Nucleic acid molecule for reducing size or volume of target tissue containing adipocytes, composition containing same, or use thereof for reducing adipocytes

Abstract

The present invention provides a nucleic acid molecule comprising: (i) a first open reading frame comprising a polynucleotide encoding one or more viral replication proteins; and (ii) a second open reading frame comprising a polynucleotide encoding an antigen protein for reducing adipocytes, wherein the antigen protein for reducing adipocytes is a varicella-zoster virus (VZV) protein. In addition, provided is a use of the nucleic acid molecule of the present invention or a composition comprising same for reducing adipocytes.

Description

Nucleic acid molecules for reducing the size or volume of target tissues containing fat cells, compositions containing the same, or the use of the same for reducing fat cells

[0001] The present application claims priority based on Korean Patent Application No. 10-2025-0007347 filed on January 17, 2025, and all contents disclosed in the specification and drawings of said application are incorporated by reference into the present application. The present invention relates to a nucleic acid molecule for reducing the size or volume of fat cells, a composition containing the same, or the use of the same for reducing fat cells, and more specifically, to samRNA having the effects of causing apoptosis of fat cells, inhibiting the accumulation of fat cells, and reducing fat cells, a composition containing the same, or the use of the same for reducing fat cells.

[0002] Obesity refers not merely to having a high body weight, but to a condition in which an excessive amount of body fat accumulates within the body. It is recognized as a risk factor that increases the incidence of various chronic diseases, including metabolic disorders such as diabetes, cardiovascular diseases, and cancer. Defined by the excessive accumulation of fat, obesity is accompanied by the loss of metabolic, endocrine, and immune functions in adipose tissue; consequently, the pathological remodeling of this adipose tissue is receiving attention as a major pathophysiological factor in metabolic diseases. As an energy storage organ and a vital endocrine system, adipose tissue plays a key role in the onset and exacerbation of various metabolic diseases, such as obesity, type 2 diabetes, fatty liver disease, and cardiovascular disease. Excessive accumulation and abnormal remodeling of adipose tissue are known to induce insulin resistance, chronic inflammation, and lipid abnormalities, thereby severely impairing systemic metabolic homeostasis. Consequently, there is a continuously increasing demand for new therapeutic strategies capable of directly controlling the volume and function of adipose tissue.

[0003] Drug treatment for obesity utilizes the lipase inhibitor 'Orlistase,' which works by facilitating the elimination of some body fat; however, side effects include diarrhea and steatorrhea. Furthermore, obesity medications not only carry numerous side effects but can also break down fat in unwanted areas of a woman's body. Currently, there are no appetite suppressants available for safe use, although gastrointestinal bariatric surgery may be helpful for patients with severe obesity and complications.

[0004] Meanwhile, nucleic acid-based therapeutics, including mRNA and self-amplifying mRNA (samRNA), serve as platforms capable of transiently expressing therapeutic proteins within cells, and active efforts are being made to expand their use to various indications, such as metabolic diseases, cancer, and rare diseases, in addition to vaccines.

[0005] In addition, research is actively underway not only on technologies to address excessive fat accumulation but also on technologies for cosmetic purposes aimed at reducing localized fat in specific areas, even in individuals with normal weight. Meanwhile, while technologies to prevent fat formation are being developed, technologies to reduce the size of existing fat cells or adipose tissues are still areas requiring significant research. SamRNA-based approaches specialized for directly reducing the size of adipose tissue or the number of fat cells have not been sufficiently developed. Although some studies have reported attempts to regulate function by delivering RNA to fat cells or adipose-derived stem cells, these approaches primarily focus on improving localized functions such as wound healing or tissue regeneration, and thus have limitations as a technical means to selectively reduce the size of adipose tissue for the purpose of treating obesity.

[0006]

[0007] To solve the aforementioned problems, the present invention aims to provide a new method or composition capable of reducing the size or volume of fat cells by administering it to tissues containing fat cells. The problem that the present invention seeks to solve is to provide a new type of obesity treatment or therapeutic vaccine capable of resolving localized obesity and achieving a smooth body line.

[0008] The problem that the present invention aims to solve is to provide a novel use of the above composition capable of reducing, decreasing, or killing the size of a desired cell or tissue by administering an exogenous antigen.

[0009] One embodiment of the present invention provides a nucleic acid molecule comprising (i) a first open reading frame (ORF) comprising a polynucleotide encoding one or more viral replication proteins; and (ii) a second open reading frame (ORF) comprising a polynucleotide encoding an antigen protein for reducing fat cells, wherein the antigen protein for reducing fat cells is a varicella-zoster virus (VZV) protein; a composition in which the nucleic acid molecule and lipids are formulated; a use of the nucleic acid molecule or the composition for reducing fat cells; a method for reducing fat cells by administering the nucleic acid molecule or the composition; and a kit comprising the nucleic acid molecule or the composition.

[0010] The present invention was conceived to reduce the size or volume of fat cells in a desired area, or to reduce the number of fat cells, by locally administering a novel nucleic acid molecule.

[0011]

[0012] This is explained in detail below.

[0013]

[0014] [Nucleic acid molecule]

[0015] One embodiment of the present invention provides a nucleic acid molecule comprising (i) a first open reading frame comprising a polynucleotide encoding one or more viral replication proteins; and (ii) a second open reading frame comprising a polynucleotide encoding an antigen protein for reducing adipocytes, wherein the antigen protein for reducing adipocytes is a varicella-zoster virus (VZV) protein.

[0016] The term 'open reading frame (ORF)' refers to a sequence of nucleotides that can be translated, which generally starts with a start codon, such as ATG (AUG in RNA), and ends with a stop codon, such as TAA, TAG, or TGA (such as UAA, UAG, UGA, etc. in RNA).

[0017] The first ORF of the nucleic acid molecule comprises a polynucleotide encoding one or more viral replication proteins. Here, the viral replication proteins include viral replication proteins of alphaviruses as viral non-structural proteins (nsPs), and preferably may include a combination of nsP1, nsP2, nsP3, and nsP4 proteins derived from Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), or a combination thereof. The first ORF may include viral replication proteins derived from Venezuelan equine encephalitis virus (VEEV) capable of strongly expressing antigens with high replication power. For example, the TC-83 VEEV strain may be included in the virus. In one embodiment of the present invention, the nsP2 of the TC-83 VEEV strain of the Venezuelan equine encephalitis virus may have a mutation. Preferably, the nsP2 protein of the VEEV strain may have a Q739L mutation. In another embodiment, the nsP3 protein of the TC-83 VEEV strain of the Venezuelan equine encephalitis virus may have a mutation. Preferably, the Opal (UGA) termination codon of the VEEV nsP3 protein may be mutated into a codon encoding arginine (R). For example, arginine codons may include CGU, CGC, CGA, CGG, AGA, AGG, etc. Such mutations are advantageous for the amplification of replicons and may be advantageous for achieving the objectives of the present invention. Specifically, the Q739L mutation can reduce both Grna (genomic RNA) and sgRNA (subgenomic RNA) and reduce the cytopathic effect (CPE). Furthermore, such mutations of the nsP3 protein can increase the replication ability of the virus and induce a reduction of gRNA and sgRNA.

