Composition and method for inducing direct conversion into thermogenic adipocytes and use thereof

Abstract

The present invention relates to a composition and method for inducing direct transdifferentiation from somatic cells into thermogenic adipocytes and the use thereof, wherein differentiation from somatic cells into thermogenic adipocytes can be efficiently induced without going through the pluripotency stage of induced pluripotent stem cells through the expression of a direct transdifferentiation inducing factor.

Description

Technology Field

[0001] The present invention relates to a composition and method for inducing direct transdifferentiation from somatic cells into thermogenic adipocytes, and the use thereof. Background Technology

[0002] Obesity is a significant factor that increases the risk of developing various adult and metabolic diseases, such as hypertension, elevated blood cholesterol, diabetes, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, and various types of cancer. In fact, it has been reported that obese patients have a significantly higher incidence of these diseases compared to those with normal weight. Obesity is caused by the excessive accumulation of adipose tissue, and these accumulated fat cells affect metabolic and endocrine functions, directly contributing to the development and progression of various diseases. Furthermore, numerous clinical and epidemiological studies have confirmed that weight loss can significantly lower the incidence of these diseases.

[0003] The aforementioned adipocytes include white adipocytes, brown adipocytes, and beige adipocytes; among these, brown and beige adipocytes play a beneficial role in improving obesity and metabolic diseases by consuming energy through thermogenesis. In particular, as it has been revealed that beige or brown adipocytes are the beneficial adipocytes found in humans, interest is growing in technologies that convert or induce the differentiation of unhealthy white adipocytes into beige or brown adipocytes.

[0004] However, existing technologies for inducing conversion or differentiation into beige / brown adipocytes require the complex introduction of various genes or external factors, resulting in complex differentiation mechanisms and limitations in actual clinical application. Furthermore, a method for directly transdifferentiating somatic cells into beige and brown adipocytes by introducing only specific gene combinations has not yet been clearly identified.

[0005] Accordingly, the inventors conducted research on the direct conversion of somatic cells into adipocytes of a different lineage without passing through the pluripotent stem cell stage, in order to eliminate the risk of teratoma formation and to produce thermogenic adipocytes efficiently and safely. As a result, they confirmed that somatic cells can be efficiently directly converted into thermogenic adipocytes by introducing a specific gene into somatic cells to induce ectopic expression, and based on this, the present invention was completed. The problem to be solved

[0006] The objective of the present invention is to provide a composition for inducing direct transdifferentiation from somatic cells into thermogenesis adipocytes through the introduction of a specific gene into somatic cells or the expression of a specific protein.

[0007] Another objective is to provide a method for directly transdifferentiating somatic cells into thermogenic adipocytes using the above composition.

[0008] Another objective is to provide thermogenic adipocytes directly induced by transdifferentiation by the above method. means of solving the problem

[0009] To achieve the above objective, one aspect provides a composition for inducing direct transdifferentiation from somatic cells into thermogenic adipocytes, comprising (1) proteins Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5); (2) nucleic acid molecules encoding said proteins; and (3) one or more selected from the group consisting of a vector into which said nucleic acid molecules are introduced.

[0010] The term "Direct Conversion" in this specification refers to a process of inducing conversion between mature (completely differentiated) cells of completely different cell types in higher organisms. Unlike conventional techniques that require reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and redifferentiating them to produce target cells, this method differs in that it induces conversion directly into target cells without going through pluripotent stem cell stages such as embryonic stem cells and induced pluripotent stem cells. Currently, direct conversion is recognized for its potential in disease modeling and new drug development, and is known as a technology that can be applied to gene therapy and regenerative medicine.

[0011] In one embodiment, a vector containing nucleic acid molecules encoding Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) proteins can be introduced into somatic cells to directly confirm cross-differentiation into thermogenic adipocytes.

[0012] The above Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) proteins may include all proteins derived from mammals such as humans, horses, sheep, pigs, goats, camels, antelopes, and dogs.

[0013] Additionally, available Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) proteins may include proteins having their wild-type amino acid sequences as well as their variants (e.g., subtypes of each protein).

[0014] The above variant refers to a protein that maintains the inherent biological function of the wild-type protein while having a sequence different from the wild-type protein by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues of the wild-type protein's natural amino acid sequence. The above variant may be a functional equivalent exhibiting the same biological activity as the wild-type protein, or a variant in which the physicochemical properties of the protein are modified as needed, or a variant in which structural stability against physical and chemical environments is increased or physiological activity is increased.

[0015] The above-mentioned Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) proteins or variants thereof may be isolated from nature or produced recombinantly or synthetically (non-naturally occurring).

