltage of 0 mV. Currents were sampled at 5 kHz. All data were low-pass filtered at 1 kHz. CFTR currents were activated by cAMP (5 μM forskolin and 100 μM 3-isobutyl-1-methylxanine [IBMX]). The CFTR inhibitor CFTRinh-172 (10 μM) was applied to detect currents generated by CFTRs. Data were collected and command pulses were applied using pClamp 10.2 and Digidata 1440A (Molecular Devices). Voltage and current traces were recorded using pClamp 10.2 and Origin 8.0 (OriginLab Corp., Northampton, MA, USA) were used to store and analyze the data.

[0115]

[0116] Statistical analysis

[0117] The results of multiple experiments are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple comparison test, where appropriate. This was performed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). A p < 0.05 was considered statistically significant.

[0118]

[0119] Experimental results

[0120] Effect on ΔF508-CFTR

[0121] Cystic fibrosis is a disease caused by mutations in the CFTR gene. Specifically, in cases of misfolding mutations, the protein fails to reach the cell membrane and is degraded in the endoplasmic reticulum (ER). These mutant proteins, if they reach the cell membrane through unconventional protein secretion (UPS), can function as a channel to some extent, although with lower activity than the normal protein. This study confirmed the involvement of OSBPL1 / OSBPL7 / OSBPL9 in the UPS process of CFTR and demonstrated that overexpression of these genes can increase the UPS of CFTR.

[0122] In the CFTR UPS environment (ARF1-Q71L overexpression), changes in the UPS were confirmed by a surface biotinylation assay when the expression of the OSBP gene family (Fig. 1) was inhibited. As a result, it was confirmed that the UPS of CFTR was reduced when the expression of OSBPL1 / OSBPL7 / OSBPL9 was inhibited (Fig. 2). In addition, in the CFTR UPS environment, changes in the UPS of CFTR were confirmed by a surface biotinylation assay. As a result, it was confirmed that the overexpression of OSBPL9 significantly increased the UPS of CFTR (Fig. 3). Although the UPS of CFTR was increased when OSBPL1 and OSBPL7 were overexpressed, the results were not statistically significant. Therefore, it was confirmed that OSBPL9 is the most important gene in the atypical membrane transport among the OSBP gene family.

[0123] The activity of CFTR, which reached the cell membrane through OSBPL9 overexpression, was measured using patch clamp. This confirmed that the current flow of mutant CFTR increased in OSBPL9-overexpressing cells (Fig. 4).

[0124] To confirm the superior therapeutic effect of OSBPL9 compared to VX-809, a conventional treatment for cystic fibrosis, CFTR channel activity was measured in HEK 293T cells and HeLa cells. CFTR currents were activated by cAMP (5 μM forskolin and 100 μM 3-isobutyl-1-methyljanine [IBMX]), and the current generated by CFTRs was confirmed by applying CFTRinh-172 (10 μM), a CFTR inhibitor. When ΔF508-CFTR-overexpressing cells were treated with OSBPL9 for 48 hours (Fig. 5a), the current flow increased approximately 20-fold compared to when VX-809 was treated for 24 hours (Fig. 5b). Through this, we were able to confirm the superior therapeutic effect of OSBPL9 compared to the conventional treatment VX-809 for cystic fibrosis.

[0125]

[0126] In order to confirm the superior cystic fibrosis treatment effect of ORP9 compared to the existing treatments VX-809, VX-445+VX661, and PI4K2α, the existing treatments and ORP9 were treated in ΔF508-CFTR overexpressing cells, and the current of CFTR was compared. Compared to the existing cystic fibrosis treatments including VX-809, the highest current was measured when ORP9 was treated, confirming that ORP9 has an excellent cystic fibrosis treatment effect by increasing UPS (Fig. 8a). In the case of cystic fibrosis caused by N1303K-CFTR overexpression as well as ΔF508-CFTR, the UPS increased when ORP9 was treated, confirming that there was CFTR rescue in the cell membrane, confirming the therapeutic effect on cystic fibrosis (Fig. 8b).

[0127]

[0128] Effects on other CFTR mutations that cause cystic fibrosis

[0129] Among the various mutations reported to cause cystic fibrosis, we selected mutations with folding defects and treated them with ORP9 to determine whether ORP9 is also effective in treating cystic fibrosis caused by mutations other than ΔF508-CFTR. Mutations known to cause CF in the CFTR2 database were sorted in order of allele frequency, and mutations known to have folding defects were selected for testing. As a result, it was confirmed that the UPS was increased by ORP9 for all N1303K-CFTR, ΔI507-CFTR, R560T-CFTR, R1066C-CFTR, V520F-CFTR, and A561E-CFTR mutations, thereby restoring the amount of CFTR delivered to the cell membrane (Fig. 6).