[0018]

[0019] The second ORF of the above nucleic acid molecule contains a polynucleotide encoding an antigen protein for adipocyte reduction, and the antigen protein for adipocyte reduction may include the varicella-zoster virus (VZV). The varicella-zoster virus (VZV) is a double-stranded DNA virus belonging to human herpesvirus 3 (HHV-3), has a linear double-stranded DNA genome of approximately 125 kbp, and expresses various structural and non-structural proteins through more than 70 ORFs. VZV glycoprotein E (gE) is a major envelope glycoprotein encoded by ORF68 and is an immune-dominant antigen most abundantly expressed on the surface of infected cells. Anti-gE antibodies and gE-specific T-cell responses are known to play an important role in protective immunity against VZV, and recombinant gE proteins or gE-based vaccines are used for the prevention of herpes zoster. The inventors of the present invention have for the first time discovered that VZV glycoprotein E, used for the prevention of herpes zoster, is particularly effective in reducing adipocytes by optimizing its codons. In particular, they have for the first time discovered that VZV glycoprotein E exhibits an excellent effect in reducing adipocytes when provided as mRNA, more preferably as self-replicating RNA. The VZV glycoprotein E has excellent stability.

[0020] The second ORF above may provide a polynucleotide composed of or containing a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO. 8.

[0021] The term "codon optimization" as used herein refers to the substitution of a polynucleotide, nucleic acid sequence, or coding sequence to optimize protein expression while maintaining the amino acid sequence of the translated protein identical to that of the wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence, by selecting a different codon without altering the amino acid sequence of the coded protein. Methods for codon optimization may utilize conventional methods in the industry and may be used without limitation to the extent that they do not impede the purpose of the present invention.

[0022] In one embodiment, the nucleic acid molecule comprises a 5'-untranslated region (UTR). The 5'-UTR may comprise a viral 5'-UTR, a non-viral 5'-UTR, or a combination of viral and non-viral 5'-UTR sequences, and a sequence having at least 80% sequence identity with these, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the 5'-UTR may comprise an alphavirus 5'-UTR, or a 5'-UTR having at least 80% sequence identity with it. Preferably, the alphavirus may include the 5'-UTR of the Venezuelan equine encephalitis virus (VEEV) of SEQ ID NO. 5 or a 5'-UTR having at least 80% sequence identity with it. Preferably, the 5'-UTR may include an a3g variant in the polynucleotide of the Venezuelan equine encephalitis virus (VEEV), and such a variant may be advantageous for evading the interferon (IFN) response, inducing an increase in sgRNA to increase the stability of the nucleic acid molecule, and increasing the replication of the nucleic acid molecule. That is, it is less sensitive to IFN, which is advantageous for immune evasion, and more subgenome RNA is produced, leading to increased structural gene expression.

[0023] In one embodiment, the nucleic acid molecule comprises a 3'-untranslated region (UTR). In one embodiment, the 3'-UTR may comprise the sequence of SEQ ID NO. 6, or a sequence having at least 80% sequence identity with these, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. The 3'-UTR sequence comprises a poly-A sequence at its end.

[0024]

[0025] The RNA molecule comprises a 5' untranslated region (5'-UTR), the first open reading frame, the second open reading frame, a 3'-untranslated region (3'-UTR), and a poly-A sequence, which can be operably connected in the 5'→3' direction. Preferably, additional sequences may be included between the first ORF and the second ORF, for example, an intergenic region (IGR) and a Kozak sequence may be further included. The additional sequences may be included to increase the transcription and translation efficiency of the target gene and to improve the protein expression rate, and may be included without limitation within a range that does not impede the purpose of the present invention.

[0026] For example, the intergene region may include polynucleotides represented by SEQ ID NO. 10, polynucleotides with sequence homology of 80% or more, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and the Kozak sequence may be a sequence commonly known in the industry, for example, the Kozak sequence mentioned in Mol Vis. 2021 May 8;27:233-242., or a sequence with sequence homology of 80% or more.

[0027]

[0028] The nucleic acid molecule may be an RNA molecule that is an RNA replicon, self-replicating RNA, or self-amplifying RNA, or a DNA molecule; preferably, it may be an RNA molecule, and more preferably, self-amplifying RNA. In particular, when administered to fat cells as samRNA, the effect of reducing fat cells may be sustained.

[0029] The above nucleic acid molecule is an RNA molecule, and provides a nucleic acid molecule comprising or composed of a nucleic acid sequence that is at least 80%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO. 4. The RNA molecule may comprise a 5' cap, and the 5' cap may have a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, a Cap 0 structure, or any combination thereof. For example, a sequence having at least 80% homology with SEQ ID NO. 4 may comprise or be composed of any one polynucleotide sequence selected from the group consisting of SEQ ID NOs 11 to 16.

[0030] In another embodiment, the nucleic acid molecule may be a DNA molecule. The DNA molecule further comprises a promoter, and the DNA molecule may comprise or be composed of a nucleic acid sequence identical to SEQ ID NO. 2 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereof. Here, the promoter is located at the 5' end of the 5'-UTR. The promoter may comprise a T7 promoter, a T3 promoter, or an SP6 promoter, and preferably may comprise a T7 promoter.

[0031] The nucleic acid molecules of the present invention may include fragments, variants, or derivatives thereof.

[0032] Herein, the term ‘fragment, variant, or analog thereof’ may be understood to mean a part of a protein, peptide, polynucleotide, nucleic acid, or nucleic acid molecule that achieves the purpose intended by the protein, peptide, polynucleotide, nucleic acid, or nucleic acid molecule of the present invention, and may have sequence homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0033] The above nucleic acid molecule may have an excellent effect on target tissues, preferably on the induction of adipocytes and the reduction of tissue size containing said adipocytes. Furthermore, it exhibits excellent sustained effect in reducing adipocytes and good protein expression efficiency, while having few side effects. In addition, there is little risk of deterioration even when stored as an injectable for a long period.

[0034]

[0035] [use]

[0036] One embodiment of the present invention aims to provide a new use for reducing fat cells by administering the nucleic acid molecule to a target tissue. Another embodiment of the present invention aims to provide a composition for reducing fat cells, specifically a pharmaceutical composition, comprising the nucleic acid molecule. Another embodiment of the present invention aims to provide a method for reducing fat cells by administering the nucleic acid molecule to an individual in need thereof. The target tissue comprises fat cells. One embodiment of the present invention provides a composition for reducing the size or volume of a tissue containing fat cells (preferably a vaccine composition), and a composition for improving, suppressing, or treating obesity, wherein when an antigen gene is expressed in the cells constituting the target tissue by the administered nucleic acid molecule, a pre-existing immunity acts upon it to kill the corresponding cells.

[0037] More specifically, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of obesity, a composition for the removal of subcutaneous fat cells, a composition for reducing tissue size or volume (preferably a vaccine composition), and a composition for improving, inhibiting, or treating obesity, comprising the above-mentioned nucleic acid molecule. Preferably, the composition is a pharmaceutical composition and may include a vaccine composition.

[0038] One embodiment of the present invention confirms that a virus or a composition containing a virus is administered to a target tissue, and that antigen genes are expressed in the cells constituting the target tissue by the administered nucleic acid molecules, thereby reducing the size of fat, particularly subcutaneous fat cells. Furthermore, the invention provides a novel concept of a method for reducing tissue size or volume, and a method for improving, suppressing, or treating obesity, wherein a preexisting immune response acts to kill the corresponding cells after the composition is administered.

[0039] One embodiment of the present invention aims to provide a novel use of a composition comprising said nucleic acid molecule, wherein said nucleic acid molecule is administered to a target tissue, and an antigen gene is expressed in the cells constituting said target tissue by the administered nucleic acid molecule, thereby reducing the size of fat, particularly subcutaneous fat cells. The said use provides a use for reducing tissue size or volume by causing the corresponding cells to die through the action of a pre-existing immune response (pre-existing immunity) after the composition is administered, and a use for improving, suppressing, or treating obesity.