[0016] In addition, the nucleic acids encoding the proteins Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) are base sequences encoding each protein in the wild type or variant form as described above, which may be modified by substitution, deletion, insertion, or a combination thereof, and may be isolated from nature or prepared using chemical synthesis methods. The nucleic acid having a base sequence encoding each of the above proteins is a base sequence encoding Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) proteins, which may be single-stranded or double-stranded, and may be a DNA molecule (genome, cDNA) or an RNA molecule (mRNA).

[0017] The term "white adipocyte" in this specification refers to a fat cell that has the function of storing large amounts of triglycerides in the body and is primarily involved in energy storage. It proliferates intensively during specific periods, such as late pregnancy, infancy, and puberty, and its cell size can increase up to 15 times due to the excessive accumulation of fat. Generally, the term "adipocyte" often refers to white adipocytes.

[0018] The term "thermogenic adipocytes" in this specification refers to adipocytes that consume energy by performing the function of thermogenesis. For example, the thermogenic adipocytes include, but are not limited to, brown adipocytes and beige adipocytes. The thermogenic adipocytes are rich in mitochondria and can contribute to regulating the body's energy balance and improving metabolic diseases by converting stored energy into heat through the expression of proteins such as UCP-1 (uncoupling protein-1).

[0019] The term "beige adipocyte" in this specification refers to a cell that can be induced primarily within white adipose tissue and has a beige color due to the presence of iron in its mitochondria. Beige adipocytes have the function of consuming energy to generate heat and express UCP-1 (uncoupling protein-1) in response to cold or specific hormonal stimulation. Unlike white adipocytes, they are characterized by their involvement in energy consumption and heat generation.

[0020] The term "brown adipocyte" in this specification refers to a fat cell that appears yellowish-brown or reddish-brown to the naked eye due to its abundance of mitochondria, and performs heat generation by converting fat into energy. Brown adipocytes originate from stem cell lineages that differentiate primarily into muscle cells and, like beige adipocytes, contribute to energy expenditure and body fat reduction. The more brown adipocytes there are, the lower the body fat becomes, and the amount tends to decrease as one reaches adulthood.

[0021] In one embodiment, the composition for direct cross-differentiation induction may comprise, but is not limited to, one or more selected from the group consisting of a Pparγ (Peroxisome Proliferator-Activated Receptor Gamma) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; one or more selected from the group consisting of a MYF5 (Myogenic Factor 5) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; and one or more selected from the group consisting of a UCP1 (Uncoupling Protein 1) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced.

[0022] In one embodiment, the composition for direct cross-differentiation induction may comprise, but is not limited to, one or more selected from the group consisting of a PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; one or more selected from the group consisting of a MYF5 (Myogenic factor 5) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; and one or more selected from the group consisting of a UCP1 (Uncoupling protein 1) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced.

[0023] In one embodiment, the somatic cell is one or more selected from the group consisting of fibroblasts, epithelial cells, adipocyte precursors, white adipocytes, myocytes, neurons, hair cells, hair bulb cells, hair follicle cells, oral epithelial cells, urine-derived somatic cells, gastric mucosal cells, goblet cells, G cells, B cells, periderm cells, astrocytes, blood cells, neural stem cells, oligodendrocyte precursor cells, hematopoietic stem cells, cord blood stem cells, and mesenchymal stem cells. It could be.

[0024] The term "somatic cell" in this specification may mean any cell excluding germ cells, and may be of origin or isolated from mammals such as humans, horses, sheep, pigs, goats, camels, antelopes, and dogs, for example.

[0025] In one embodiment, the vector may be one or more selected from the group consisting of a plasmid vector, a cosmid vector, a virus vector, a lentivirus vector, a retrovirus vector, an HIV (Human immunodeficiency virus) vector, an MLV (Murineleukemia virus) vector, an ASLV (Avian sarcoma / leukosis) vector, an SNV (Spleen necrosis virus) vector, an RSV (Rous sarcoma virus) vector, a MMTV (Mouse mammary tumor virus) vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, and an episomal vector.

[0026] The term "vector" in this specification may refer to an expression vector capable of expressing a target protein in a host cell, and may mean a gene delivery vehicle comprising an essential regulatory element operably linked to allow the expression of a gene insert.

[0027] The above vector may be prepared in various ways depending on the purpose, including signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers. The promoter of the vector may be constitutive or inducible. Additionally, the expression vector includes selectivity markers for selecting host cells containing the vector, and, if it is a replicable expression vector, includes a replication origin. The vector may self-replicate or be incorporated into host DNA.

[0029] Another aspect provides a method for directly transdifferentiating somatic cells into thermogenic adipocytes, comprising one or more selected from the group consisting of (1) proteins Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1) or MYF5 (Myogenic factor 5); (2) nucleic acid molecules encoding said proteins; and (3) vectors into which said nucleic acid molecules are introduced.

