Genetically engineered mesenchymal stem cells and uses thereof

Abstract

This article discloses a genetically engineered mesenchymal stem cell (MSC) containing genetic variations that enhance adipogenic differentiation, mitochondrial biosynthesis, and gene expression of brown adipocyte markers. This article also discloses a pharmaceutical composition and its use in the prevention, relief, and / or treatment of metabolic disorders.

Description

Technical Field

[0001] This disclosure generally pertains to the field of engineered stem cells and their applications. Specifically, these engineered stem cells can be used to treat or alleviate metabolic disorders. Prior Technology

[0002] Obesity and related metabolic disorders are increasingly prevalent globally, leading to rising morbidity and mortality. While obesity-related hepatic steatosis is often clinically benign, it can rapidly progress to fatty liver disease associated with metabolic dysfunction (MASLD), a severe metabolic liver condition characterized by persistent inflammation, liver damage, and fibrosis. MASLD significantly increases the risk of advanced liver disease, including cirrhosis and hepatocellular carcinoma (HCC). Despite significant efforts to develop pharmacological therapies targeting MASLD, many of these therapies have failed in clinical trials. Summary of the Invention

[0003] The following is a simplified overview of this disclosure to provide the reader with a basic understanding. This overview is not a comprehensive summary of this disclosure, nor does it identify key / essential elements of the invention or define its scope. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as a prelude to the more detailed description that follows.

[0004] As illustrated and broadly described herein, one aspect of this disclosure relates to genetically engineered mesenchymal stem cells (MSCs). In some embodiments of this disclosure, MSCs contain genetic variations that enhance the expression of genes involved in adipogenic differentiation, mitochondrial biosynthesis, and brown adipocyte markers. In some embodiments of this disclosure, MSCs contain exogenous genes including those involved in adipogenic differentiation, mitochondrial biosynthesis, and brown adipocyte markers, wherein the expression of these genes is enhanced.

[0005] In some embodiments disclosed herein, gene mutations enhance gene expression of peroxisome proliferation factor activating receptor γ (PPARγ).

[0006] In some embodiments disclosed herein, gene mutations enhance gene expression of peroxisome proliferation factor activator receptor-γ coactivator 1-α (PGC1α).

[0007] In some embodiments disclosed herein, gene mutations enhance gene expression of mitochondrial uncoupling protein 1 (UCP1).

[0008] In one embodiment, this disclosure provides a genetically engineered mesenchymal stem cell (MSC) containing one, two, or three exogenous genes selected from perosome proliferator-activated receptor γ (PPARγ), perosome proliferator-activated receptor-γ coactivator 1-α (PGC1α), and mitochondrial uncoupling protein 1 (UCP1), wherein the gene expression of PPARγ, PGC1α, and UCP1 is enhanced.

[0009] In some embodiments, the genetically engineered MSCs include an exogenous promoter, an exogenous nucleic acid region encoding PPARγ, an exogenous nucleic acid region encoding PGC1α, and an exogenous nucleic acid region encoding UCP1.

[0010] In one embodiment, this disclosure provides a method for preparing a population of genetically engineered MSCs, comprising: preparing a vector containing a multi-gene activated plasmid based on an integrated CRISPR, wherein the plasmid is prepared as follows: providing a gRNA selection vector containing a recombination site and PPARγ, PGC1α and UCP1 respectively, and mixing the gRNA selection vector with a transposon backbone vector containing CRISPR / nuclease to generate a multi-gene activated plasmid based on an integrated CRISPR; transfecting MSCs with the vector to generate genetically engineered MSCs; culturing the genetically engineered MSCs in a medium suitable for the growth and proliferation of genetically engineered MSCs; and harvesting the genetically engineered MSC population.

[0011] In one embodiment, this disclosure provides a population of engineered mesenchymal stem cells as described herein.

[0012] Examples of MSCs include (but are not limited to) umbilical cord mesenchymal stem cells (UMSC), adipose-derived mesenchymal stem cells (ADSC), or bone marrow mesenchymal stem cells (BMSC).

[0013] In one embodiment, the genetically engineered MSCs described herein include a U6 promoter operatively linked to a foreign gene.

[0014] In some embodiments disclosed herein, gene variants and / or exogenous gene lines are introduced into MSCs using a vector containing genes for adipogenic differentiation, mitochondrial biosynthesis, and brown adipocyte markers.