[0040] The inventors of the present invention have completed the invention by confirming that the tissue composed of said cells can be reduced by decreasing the cells constituting the target site using an immune response induced by the nucleic acid molecules of the present invention. In particular, through experiments, it has been confirmed that the present invention enables the effective removal and / or reduction of target cells, thereby demonstrating body shape correction and obesity treatment effects.

[0041] The above-mentioned reduction of fat cells may refer to a reduction in the volume or mass of fat cells, and may also include a reduction in the number of fat cells. Here, the reduction of fat cells may result in a decrease in the volume or size of the target tissue containing the fat cells. The tissue may be understood as a tissue containing fat cells. The immune action of the present invention may reduce the size of fat cells or adipose tissue. For example, the fat cells or adipose tissue may more preferably be subcutaneous adipose tissue. The composition may induce a reduction, shrinkage, or death of fat cells, particularly those distributed in subcutaneous fat.

[0042] The inventors of the present invention provide a composition or kit that has fewer side effects compared to existing liposuction surgeries, drugs, etc., and can be expected to provide a body shape correction effect by targeting only a local target area (e.g., adipose tissue in a specific area). The present invention provides a pharmaceutical composition that acts locally on a target area and reduces the size or volume of the target tissue using immunotherapy. The present invention may preferably provide a pharmaceutical composition, a vaccine composition, or a therapeutic vaccine composition that induces the death of fat cells, particularly subcutaneous fat cells. The present invention may provide a pharmaceutical composition, a vaccine composition, or a therapeutic vaccine composition that acts locally on a target area and improves, suppresses, or treats obesity using immunotherapy.

[0043] As used in this specification, "target tissue or target site" refers to a group of cells having the same structure and function for reducing size or volume. The "body shape correction or body contouring" means killing or reducing cells constituting tissue (e.g., adipose tissue) accumulated in unwanted areas, thereby reducing the size or volume of the tissue and achieving an externally slim and balanced effect. As used in this specification, "acts locally" means bringing about the effect of cell death or reduction of tissue size or volume within a radius of 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm relative to the injection site, and preferably within a range of 3 cm.

[0044] When an individual is exposed to a virus-derived antigen, immune cells functionally perform the functions of phagocytosis and antigen presentation. In one embodiment, after an immune environment is established by intentionally or unintentionally introducing a foreign substance into the body to induce an immune response, the nucleic acid molecule of the present invention, or a composition containing the nucleic acid molecule, may be administered. In another embodiment, the nucleic acid molecule or composition may be repeatedly administered into the body. In another embodiment, the nucleic acid molecule of the present invention is administered to an individual to induce pre-existing immunity, and subsequently, through repeated administration, the fat cells to which the antigen was presented may die due to substances secreted by immune action, such as monocytes, neutrophils, natural killer cells (a type of lymphocyte), cytotoxic T cells, physiologically active substances, antibodies, and complement, thereby reducing the size or volume of the adipose tissue. The above composition can be used for body shape correction or body shape correction purposes, and can be used for body shape correction of localized areas.

[0045]

[0046] [Composition]

[0047] The above nucleic acid molecule may be provided in a formulation for intracellular delivery to target tissues. Suitable carriers may be provided for sufficient delivery and expression of the nucleic acid molecule, and may include, for example, ionic liposomes, pH-responsive liposomes, lipid nanoparticles, nanostructured lipid carriers (NLC), cationic polymers (PEI, PLL, PAMAM, poly(β-amino ester), PLGA-based polymers, etc.). Preferably, the nucleic acid molecule of the present invention may be provided in a complex with lipid nanoparticles (LNP). In one embodiment, the LNP complexed with the nucleic acid molecule may comprise a mixture of (i) at least one cationic lipid, (ii) distearoylphosphatidylcholine (DSPC); (iii) cholesterol; and (iv) PEG-lipid. The cationic lipid is an ionizable cationic lipid. In another aspect, the lipid nanoparticle comprises (i) at least one cationic lipid, (ii) distearoylphosphatidylcholine (DSPC); and (iii) cholesterol; (iv) The PEG-lipid may be composed of approximately 40 mol% to approximately 60 mol% of the ionizable cationic lipid, approximately 4 mol% to approximately 16 mol% of DSPC, approximately 30 mol% to approximately 47 mol% of cholesterol, and approximately 0.5 mol% to approximately 5 mol% of PEG-lipid. In another embodiment, it may be composed of approximately 40-70 mol% of the ionizable cationic lipid; approximately 2-15 mol% of DSPC; approximately 20-45 mol% of cholesterol; and approximately 0.5-3 mol% of PEG-lipid. In one embodiment, to achieve the purpose of the present invention and ensure the stability of the nucleic acid molecule, the composition has an N / P molar ratio of nucleic acid molecules to lipids (molar ratio of the number of moles of protonable amine nitrogen N of cationic lipids to the number of moles of negatively charged phosphate P of mRNA nucleotides) of 0.1 to 10:1, preferably 0.5 to 9:1, preferably 1 to 8:1, and preferably 0.It may be included in a ratio of 5 to 7:1.

[0048] The above composition may further include a nucleic acid molecule encoding one or more cytokines selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-alpha, INF-beta, TNF-alpha, and TNF-beta, and it may be preferable to include IL-12 to further maximize the immune-stimulating effect by the antigen expressed by the nucleic acid molecule.

[0049] The above composition may be provided by being included in a vector that delivers the nucleic acid molecule to an animal cell. The nucleic acid may be provided by being included in an expression vector, such as a plasmid, and preferably, the nucleic acid may include transcription factors suitable for expression in mammalian cells, such as human cells. The "vector" as used herein may transport genetic material to be administered into a cell. The vector may be used for the delivery of the nucleic acid molecule. Preferably, such a vector may include a coding sequence operably linked to an expression control sequence. Preferably, the nucleic acid molecule may be operably linked to the vector.

[0050] In another embodiment, the composition may further enhance the effects of adipocyte apoptosis or target tissue reduction by including an adjuvant capable of enhancing a cellular immune response. The composition may further include a nucleic acid molecule encoding an adjuvant capable of enhancing the immune-enhancing effect, for example, an auxiliary nucleic acid molecule encoding interleukin 12 (IL-12). The auxiliary nucleic acid molecule may be provided, for example, by inserting a coding region into a pSF-CMV-CMV-Sbfl vector.

[0051] The composition of the present invention may be used with the cytokine, the gene encoding it, or other adjuvants, as well as with pharmaceutical carriers or excipients as widely used in the art. The composition may be formulated for human or veterinary use and administered via various routes. Routes of administration may include, but are not limited to, oral, transdermal, intramuscular, peritoneal, intravenous, and subcutaneous routes. More preferably, transdermal, intramuscular, peritoneal, and subcutaneous routes may be used. Furthermore, the composition may be administered by any device or route capable of delivering the active substance to target cells, and the method of administration is not particularly limited as long as the composition of the present invention or nucleic acid molecules contained in the composition can be delivered to target tissues or cells constituting the tissues. For example, it may be provided in the form of an injection, a microneedle patch, etc. For example, when used as an injection, the injectable formulation may be prepared using aqueous solvents such as physiological saline solution or Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), within a range that does not impair the effect of the nucleic acid molecule, and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.). When used as a microneedle patch, the microneedle may include both insoluble and soluble microneedle.

[0052] In addition, nucleic acid molecules and / or auxiliary genetic material included in the above composition may be delivered into the body according to conventional gene delivery methods in the industry. Non-limiting examples include physical delivery methods such as electroporation or gene guns, and chemical delivery methods such as lipid-DNA complexes (Lipoplex), polymer-DNA complexes (Polyplex), liposomes, dendrimers, nanoparticles, or other suitable transfer vectors.