[0030] The above direct transdifferentiation method may include the steps of: culturing somatic cells in a culture medium; transforming the cultured somatic cells with a vector into which the genes Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) have been inserted; and culturing the infected somatic cells under culture conditions capable of inducing direct transdifferentiation.

[0031] The culture medium used for the above somatic cells includes all media conventionally used for somatic cell culture in the field. The culture medium generally contains carbon sources, nitrogen sources, and trace element components.

[0032] In addition, the culture conditions capable of inducing direct transdifferentiation of the above somatic cells into thermogenic adipocytes may include media and / or conventional culture conditions commonly used in the field to induce direct transdifferentiation of somatic cells.

[0033] By introducing the above-mentioned composition for direct transdifferentiation induction into somatic cells, ectopic expression of direct transdifferentiation inducing factors such as Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1), or MYF5 (Myogenic factor 5) can be induced. Ectopic expression refers to the expression of a gene outside the tissue or cell in which it is originally expressed, or expression at a time different from the time of original expression. According to one aspect of the method, thermogenic adipocytes can be effectively produced from somatic cells.

[0035] Another aspect provides a thermogenic adipocyte directly induced by cross-differentiation by contacting or inserting into a somatic cell a composition selected from the group consisting of (1) proteins Pparγ (Peroxisome proliferator-activated receptor gamma), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), C / EBPβ (CCAAT / enhancer-binding protein beta), PRDM16 (PR / SET domain 16), UCP1 (Uncoupling protein 1) or MYF5 (Myogenic factor 5); (2) a nucleic acid molecule encoding said protein; and (3) a vector into which said nucleic acid molecule is introduced.

[0036] Thermogenic adipocytes induced by a composition or method according to one aspect may be characterized by expressing armogenic adipocyte-specific markers without expressing the original somatic cell-specific markers from which they originated.

[0037] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs. Effects of the invention

[0038] By using a composition for inducing direct transdifferentiation from somatic cells to thermogenic adipocytes according to one aspect, differentiation from somatic cells to thermogenic adipocytes can be efficiently induced through the expression of a direct transdifferentiation inducing factor without going through the pluripotency stage of induced pluripotent stem cells. Brief explanation of the drawing

[0039] Figure 1 is a schematic diagram showing the process of converting white fat cells into brown fat cells and beige fat cells through direct conversion according to the pattern. Figure 2 is a photograph showing the results of evaluating changes in the amount of fat accumulated in adipocytes through Oil Red O staining according to treatment with direct transdifferentiation inducing factors according to the pattern. Figure 3 is a graph showing the results of a quantitative analysis of changes in the amount of fat accumulated in adipocytes according to treatment with direct transdifferentiation inducing factors according to the pattern. Specific details for implementing the invention

[0040] Hereinafter, experimental examples and embodiments will be described in detail to aid in understanding the present invention. However, the following experimental examples and embodiments are merely illustrative of the content of the present invention and are not limited to the scope thereof. The embodiments and experimental examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0042] Example 1. Induction into thermogenic adipocytes by direct transdifferentiation-inducing factors

[0043] 3T3-L1 adipocytes were converted into beige and brown adipocytes by introducing direct cross-differentiation inducing factors such as Pparγ, PGC1α, C / EBPβ, PRDM16, UCP1, and MYF5, either alone or in various combinations, using AAV8 or AAV9 viruses.

[0044] Specifically, 3T3-L1 adipocytes were seeded into a 6-well plate at a concentration of 1 x 10^5 cells / 2 ml and cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% Bovine calf serum (BCS), 4 mM L-glutamine, and 1% penicillin streptomycin until confluent. To induce white adipocyte differentiation, DMEM medium containing DMI (500 µM IBMX, 1 µM dexamethasone, 10 µg / ml Insulin, 10% Fatal bovine serum (FBS)) was added to D0 to induce differentiation. After 2 days, the DMEM medium was changed to one containing DMII (10 µg / ml Insulin, 10% FBS). After 2 days, the DMEM medium was changed to one containing MM (10% FBS) to induce maturation of white adipocytes. On Day 7, direct transdifferentiation inducers Pparγ, Pgc1α, C / ebpβ, Prdm16, Ucp1, or Myf5 were introduced individually or in combination. Subsequently, conversion to beige and brown adipocytes was induced until Day 28.

[0045] Experimental Example 1. Confirmation of reduction in fat accumulation in induced thermogenic adipocytes

[0046] To confirm the effect of reducing fat accumulation in beige and brown adipocytes induced in Example 1, the degree of intracellular triglyceride accumulation was evaluated under each condition through Oil Red O staining.