[0015] In some embodiments disclosed herein, genetic variations and / or exogenous gene lines are introduced into MSCs using a vector containing the PPARγ, PGC1α, and UCP1 genes.

[0016] In some embodiments disclosed herein, the carrier is a transposable carrier.

[0017] In some embodiments disclosed herein, genetic variations and / or exogenous gene lines are introduced into MSCs using clustered regularly spaced short palindromic repeats (CRISPR) / nucleases.

[0018] In some embodiments disclosed herein, the vector comprises dCas9-VP64 and MS2-p65-HSF1 fragments.

[0019] In some embodiments disclosed herein, the vector comprises a U6 promoter operatively linked to a foreign gene.

[0020] In some embodiments disclosed herein, the vector is a multi-gene activator plasmid based on integrated CRISPR, which is constructed by the following steps: Provide gRNA selection vectors containing recombination sites and PPARγ, PGC1α, and UCP1 respectively, and The gRNA selection vector was mixed with a transposon backbone vector containing CRISPR / nuclease to generate multi-gene activators based on integrated CRISPR.

[0021] As illustrated and broadly described herein, another aspect of this disclosure relates to a pharmaceutical composition comprising genetically engineered MSCs as described herein and, where applicable, a pharmaceutically acceptable carrier.

[0022] As embodied and broadly described herein, another aspect of this disclosure relates to the use of the pharmaceutical composition described herein for the manufacture of an agent for the prevention, relief, and / or treatment of metabolic disorders in an individual in need. Alternatively, another aspect of this disclosure relates to a method for the prevention, relief, and / or treatment of metabolic disorders in an individual in need, comprising administering to the individual the pharmaceutical composition described herein. Still another aspect of this disclosure relates to the pharmaceutical composition described herein for the prevention, relief, and / or treatment of metabolic disorders in an individual in need.

[0023] Examples of metabolic disorders include (but are not limited to) diabetes, obesity, central obesity, insulin resistance, hypertension, insulin resistance syndrome, hepatic steatosis, heart defects, ischemic heart disease, high blood pressure, inflammation, dyslipidemia of triglycerides, dyslipidemia of HDL, dyslipidemia of cholesterol, elevated fasting plasma glucose, dysregulation of leptin, dysregulation of adiponectin, or cancer. In some embodiments disclosed herein, the metabolic disorder is fatty liver disease (MASLD) associated with metabolic dysfunction.

[0024] In some embodiments disclosed herein, the agent is used to improve insulin sensitivity, promote efficient energy utilization, remodel immune cell populations, and / or improve lipid accumulation, inflammation, or fibrosis in the liver.

[0025] In some embodiments disclosed herein, the effective amount ranges from about 1×10⁴ cells to about 1×10⁸ cells; about 2×10⁴ cells to about 8×10⁷ cells; about 5×10⁴ cells to about 5×10⁷ cells; about 8×10⁴ cells to about 2×10⁷ cells; about 1×10⁵ cells to about 1×10⁷ cells; about 2×10⁵ cells to about 8×10⁶ cells; about 5×10⁵ cells to about 5×10⁶ cells; about 8×10⁵ cells to about 2×10⁶ cells; and about 1×10⁶ cells.

[0026] In some embodiments disclosed herein, the drug is administered via intravenous injection.

[0027] The various incidental features and advantages of this disclosure will be better understood by referring to the following embodiments in conjunction with the accompanying drawings. Simple Explanation of the Diagram

[0028] Figures 1a and 1b illustrate the generation of a one-step CRISPR-based multi-gene activation system. Figure 1a shows the selection of three gRNAs designed for different gene promoters into three vectors. Using multi-site gateway selection technology, all three gRNAs, along with CRISPR-based activation mechanisms (dCas9-VP64 and MS2-p65-HSF1), can be selected into transposon vectors to create a monoplast. Figure 1b shows that when the monoplast is delivered to the cell and incorporated into the DNA by transposases, the CRISPR-SAM system simultaneously activates the three different genes corresponding to the three gRNAs.

[0029] Figures 2a and 2b illustrate the generation of BATMEns using CRISPR-based multi-gene activation. Figure 2a shows the delivery of transposase-expressing vectors and monoclonal plastids to human mesenchymal stem cells (BATMen and CTL-hMSCs), with or without three different gRNAs targeting UCP1, PPARγ, and PGC1α. Figure 2b shows the expression of MS2, UCP1, PPARγ, and PGC1α in primitive hMSCs, CTL-hMSCs, and BATMEns.