[0053] The above composition may include, without limitation, ingredients commonly used in the industry for formulation, to the extent that such inclusion does not impede the purpose of the present invention. For example, to be provided as a freeze-dried composition, it may include a freeze-drying protective agent and may include conventional buffers, etc.

[0054] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means a sufficient amount of the composition to produce a therapeutic, corrective, or preventive effect, or a vaccine effect, and also means an amount that does not cause side effects or serious or excessive immune responses. Due to its characteristics intended to induce an immune response and induce apoptosis, the composition of the present invention may administer nucleic acid molecules (preferably samRNA). Such administration may be systemic or topically administered. Such administration may include application to the skin. The subject to which the administration is performed may be a subject requiring reduction of adipose tissue. Such subject may include, but is not limited to, mammals, specifically, humans, dogs, cats, rabbits, mice, cattle, horses, sheep, etc. The administration of the composition according to one embodiment is 0.1 ug to 1,000 ug, for example, 0.1 ug to 500 ug, 0.1 ug to 100 ug, 0.1 ug to 50 ug, 0.1 ug to 25 ug, 1 ug to 1,000 ug, 1 ug to 500 ug, 1 ug to 100 ug, 1 ug to 50 ug, 1 ug to 25 ug, 1 ug to 20 ug, 1 ug to 15 ug, 1 ug to 10 ug, 1 ug to 8 ug, 2 ug to 1,000 ug, 2 ug to 500 ug, 2 ug to 100 ug, 2 From ug to 50 ug, from 2 ug 25 µg, 2 µg to 1,000 µg, 2 µg to 500 µg, 2 µg to 100 µg, 2 µg to 50 µg, or 2 µg to 25 µg may be administered. However, the dosage may vary depending on factors such as the formulation method, method of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors.The administration frequency may be once a day or two or more times within the range of clinically acceptable side effects. Regarding the injection site, it may be administered to one or two or more sites. For example, it may be administered to localized areas such as submental fat, abdominal fat, and forearm fat, and may be administered to one or more sites simultaneously. The administration interval may be daily, or at intervals of 2 to 5 days, at intervals of 1 week or more, 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, 6 months or more, or 1 year or more. If necessary, the same administration may be repeated after the appropriate period. The appropriate dosage may be adjusted for each site.

[0055] Another embodiment of the present invention provides a method for reducing or decreasing the size or volume of tissue, comprising the step of administering the nucleic acid molecule, the vector containing the nucleic acid molecule, or the composition containing the same to a target tissue of an individual. The method may preferably kill cells constituting subcutaneous fat tissue, reduce fat weight, or reduce the size of cells. The method i); or ii) the nucleic acid molecule, the vector containing the nucleic acid molecule, or the composition containing the same, or the kit containing the same may be used for the purpose of improving or treating obesity in an individual, for the purpose of removing fat pockets under the eyes for cosmetic purposes, and thereby for the purpose of alleviating dark circles.

[0056] Another embodiment of the present invention may further include a step of creating an immune environment within the body before administering the nucleic acid molecule, or a composition containing the same, to a target tissue. The step of creating an immune environment within the body may include a step of administering an external substance capable of inducing an immune response. The external substance may be included without limitation as long as it is a substance that utilizes a protein as an antigen. It is clearly understood that the step of creating an immune environment is not an essential process for implementing the present invention.

[0057] One embodiment of the present invention may be provided for the treatment, improvement, or prevention of various indications, as well as for cosmetic purposes to improve appearance.

[0058] One embodiment of the present invention may be provided as a vaccine for treating obesity. The therapeutic vaccine can be defined as a vaccine in that it utilizes an immune response within the body, and can be defined as a therapeutic agent in that it affects already formed fat, fat cells, or adipose tissue to reduce their size or cause cell death. Therefore, the obesity treatment vaccine can be understood as a concept distinct from a preventive agent that merely inhibits the formation of fat cells or inhibits the accumulation of fat.

[0059] Another embodiment of the present invention provides a kit comprising (a) the nucleic acid molecule described above, or a composition comprising said nucleic acid molecule; and (b) an administration guide for administering said nucleic acid molecule, or the composition of (a). The kit may further comprise (c) a composition comprising a protein antigen or an epitope thereof for creating an immune environment prior to administration of said composition of (a). Herein, said (c) may be understood as at least one pre-vaccine or composition for inducing an immune response.

[0060] The "pre-vaccine" referred to herein may be understood as a vaccine composition administered prior to the administration of the "pharmaceutical composition for improving, suppressing, or treating obesity" or the "composition for reducing the size or volume of a target tissue" of the present invention in order to obtain pre-existing immunity. The said pre-vaccine may include, but is not limited to, an antigenic material identical to the nucleic acid molecule contained in the "pharmaceutical composition for improving, suppressing, or treating obesity" or the "composition for reducing the size or volume of a target tissue" of the present invention, and may include, for example, a vaccine administered during infancy to induce the possession of antibodies in the body.

[0061] The above composition (a) may further include a nucleic acid molecule encoding a cytokine, and a substance that can be used to enhance the immune effect of the above composition (a) may be included without limitation. The nucleic acid molecule encoding the cytokine may be replaced with a protein expressed by the above nucleic acid molecule. The cytokine may include one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-alpha, INF-beta, TNF-alpha, and TNF-beta, and in addition to the above cytokine, cytokines known in the industry may be included without limitation.

[0062] In the kit of the present invention, at least one additional agent as defined herein in relation to the pharmaceutical composition, antimicrobial agent, DNase inhibitor, RNase inhibitor, solubilizer, etc. may be included. The kit may consist of, for example, one or more kit components (e.g., containers). For example, each container may be a syringe, pre-filled syringe, vial, bottle, jar, sealed sleeve, envelope or pouch, tube or blister package, or any other suitable form configured such that the container prevents premature mixing of the components. Each of the different components may be provided individually, or some of the different components may be provided together (i.e., within the same container). The kit may also include a dosing instruction sheet having information on the administration, method of administration, and dosage of any component.

[0063] Another embodiment of the present invention provides a syringe filled with the nucleic acid molecule or a composition for improving, inhibiting, or treating obesity comprising the nucleic acid molecule, preferably a vaccine composition. Another embodiment of the present invention provides a syringe filled with a pharmaceutical composition for reducing the size or volume of a target tissue, preferably a vaccine composition, comprising the nucleic acid molecule of the present invention. A composition for improving, inhibiting, or treating obesity, preferably comprising the nucleic acid molecule of the present invention; or a syringe filled with a vaccine composition for reducing the size or volume of a target tissue. The syringe may further comprise a nucleic acid molecule encoding a cytokine. The cytokine may comprise one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-alpha, INF-beta, TNF-alpha, and TNF-beta.

[0064] One embodiment of the present invention aims to provide a pharmaceutical composition for improving, inhibiting, or treating obesity, comprising a nucleic acid molecule of the present invention.

[0065] The above pharmaceutical composition can reduce the size or volume of a target tissue.

[0066] The nucleic acid molecules of the present invention or compositions containing the same can induce an intracellular immune response.

[0067] The above composition acts locally, and the composition may be applied after at least one antigen for creating an immune environment has been applied to the individual first, but is not limited thereto.

[0068] The nucleic acid molecules of the present invention or compositions containing the same may be administered to target tissues by injection via a transdermal, muscular, peritoneal, intravenous, or subcutaneous route, or via electroporation, a gene gun, liposomes, dendrimers, nanoparticles, or a transfer vector.

[0069] The above composition is administered to an individual for the purpose of reducing, shrinking, or eliminating the size of adipose tissue, and the above composition may be administered at least once to a site for the purpose of reducing, shrinking, or eliminating the size of adipose tissue.