[0130] To further investigate the therapeutic effect of ORP9 in cystic fibrosis-causing mutations, CFTR activity was measured by patch clamp in HEK 293T cells expressing N1303K-CFTR, an exemplary mutant. Similarly, CFTR currents were activated by cAMP (5 μM forskolin and 100 μM 3-isobutyl-1-methylxanine [IBMX]), and the CFTR inhibitor CFTRinh-172 (10 μM) was applied to confirm the currents generated by CFTRs. In cells overexpressing N1303K-CFTR (700 ng) for 20 hours, no CFTR current was observed (7a), whereas in cells coexpressing N1303K-CFTR (800 ng) and ORP9 (300 ng) for 24 hours, a CFTR current of approximately 250 pA was measured (Fig. 7b). This suggests that ORP9 also acts as a therapeutic agent for cystic fibrosis by increasing UPS in N1303K-CFTR.

[0131]

[0132] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing or treating a disease related to the regulation of unconventional protein secretion (UPS), comprising as an active ingredient an agent for regulating the expression of OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

2. A composition according to claim 1, characterized in that the expression regulating agent is an inhibitor or activator of the OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

3. A composition according to claim 2, wherein the disease related to regulation of the unconventional protein secretion (UPS) is fibrosis, and wherein the expression regulating agent is an expression activator.

4. A composition according to claim 2, wherein the disease related to regulation of the unconventional protein secretion (UPS) mechanism is an infection of a virus that utilizes the unconventional protein secretion mechanism, and wherein the expression regulating agent is an expression inhibitor.

5. A composition according to claim 3, characterized in that the fibrosis is cystic fibrosis.

6. A composition according to claim 5, wherein the cystic fibrosis is caused by a mutation selected from the group consisting of ΔF508-CFTR, N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR, and V520F-CFTR.

7. A composition according to claim 4, wherein the virus is SARS-CoV-2.

8. A composition for diagnosing a disease related to the regulation of unconventional protein secretion (UPS), comprising as an active ingredient a preparation for measuring the expression level of OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same.

9. A composition according to claim 8, wherein the disease related to regulation of the unconventional protein secretion (UPS) mechanism is fibrosis or an infection by a virus that utilizes the unconventional protein secretion (UPS).

10. A composition according to claim 9, characterized in that the fibrosis is cystic fibrosis.

11. A composition according to claim 10, wherein the cystic fibrosis is caused by a mutation selected from the group consisting of ΔF508-CFTR, N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR, and V520F-CFTR.

12. A composition according to claim 9, wherein the virus is SARS-CoV-2.

13. A method for providing information necessary for diagnosing a disease related to regulation of unconventional protein secretion (UPS), comprising the step of measuring the expression level of OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same in a biological sample isolated from an individual.

14. A method according to claim 13, wherein the disease related to regulation of the unconventional protein secretion (UPS) mechanism is characterized by being an infection of a virus that utilizes fibrosis or unconventional protein secretion (UPS).

15. A method according to claim 14, characterized in that the fibrosis is cystic fibrosis.

16. A method according to claim 15, wherein the cystic fibrosis is caused by a mutation selected from the group consisting of ΔF508-CFTR, N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR, and V520F-CFTR.

17. A method according to claim 14, wherein the virus is SARS-CoV-2.

18. A method for screening a composition for preventing or treating a disease related to the regulation of unconventional protein secretion (UPS), comprising the following steps: (a) a step of contacting a test substance with a biological sample containing OSBPL9 (Oxysterol binding protein-like 9) protein or a gene encoding the same; (b) a step of measuring the expression level of the protein or the gene in the biological sample; Diseases related to the regulation of the above unconventional protein secretion (UPS) mechanism include fibrosis or infection by a virus that utilizes the unconventional protein secretion (UPS). If the activity or expression level of the protein or gene in the biological sample increases, the test substance is determined to be a composition for preventing or treating fibrosis. If the activity or expression level of the protein or gene in the biological sample decreases, the test substance is determined to be a composition for preventing or treating infection by a virus that uses an unconventional protein secretion (UPS) mechanism.

19. A method according to claim 18, characterized in that the fibrosis is cystic fibrosis.

20. A method according to claim 19, wherein the cystic fibrosis is caused by a mutation selected from the group consisting of ΔF508-CFTR, N1303K-CFTR, I507del-CFTR, R560T-CFTR, R1066C-CFTR, A561E-CFTR, and V520F-CFTR.

21. A method according to claim 18, wherein the virus is SARS-CoV-2.

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