[0030] Figures 3a to 3h show how BATMEn improves glucose tolerance and energy expenditure in MASLD mice. Figure 3a shows the cell therapy regimen (including CTL-hMSCs and BATMEn) used to treat MASLD. Figure 3b shows the distribution of BATMEn stained with DiR after tail vein injection. Figures 3c to 3e show the measurement of body weight (Figure 3c), lean body mass (Figure 3d), and fat mass (Figure 3e) of mice after four injections of PBS or cells. Figures 3f to 3h show the determination of metabolic parameters in the MASLD mouse model after four injections of PBS or cells, including glucose tolerance test (GTT, Figure 3f), insulin tolerance test (ITT, Figure 3g), and energy expenditure (Figure 3h).

[0031] Figures 4a to 4d show that BATMEn ablated lipid accumulation, fibrosis, and damage in the liver of MASLD mice. Figure 4a shows the staining of lipid droplets and collagen fibers in liver sections from normal-diet mice and MASLD mice treated with hMSCs or BATMEn, respectively, using Oil Red O and Masson trichrome stains. (Figures 4b and 4c) mRNA expression levels of genes in mouse liver, including Col3a1 (Figure 4b) and TNFα (Figure 4c). Figure 4d shows ALT activity in mouse plasma.

[0032] Figures 5a to 5d show the regulation of immune cell infiltration in the liver of MASLD mice by BATME. Figure 5a shows different immune cell populations in the liver of normal-diet mice and MASLD mice infected with hMSCs or BATME using CyTOF analysis. Figure 5b shows the quantification of different immune cell populations. Figure 5c shows the tSNE atlas of CD11b+ monocytes / macrophages in the liver of mice receiving different therapies. Figure 5d shows the performance of MASLD-associated macrophage (NAM) markers (including GPNMB, TREM2, and APOE) in non-parenchymal cells (NPCs) isolated from mouse liver. Implementation

[0033] The following detailed description, provided in conjunction with the accompanying drawings, is intended to describe examples of the invention and not to represent a simple form in which the examples of the invention can be constructed or used. This description elucidates the function of the examples and the sequence of steps for constructing and operating the examples. However, different examples may perform the same or equivalent functions and sequences.

[0034] Unless otherwise defined, all scientific or technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Those skilled in the art can understand and practice this invention using any methods and materials similar to or equivalent to those described herein.

[0035] Unless otherwise specified, all figures used in the specification and claims that indicate the quantity of ingredients, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless stated to the contrary, the numerical parameters set forth in the specification and claims of this invention are approximate values ​​and may vary depending on the desired characteristics sought by the invention.

[0036] The term "a / an" shall mean one or more objects described in this invention. The term "and / or" means one or both of the alternatives. The term "a cell" or "the cell" may include a plurality of cells.

[0037] The term "and / or" is used to refer to two things or either of the two things.

[0038] As used herein, the term "stem cell" refers to cells in an undifferentiated or partially differentiated state that possess self-renewal properties and the developmental potential to naturally differentiate into more differentiated cell types. This developmental potential has no specific implied meaning (i.e., totipotency, pluripotency, etc.). Self-renewal means that stem cells can proliferate and produce more of these stem cells while maintaining their developmental potential. Therefore, the term "stem cell" refers to any subpopulation of cells that, under specific conditions, possess the developmental potential to differentiate into more specific or higher-differentiated phenotypes and, under certain conditions, maintain proliferation without substantially differentiating.

[0039] The term "genetically engineered / genetic engineering" in cells refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels within a cell. Genetic material can be in the form of DNA or RNA. It can be transferred into cells via various methods, including viral transduction and non-viral transfection. Following genetic engineering, the expression levels of certain genes within a cell can be permanently or temporarily altered.

[0040] As used herein, the term "exogenous" means any material introduced or produced outside of an organism, cell, tissue, or system.

[0041] As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid that has been linked to it.

[0042] The term "expression vector" refers to a drug that delivers foreign genes into cells for expression without degradation. In this invention, the expression vector can be a plastid, a viral vector, or an artificial chromosome.