[0070] The above composition may be provided as a vaccine, such as a therapeutic agent, a therapeutic vaccine, or a preventive vaccine.

[0071] Another embodiment may provide a kit for providing the above composition. In addition to the composition, the kit may include instructions for administration of the composition.

[0072] The above kit may further include an additional composition comprising an antigen for establishing an immune environment in the body prior to administering the above composition. The above kit may further include a nucleic acid molecule encoding a cytokine.

[0073] Another embodiment may provide a pharmaceutical composition for reducing the size or volume of a target tissue, comprising a nucleic acid molecule of the present invention.

[0074] The above pharmaceutical composition can reduce the number of fat cells or the size of adipose tissue, or induce apoptosis of said cells or tissue.

[0075] According to one embodiment, the present invention provides a method for reducing the size or volume of a target tissue by administering the composition to an individual in need thereof. Another embodiment provides a method for suppressing, improving, or treating obesity by administering the composition to an individual in need thereof. Herein, the individual may include mammals without limitation, and may include, for example, dogs, cattle, pigs, horses, humans, etc., and preferably humans.

[0076] In one embodiment, the method may provide a method for improving, inhibiting, or treating obesity, or a method for reducing, shrinking, or killing adipose tissue, comprising the step of administering the nucleic acid molecule or a composition containing the nucleic acid molecule to an individual intended for reducing, shrinking, or killing the size of adipose tissue.

[0077] The above method may repeat the administration step one or more times, two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times, preferably two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times, more preferably three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times. Preferably, the administration step may be repeated at intervals of 1 to 10 days, 2 to 8 days, or 3 to 7 days.

[0078] The composition used in the above administration step may further include one or more cytokines, and preferably, said cytokines may include any one or more cytokines selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-alpha, INF-beta, TNF-alpha, and TNF-beta. Alternatively, the method may further include a step of administering a composition containing one or more cytokines after the administration of said nucleic acid molecule or composition, and preferably, said cytokines may include any one or more cytokines selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-alpha, INF-beta, TNF-alpha, and TNF-beta. That is, said cytokines may be administered together with or sequentially with the nucleic acid molecule of the present invention or the composition containing said nucleic acid molecule.

[0079] One embodiment of the present invention provides a use for improving, inhibiting, or treating obesity with said nucleic acid molecule or a composition containing said nucleic acid molecule. Another example provides a use for reducing the size or volume of a target tissue, including fat cells, with a composition comprising one or more viruses selected from the group consisting of yellow fever, herpes zoster, and rubella viruses; said virus-derived proteins; or a nucleic acid molecule encoding said virus or protein.

[0080] The above application may be used to reduce the size or volume of a target tissue. Preferably, the target tissue may include a tissue containing fat cells.

[0081] The above composition may be administered to act locally, and the composition may be applied after at least one antigen for creating an immune environment has been applied to the individual first.

[0082] In one embodiment, the nucleic acid molecule or a composition containing the same is administered to an individual intended for the reduction, shrinkage, or elimination of the size of adipose tissue, and may be administered at least once to a site intended for the reduction, shrinkage, or elimination of the size of adipose tissue.

[0083] The present invention can bring about an effect of reducing the size or volume of tissue in a specific targeted area. The present invention provides a novel composition for improving or treating obesity, or a therapeutic vaccine composition, capable of killing cells in the target tissue and reducing or decreasing the size or volume of the tissue. By administering the composition, a body shape correction effect or a body shape improvement effect can be obtained.

[0084] The present invention can provide a new type of obesity treatment that can resolve localized obesity and achieve a smooth body line.

[0085] The composition of the present invention for improving, inhibiting, or treating obesity is effective for selective local reduction, capable of selectively reducing only the tissue of a desired area without affecting the tissue or cells constituting the unwanted area. Additionally, the composition of the present invention can also be used for cosmetic purposes to improve appearance.

[0086] The present invention has excellent sustained expression of antigen proteins and can gradually reduce fat cells. When cell death occurs due to a strong and radical reaction such as necrosis, severe side effects such as edema or inflammation may occur. Additionally, when remodeling occurs after the destruction of fat cells or tissues, fibrosis may develop, causing problems with the regeneration of normal tissues and resulting in an uneven skin surface. The present invention can reduce such side effects through a gradual reaction.

[0087] The present invention has an excellent antigen protein expression rate and an outstanding fat cell reduction effect.

[0088] Figure 1 is a schematic gene map of the samRNA construct of the present invention.

[0089] Figure 2 shows the results of confirming the expression test of samRNA in a target tissue obtained according to an embodiment of the present invention using an immunofluorescence assay (IFA). It can be confirmed that VZVgE protein expression occurs in the target tissue.

[0090] Figure 3 is a graph showing the results of confirming the number of VZVgE proteins expressed in Vero cells by construct. The horizontal axis represents days, and the vertical axis represents the Object sum integral (au (arbitrary units)).

[0091] Figure 4 shows the results of confirming the in vitro expression levels of conventional mRNA (mRNA) and self-amplifying mRNA (samRNA). The levels of fluorescence expression within cells were quantified after treatment with mRNA at doses of 500 ng and 50 ng, and samRNA at doses of 500 ng, 250 ng, and 50 ng.

[0092] Figure 5 shows the results of photographing the fluorescence brightness on day 1, day 2, day 3, and day 4 after treatment with mRNA at doses of 500 ng and 50 ng, and samRNA at doses of 500 ng, 250 ng, and 50 ng.

[0093] Figure 6 shows the results of confirming the degree of weight reduction in adipose tissue after administering 2 μg each of mRNA and samRNA to the adipose tissue of obese mice. Even though a high-fat diet was continued after administration, the fat weight was not completely recovered and the high efficacy was continuously maintained.

[0094] Figure 7 shows the results of examining adipose tissue by necropsying obese mice after substance administration. Left inguinal fat and right inguinal fat isolated within a single individual were observed on each day.

[0095] Figure 8 shows changes in adipose tissue after injecting VZV-gE samRNA into the inguinal fat pad. This is the result of observing changes in protein expression and cell morphology in adipose tissue isolated by necropsy of obese mice after water administration. Histological findings of the adipose tissue and the expression of Varicella-Zoster Virus gE protein were confirmed by immunohistochemistry using DAB (3,3'-diaminobenzidine). Individual adipocytes within the tissue were classified and analyzed according to size. 2 μg was injected into two divided sites on the right pad of the inguinal fat pad.

[0096] Figure 9 shows the results of analyzing adipocytes in adipose tissue isolated by necropsy of obese mice after substance administration. Cell size was used as the classification criterion, and the ratio of the number of cells to the area for each size was indicated. Small: reduced fat accumulation or reduced number of adipocytes; Normal: based on DIO, fat accumulation; Hypertrophic: hypertrophic cells, indicating metabolic stress, and the possibility of inducing apoptosis or lipolysis.

[0097]

[0098] The present invention will be described in more detail below.

[0099]

[0100] Example 1. SamRNA Platform Design

[0101] A platform was selected through the comparison of GOI (VZV-gE) protein expression and gRNA (genomic RNA) and sgRNA (subgenomic RNA) amplification.

[0102] Based on the design of Fig. 1, various constructs in Table 1 below were produced.

[0103] Sequence number R0214R02311R02412R02513R02614R02715R02816

[0104] A total of 7 constructs were constructed, and expression tests were performed on each replicon.

[0105] < Protocol >

[0106] 1. In vitro transcription (IVT) - samRNA production

[0107] Plasmid DNA linearization was performed on the constructs of Table 1 using a restriction enzyme. The linearized DNA was purified using a silica membrane spin column.