[0047] Specifically, to confirm the effect of reducing lipid accumulation in induced beige and brown adipocytes, the degree of triglyceride accumulation in the cells under each condition was evaluated using Oil Red O staining. Samples were fixed by treating with 4% paraformaldehyde for 1 hour, followed by treatment with 60% isopropanol. To prepare the Oil Red O working solution, the stock was mixed with ddH2O in a 6:4 ratio, maintained at room temperature for 20 minutes, and then filtered through a 0.2 µm filter. The prepared Oil Red O working solution was applied to the samples for 10 minutes, followed by washing, and the staining intensity was observed under a microscope. Subsequently, 100% isopropanol was applied to the samples to elute the Oil Red O reagent. The absorbance at a wavelength of 510 nm was measured using a spectrophotometer (SPECTRAmax) to determine the Oil Red O staining intensity, thereby quantitatively evaluating the degree of intracellular triglyceride accumulation under each condition.

[0048] The results are shown in Figures 2 and 3.

[0049] Figure 2 is a photograph showing the results of evaluating changes in the amount of fat accumulated in adipocytes through Oil Red O staining according to treatment with direct cross-differentiation inducing factors according to the pattern.

[0050] Figure 3 is a graph showing the results of a quantitative analysis of changes in fat accumulation within adipocytes according to treatment with direct cross-differentiation inducing factors according to a specific pattern.

[0051] As shown in Figure 2, Oil Red O staining results revealed distinct differences in the degree of fat accumulation within 3T3-L1 adipocytes following the introduction of direct cross-differentiation inducers. Compared to the group without direct cross-differentiation inducers (No TF), fat accumulation was significantly reduced in the M5+U1+PG and M5+U1+PP combination groups, resulting in the weakest staining intensity.

[0052] As shown in Figure 3, quantitative analysis of Oil Red O staining confirmed that the degree of fat accumulation in 3T3-L1 adipocytes changed numerically depending on the combination of direct cross-differentiation inducible factors. Fat accumulation decreased in the M5+U1+PG and M5+U1+PP combinations, showing a prominent fat consumption effect compared to the group without direct cross-differentiation inducible factors.

[0053] These results indicate that introducing specific direct transdifferentiation-inducing factors into somatic cells effectively converts them into thermogenic adipocytes, resulting in reduced fat accumulation and enhanced thermogenic activity.

Claims

Claim 1 A composition for inducing direct transdifferentiation from somatic cells to thermogenic adipocytes, comprising: one or more selected from the group consisting of MYF5 (Myogenic factor 5) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; one or more selected from the group consisting of UCP1 (Uncoupling protein 1) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; and one or more selected from the group consisting of Pparγ (Peroxisome proliferator-activated receptor gamma) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced. Claim 2 A composition for inducing direct transdifferentiation from somatic cells to thermogenic adipocytes, wherein the composition further comprises: one or more selected from the group consisting of a PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; or one or more selected from the group consisting of a C / EBPβprotein beta) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced. Claim 3 delete Claim 4 In claim 1, the somatic cell is one or more selected from the group consisting of fibroblasts, epithelial cells, adipocyte precursors, white adipocytes, myocytes, neurons, hair cells, hair bulb cells, hair follicle cells, oral epithelial cells, urine-derived somatic cells, gastric mucosal cells, goblet cells, G cells, B cells, periderm cells, astrocytes, blood cells, neural stem cells, oligodendrocyte precursor cells, hematopoietic stem cells, cord blood stem cells, and mesenchymal stem cells. A composition for inducing direct cross-differentiation from somatic cells into thermogenic adipocytes. Claim 5 A composition for inducing direct transdifferentiation from somatic cells to thermogenesis adipocytes, wherein the vector is one or more selected from the group consisting of a plasmid vector, a cosmid vector, a virus vector, a lentivirus vector, a retrovirus vector, an HIV (Human immunodeficiency virus) vector, an MLV (Murineleukemia virus) vector, an ASLV (Avian sarcoma / leukosis) vector, an SNV (Spleen necrosis virus) vector, an RSV (Rous sarcoma virus) vector, an MMTV (Mouse mammary tumor virus) vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, and an episomal vector. Claim 6 A method for directly transdifferentiating somatic cells into thermogenic adipocytes, comprising: one or more selected from the group consisting of MYF5 (Myogenic factor 5) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; one or more selected from the group consisting of UCP1 (Uncoupling protein 1) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; and one or more selected from the group consisting of Pparγ (Peroxisome proliferator-activated receptor gamma) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced. Claim 7 Thermogenic adipocytes directly induced to undergo transdifferentiation by contacting or inserting a composition comprising: one or more selected from the group consisting of MYF5 (Myogenic factor 5) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; one or more selected from the group consisting of UCP1 (Uncoupling protein 1) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced; and one or more selected from the group consisting of Pparγ (Peroxisome proliferator-activated receptor gamma) protein, a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule is introduced;

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