[0108] In vitro transcription (IVT) was performed using linearized DNA as a template. Subsequently, after DNase treatment, RNA was precipitated and purified using LiCl and dissolved in nuclease-free water.

[0109] 2. Transfection

[0110] Vero cells were seeded in a 24-well plate at a density of 5 E4 / well. 24 hours after seeding, 125 ng of samRNA was mixed with lipofectamine to form a complex, and the complex was applied to the cells. Protein expression was confirmed by performing IFA as a time-course 24 to 72 hours after treatment.

[0111] 3. Immunofluorescence assay (IFA) - Confirmation of VZVgE protein expression

[0112] (1) After removing the media, wash once with DPBS.

[0113] (2) Add 4% PFA to the well and fix the cells at room temperature for 20 minutes.

[0114] (3) After removing 4% PFA, wash twice with DPBS.

[0115] (4) Add blocking buffer (5% BSA in DPBS) to the well and block for 1 hour at room temperature.

[0116] (5) After removing the blocking buffer, wash once with DPBS. (1 mL / well)

[0117] (6) Add the anti-VZV gE antibody diluted in dilution buffer (0.5% BSA in 0.05% PBS-T) to the wells and incubate at room temperature for 1 hour. (1:1500)

[0118] (7) After removing the antibody, wash twice with DPBS.

[0119] (8) Add the anti-mouse IgG H&L Alexa488 antibody diluted in dilution buffer to the wells and incubate at room temperature for 1 hour. (1:2000)

[0120] (9) After removing the antibody, wash twice with DPBS.

[0121] (10) Measure fluorescence using Cytation7 with DPBS (1 mL / well) added.

[0122]

[0123] Figures 2 and 3 show the results of confirming VZVgE expression in vero cells. The fat-reducing effect was confirmed in the construct showing the lowest protein expression, and since R021 showed the lowest protein expression rate, it can be seen that it has the highest fat-reducing effect. It was confirmed that VZVgE protein was expressed in all replicons, and additional experiments were conducted on R021, which had the highest fat-reducing effect among them.

[0124]

[0125] Example 2. In Vitro Transcription (IVT)

[0126] 1.1 Plasmid DNA Linearization

[0127] Plasmid DNA containing mRNA and samRNA expression cassettes was fully linearized using a suitable restriction enzyme (e.g., NotI, XbaI, sapI, esp3I, or a unique site located at the 3' end of the expression cassette). The reaction was performed under the manufacturer's recommended buffer conditions and induced at 37 °C for 3-4 hours.

[0128] To remove DNA fragments and enzyme / buffer residues generated after restriction enzyme treatment, the DNA was purified using a silica membrane-based spin column purification kit (Qiagen or equivalent). The purity of the purified linearized DNA was confirmed by measuring the 260 / 280 ratio, and it was used as a template for the IVT reaction.

[0129] 1.2. Performing In vitro Transcription (IVT) Reaction

[0130] SamRNA was synthesized using a T7 RNA polymerase-based IVT system (MEPIVS, including ARCA cap analog, or CleanCap® system) with purified linearized DNA as a template. The reaction included an NTP mix, reaction buffer, RNA polymerase, and a cap analog, and was carried out at 37 °C for 4–5 hours under the manufacturer's recommended conditions.

[0131] 1.3. DNase Treatment and RNA Recovery (Purification)

[0132] After the completion of IVT, DNase I was added to remove residual template DNA from the reaction solution and treated at 37 °C for 15–30 minutes. Subsequently, RNA was precipitated using the LiCl precipitation method.

[0133] After adding and mixing a 7.5 M LiCl solution to the reaction mixture, the mixture was precipitated at -20 °C for at least 30 minutes, and the RNA pellet was recovered by centrifuging at 17,000 KJ for 15-20 minutes.

[0134] The recovered pellet was washed once with 70% ethanol, completely dried, and dissolved in RNase-free water. The concentration and integrity of the RNA were verified using NanoDrop and Agilent Bioanalyzer (to check RIN values).

[0135]

[0136] Example 2. Lipid Nanoparticle (LNP) Formulation

[0137] 2.1 Preparation of Lipid Mixture

[0138] Ionizable lipid (Cayman Chemical: "SM-102 (Cat. 33474)"), 1,2-DSPC, cholesterol, and PEG-lipid (DMG-PEG (2000)) were dissolved in ethanol and mixed in a molar ratio e.g., 50:10:38.5:1.5 (typical compositions based on raw materials from Acuitas or Precision Nanosystems). The final concentration of the lipid solution was adjusted to a range of 10-20 mg / mL to be suitable for microfluidic mixing.

[0139]

[0140] 2.2 Preparation of RNA Solution

[0141] samRNA synthesized with IVT in sodium acetate buffer (final concentration 25 mM, pH It was prepared at a concentration suitable for LNP preparation by diluting it in 5.0 (samRNA concentration approximately 0.2-1 mg / mL). The volume was prepared to be approximately 4 mL.

[0142]

[0143] 2.3 Microfluidic-based LNP Manufacturing

[0144] LNPs were formed by mixing an ethanol-phase lipid solution and an aqueous-phase RNA solution at a predetermined flow rate ratio (e.g., aqueous:ethanol = 3:1 or platform-based ratio). Mixing was performed using a microfluidic mixer (NanoAssemblr Ignite or Blaze). After preparation, the size, PDI, and encapsulation efficiency of the LNPs were confirmed using DLS and RiboGreen assays.

[0145]

[0146] 2.4 Buffer Exchange and Purification

[0147] The generated LNP was filtered using an MWCO filter (Amicon Ultra, 100 kDa, etc.) to remove residual ethanol and sodium acetate buffer, and then exchanged with DPBS as the final formulation buffer.

[0148] If necessary, the final LNP formulation (mRNA-LNP or samRNA-LNP) was secured for administration through a concentration step.

[0149]

[0150] Example 3. Immunofluorescence Assay (IFA)

[0151] 3.1 Cell Culture and LNP Treatment

[0152] C2C12 murine myoblast cells in 2x10 wells per well of a 24-well plate 4 Cells were uniformly inoculated. After incubating for 6 hours for cell attachment and stabilization, mRNA-LNP and samRNA-LNP were diluted in DPBS and treated at final concentrations ranging from 50 to 500 ng / well.

[0153] After LNP treatment, samples were collected in a time-course manner over 20 to 90 hours to perform IFA, and the persistence and intensity of expression between treatment groups were compared quantitatively and qualitatively.

[0154]

[0155] 3.2 Immunofluorescence Assay (IFA) Procedure

[0156] (1) Cell washing

[0157] After completely removing the culture medium, each well was washed once with DPBS to remove residual serum protein and non-specific reaction-inducing factors.

[0158] (2) Cell fixation

[0159] 4% paraformaldehyde (PFA) solution was added to each well and reacted at room temperature for 20 minutes to fix the cell structure.

[0160] (3) Wash after fixing

[0161] The PFA solution was removed and washed twice with DPBS to completely remove the residual fixative.

[0162] (4) Permeabilization

[0163] The washed cells were mixed with a 0.1% Triton X-100 solution and reacted at room temperature for 20 minutes to permeabilize the cells.

[0164] (5) Wash after permeabilization

[0165] The 0.1% Triton X-100 solution was removed, and the residue was completely removed by washing twice with DPBS.

[0166] (6) Non-specific binding blocking

[0167] DPBS-based blocking buffer containing 5% BSA was added to each well and incubated at room temperature for 1 hour to block non-specific binding of antibodies.

[0168] (7) Preparation for primary antibody treatment after washing

[0169] After removing the blocking buffer, the well was washed once with DPBS (approx. 1 mL / well).

[0170] (8) Primary antibody response (anti-VZV gE antibody)

[0171] The anti-VZV gE antibody was diluted at a ratio of 1:1500 in a dilution buffer prepared with 0.5% BSA / 0.05% PBS-T. After adding the prepared primary antibody solution to each well, the mixture was reacted at room temperature for 1 hour to induce specific binding to the target protein.

[0172] (9) 1st antibody wash

[0173] The primary antibody was removed and washed twice with DPBS.

[0174] (10) Secondary antibody response (Alexa Fluor 488-conjugated anti-mouse IgG)

[0175] The anti-mouse IgG H&L (Alexa Fluor 488) antibody was diluted 1:2000 in the same dilution buffer. It was added to each well and reacted at room temperature for 1 hour.

[0176] (11) Second antibody wash

[0177] After the reaction was finished, the antibody solution was removed and washed twice with DPBS.

[0178] (12) Fluorescence signal measurement

[0179] Alexa488-based fluorescence signals were measured using a Cytation 7 imaging system with 1 mL of DPBS maintained in each well. Fluorescence intensity and fluorescence pattern were used as indicators to compare the in vitro expression efficiency of mRNA-LNP and samRNA-LNP, respectively.

[0180]

[0181] 3.3 Analysis of In vitro Expression Patterns

[0182] In this example, the protein expression patterns over time in C2C12 cells treated with mRNA-LNP and samRNA-LNP were compared using immunofluorescence analysis.

[0183]

[0184] 3.3.1 Expression pattern over time (Kinetics) (Fig. 4)

[0185] After treating C2C12 cells with mRNA-LNP or samRNA-LNP, IFA was performed at each time point between 1 day (D1) and 4 days (D4) from the time of treatment to measure the fluorescence signal intensity.

[0186] In cells treated with mRNA-LNP, maximum expression was observed at time D1, but the fluorescence signal decreased rapidly during the period from D1 to D4, and it was confirmed that expression decreased rapidly starting from time D3. On the other hand, in cells treated with samRNA-LNP, maximum expression was observed at time D1, but the fluorescence signal was maintained during the period from D1 to D4, and a gradual decrease was confirmed thereafter.

[0187]

[0188] 3.3.2 Comparison of expression between mRNA-LNP and samRNA-LNP (Fig. 5)

[0189] Under conditions where an equal dose of RNA was administered, cells treated with samRNA-LNP showed higher or equivalent levels of fluorescence signal compared to cells treated with mRNA-LNP.

[0190] In addition, to compare similar molar conditions, cells treated with 50 ng of mRNA-LNP and cells treated with 250 ng of samRNA-LNP were compared, and a higher fluorescence signal was detected in the samRNA-LNP-treated cells.

[0191] From the above results, it was confirmed that samRNA-LNP tends to maintain a longer duration of intracellular protein expression and a higher fluorescence signal intensity compared to mRNA-LNP.

[0192]

[0193] Example 4. Mouse preparation, administration of therapeutic agent, autopsy, and evaluation of adipose tissue reduction

[0194] 4.1 Mouse Rearing and Establishment of Obesity Model

[0195] An obesity model was established using C57BL / 6J(M / F) mice fed a high-fat diet (HFD). All mice consumed an HFD with a composition of 60 kcal% fat or 45 kcal% fat for at least 15 weeks.

[0196] During the rearing period, body weight was measured at intervals of 1-2 weeks to continuously monitor whether obesity was induced, and individuals that reached a body weight of 35 g or more were selected as subjects for this study.

[0197]

[0198] 4.2 Conditions for Administration of Treatment Agent

[0199] During the experimental period (from the date of administration to the date of necropsy), all subjects were maintained on an HFD with a fat composition of 45 kcal%.

[0200] The therapeutic agent (mRNA-LNP, samRNA-LNP) was administered as a single dose to either the left or right side of the inguinal adipose tissue of each individual, and the dosage was set as follows.

[0201] <Dose: 2 μg / head>

[0202] The administration method was fixed via direct injection into local adipose tissue, and the effect was evaluated by excising the adipose tissue at various time points after administration.

[0203]

[0204] 4.3 Information on Experimental Group Composition and Population Size (N)

[0205] The table below summarizes the dose, post-injection time (dpi: days post injection), and number of individuals in the experimental groups administered mRNA-LNP and samRNA-LNP.

[0206] (1) mRNA-LNP administration group

[0207] Classification Dose Time of Autopsy Number of Individuals mRNA-LNP2 μg 20 dpi 6 mRNA-LNP2 μg 50 dpi 6 mRNA-LNP2 μg 70 dpi 6

[0208] (2) samRNA-LNP administration group

[0209] Classification Dose Time of Autopsy Number of Individuals samRNA-LNP2 μg3 dpi3samRNA-LNP2 μg7 dpi3samRNA-LNP2 μg14 dpi3samRNA-LNP2 μg21 dpi3samRNA-LNP2 μg50 dpi6samRNA-LNP2 μg70 dpi6

[0210] The composition of the sample was designed to precisely measure the onset time and duration (time-dependent effect) of the adipose tissue reduction effect over time following a single administration.

[0211] 4.4 Autopsy and Adipose Tissue Excision and Weight Measurement

[0212] Autopsy was performed at set points within the 3-70 day (dpi) interval after administration. Left and right inguinal adipose tissues were excised and isolated from each individual, and after removing surface moisture, they were precisely weighed using an electronic balance.

[0213] ·X: Weight of adipose tissue treated with the therapeutic agent

[0214] ·Y: Weight of adipose tissue on the opposite side where the treatment was not administered

[0215]

[0216] 4.5 Calculation of Adipose Tissue Weight Change Rate

[0217] The fat reduction effect was calculated using the following formula based on the weight difference between the left and right adipose tissues within the same individual.

[0218]

[0219] This formula is highly valid as an internal control design to offset body weight differences between individuals and quantify local treatment effects.

[0220]

[0221] 4.6 Evaluation of efficacy in reducing adipose tissue weight (Fig. 6)

[0222] In this example, mRNA-LNP or samRNA-LNP was administered as a single dose to inguinal adipose tissue, and the effect of reducing adipose tissue was analyzed by comparing the weights of the left and right adipose tissues at the time of autopsy. The rate of weight change was calculated for each individual, and the change in efficacy over time and the relative effect of samRNA compared to mRNA were compared.

[0223]

[0224] The following Table 4 shows the rate of change in adipose tissue weight measured in each administration group.

[0225]

[0226] Classification Dose Percentage of Change in Autopsy Fat Weight (%) mean SD mRNA2 ug 20dpi -14.2 -27.8 -25.9 -36.4 -21.2 -29.2 -25.8 7.5 50dpi -19.4 -26 -10.9 -14.6 -22.8 -33.8 -21.3 8.2 70dpi -14.3 -7.1 -19.7 -13.8 -19.2 -14 -14.7 4.6 samRNA2 ug3dpi-16.2-11.6-12.3----13.42.57dpi-25.3-33.5-20.4----26.46.614dpi-38.9-43.5-40.5----412.321dpi-45.3- 40.1-38.9----41.43.450dpi-36.5-24.6-42.7-29.1-47-41.6-36.98.670dpi-14.2-13.5-35.5-38.1-38.9-19-26.512.2

[0227] As can be seen in Figure 6, in the group administered 2 μg of mRNA-LNP, the lipid reduction effect was found to be 17.4% lower at 50 days after administration (dpi 50) compared to 20 days after administration (dpi 20).

[0228] In the group administered 2 μg of samRNA-LNP, it was confirmed that the efficacy of adipose tissue weight reduction decreased by 10.9% during the period from dpi 21 to dpi 50. In the case of samRNA, the weight reduction rate was lower compared to mRNA, suggesting higher efficacy retention. This sustained expression was confirmed to be associated with the sustained efficacy of samRNA-based substances over time.

[0229]

[0230] 4.6.1 Comparative Efficacy and Visual Observation Results

[0231] As confirmed by the rate of change in adipose tissue weight and macroscopic images of adipose tissue taken during autopsy (Fig. 7), a higher fat reduction effect was observed in the self-amplifying mRNA (samRNA) administration group compared to the conventional mRNA administration group. In the samRNA-LNP administration group, a more pronounced reduction in adipose tissue was observed at the administration site (right (R) groin).

[0232]

[0233] 4.7 Histological Analysis of Adipose Tissue

[0234] In this example, VZV-gE samRNA-LNP (SM-102 formulation) was administered once to the inguinal fat pad, and protein expression and morphological changes in adipocytes within the fat tissue were analyzed using histological methods.

[0235]

[0236] 4.7.1 Analysis of Protein Expression and Adipocyte Structural Changes (Fig. 8)

[0237] After locally injecting VZV-gE samRNA-LNP (2 μg) into two sites on the same pad within the right inguinal adipose tissue of mice, immunohistochemical staining was performed on the adipose tissue excised at the time of necropsy.

[0238] VZV-gE protein expression was confirmed using DAB immunostaining (VZV-gE DAB staining), and Hematoxylin & Eosin (H&E) counterstaining was performed to observe adipocyte structure.

[0239] As a result, VZV-gE protein expression was locally detected at the site of VZV-gE samRNA-LNP administration, and changes in adipocyte structure were observed in the region where protein expression occurred. In addition, consistent with the rate of change in adipose tissue weight and visual observation results, a higher fat reduction effect was confirmed in the group administered self-amplifying mRNA (samRNA) compared to conventional mRNA.

[0240] These results indicate that VZV-gE samRNA-LNP induces protein expression in adipose tissue, and that this expression is associated with structural changes in adipocytes.

[0241]

[0242] 4.7.2 Analysis of changes in adipocyte size and cell population distribution (Fig. 9)

[0243] Changes in adipocyte size over time were analyzed in adipose tissue (right inguinal fat pad) treated with VZV-gE samRNA-LNP.

[0244] Changes in the size and composition of adipocytes were confirmed by staining tissue sections with H&E.

[0245] As a result, an increase in the small adipocyte population was observed at the early time point of administration, which was evaluated as a morphological characteristic consistent with a decrease in lipid accumulation within the adipocytes.

[0246] Meanwhile, at time D14, an increase in the large adipocyte population was observed. These cellular changes suggest the possibility that adipocyte apoptosis or lipolysis is being induced, indicating that metabolic remodeling is underway within the adipose tissue.

[0247] This analysis shows that the size distribution of adipocytes in adipose tissue changes over time due to the administration of VZV-gE samRNA-LNP, and a series of histological changes occur leading to a reduction in fat accumulation and the loss of adipocytes.

[0248]

[0249] According to the above results, localized protein expression was confirmed in adipose tissue after administration of VZV-gE samRNA-LNP, accompanied by structural changes in adipocytes, and initially, a decrease in fat accumulation (increase in small adipocytes) was observed, followed by an increase in large cell populations reflecting apoptosis or lipolysis.

[0250] Therefore, histological analysis demonstrates that the adipose tissue reduction effect of VZV-gE samRNA-LNP includes morphological changes at the cellular level beyond simple weight reduction.

[0251]

[0252] Sequence informationSequence number Features 1 DNA (2145 bp) template for mRNA production 2 DNA (9889 bp) template for samRNA production 3 synthetic mRNA (2127 bp) 4 samRNA CODE: R021 (9873 bp)55' UTR63' UTR7nsP (non-structural protein) region8GOI: Varicella zoster virus gE protein9poly A of Sequence ID: 410Intergenic region of venezuelan equine encephalitis virus11-16synthetic samRNA CODE: R023-028

[0253]

[0254] The present invention can bring about a reduction effect on adipose tissue. The nucleic acid molecule of the present invention and a composition containing it can be provided for the purpose of improving, preventing, or treating obesity.

Claims

1. (i) a first open reading frame comprising a polynucleotide encoding one or more viral replication proteins; and (ii) a nucleic acid molecule comprising a second open reading frame containing a polynucleotide encoding an antigen protein for reducing adipocytes, wherein the antigen protein for reducing adipocytes is a varicella-zoster virus (VZV) protein.

2. In claim 1, the antigen protein for reducing fat cells is a nucleic acid molecule that is glycoprotein E(gE) of the varicella-zoster virus (VZV).

3. In Paragraph 1, One or more of the above viral replication proteins are nucleic acid molecules comprising a combination of nsP1 protein, nsP2 protein, nsP3 protein, and nsP4 protein derived from Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), or a combination thereof.

4. A nucleic acid molecule according to paragraph 3, characterized in that the nsP2 protein has a Q739L mutation.

5. In paragraph 3, the nucleic acid molecule is characterized in that the nsP3 protein is mutated such that the Opal termination codon (UGA) is modified into a codon encoding arginine (R).

6. The nucleic acid molecule of claim 1, wherein the RNA molecule comprises a 5' untranslated region (5'-UTR), the first open reading frame, the second open reading frame, a 3'-untranslated region (3'-UTR), and a poly-A sequence, each of which is operably connected in the 5' to 3' direction.

7. In paragraph 1, the nucleic acid molecule is an RNA molecule that is an RNA replicon, self-replicating RNA, or self-amplifying RNA, or a nucleic acid molecule that is a DNA molecule.

8. In claim 1, the nucleic acid molecule is an RNA molecule comprising or consisting of a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.

4.

9. In claim 1, the nucleic acid molecule is a nucleic acid molecule that is a codon-optimized polypeptide composed of SEQ ID NO.

8.

10. In claim 1, the nucleic acid molecule is a DNA molecule, and is a nucleic acid molecule further comprising a promoter.

11. In claim 10, the nucleic acid molecule comprises or consists of a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.

2.

12. A composition formulated to include a nucleic acid molecule and a lipid according to any one of claims 1 to 11.

13. In claim 12, the lipid comprises a composition comprising about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 4 mol% to about 16 mol% of DSPC, about 30 mol% to about 47 mol% of cholesterol, and about 0.5 mol% to about 5 mol% of PEG-lipid.

14. A composition according to claim 12, wherein the N / P molar ratio of the nucleic acid molecule to the lipid is 0.1 to 10:

1.

15. A composition for reducing fat cells comprising a nucleic acid molecule of any one of claims 1 to 11.

16. In claim 15, the fat cell reduction is a composition for reducing fat cells that reduces the size or volume of fat cells within a target tissue.

17. In paragraph 15, the composition is injected via a transdermal, intramuscular, peritoneal, intravenous, or subcutaneous route; or A composition for reducing fat cells, characterized by being administered to a target tissue via electroporation, a gene gun, liposomes, dendrimers, nanoparticles, or a transfer vector.

18. (a) a nucleic acid molecule according to any one of claims 1 to 1, a composition according to any one of claims 12 to 14, or a composition for reducing fat cells according to any one of claims 15 to 17; and (b) A kit comprising instructions for administration of the nucleic acid molecule, composition, or fat cell reduction composition of (a) above.

19. A syringe filled with a composition for reducing fat cells comprising a nucleic acid molecule according to any one of claims 1 to 11.

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