[0043] The term "enhanced performance" in this article means that the RNA or protein of the gene of interest is more expressed in genetically engineered cells than in their non-engineered cell counterparts.

[0044] "Operationally linked" means that two nucleic acid sequences are physically or functionally related. For example, if the two sequences are operably linked, or positioned such that the regulatory DNA sequence will affect the expression of the coding or structural DNA sequence, then the promoter or regulatory DNA sequence is said to be "related" to the DNA sequence encoding RNA or protein.

[0045] As used herein, “recombining sites” (also referred to as “recombination sites” in this paper) refers to a nucleic acid sequence containing an inverted palindromic sequence separated by an asymmetric sequence, at which site-specific recombination can occur. Such recombination sites may include (but are not limited to) Lox (Sternberg et al. (1978) Cold Spring Harbor Symp. Quant. Biol. 43:1143-1146 and Hoess et al. (1990) in Nucleic Acids and Molecular Biology, Eckstein and Lilley (Springer, Berlin), Vol. 4, pp. 99-109) and FRT (reviewed in Kilby et al. (1993) Trends In Genetics, 9, 413-421).

[0046] The term "pharmaceutical composition" as used in this disclosure includes a quantity of live cells effective in treating metabolic disorders. The cellular component may be a mixture of cultured cells or isolated cell populations, such as differentiated tissue cells, precursor cells, and / or stem cells. The pharmaceutical compositions disclosed herein are in liquid form or as cell suspension buffers, and may contain pharmaceutically acceptable excipients that stabilize the liquid suspension and contribute to cell viability.

[0047] As used herein, the terms "treat," "treating," and "treatment" are interchangeable and encompass the partial or complete prevention, relief, mitigation, and / or control of symptoms, secondary conditions, or symptom associated with metabolic disorders. As used herein, the term "treatment" means the administration or delivery of the pharmaceutical composition disclosed herein to an individual suffering from symptoms, secondary conditions, or symptom associated with a metabolic disorder, with the aim of partially or completely alleviating, relieving, reducing, or diminishing one or more of the symptoms, secondary conditions, or characteristics associated with the metabolic disorder, delaying their onset, inhibiting their progression, reducing their severity, and / or reducing their incidence. For the purpose of reducing the risk of developing symptoms, secondary conditions, and / or symptom associated with a metabolic disorder, treatment may be delivered to an individual exhibiting only early signs of such symptoms, conditions, and / or symptom. Treatment is generally "effective" if one or more symptoms or clinical markers decrease, as defined herein. Alternatively, treatment is "effective" if the progression of symptoms, conditions, or symptom decreases or ceases.

[0048] The terms "preventing" or "prevention" are recognized in this technology, and when used in conjunction with a symptom, they include administering a drug before the onset of the symptom, which reduces the frequency or severity of the individual's medical symptom onset or delays the onset of the symptom compared to an individual who has not received the drug.

[0049] As mentioned herein, the term "effective amount" refers to the quantity of a component sufficient to produce the desired response. An effective amount for therapeutic purposes is also the quantity in which the beneficial therapeutic effect of the component outweighs any of its toxic or harmful effects. An effective amount of a drug is not necessary to cure a disease or condition, but rather to provide treatment to the disease or condition, thereby delaying, inhibiting, or preventing the onset of the disease or condition, or alleviating the symptoms of the disease or condition. An effective amount may be suitably divided into one, two, or more doses to be administered once, twice, or more over a specified period of time. The specific effective amount or sufficient amount will vary depending on factors such as the specific condition being treated, the patient's physical condition (e.g., body mass, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of any concurrent treatments, and the structure of the specific formulation and compound or its derivatives used. Effective amounts can be expressed, for example, in grams, milligrams, or micrograms, or in milligrams per kilogram of body weight (mg / kg). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (such as the immunoconjugate disclosed herein), such as mole concentration, mass concentration, volume concentration, weight mole concentration, mole fraction, mass fraction, and mixing ratio. Those skilled in the art can calculate the human equivalent dose (HED) of the agent (such as the immunoconjugate of this invention) based on doses determined from animal models. For example, when estimating the maximum safe dose for human individuals, industry guidance published by the U.S. Food and Drug Administration (FDA) can be followed, entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers".

[0050] As used interchangeably in this document, the terms "person," "individual," and "patient" refer to animals, preferably mammals. Examples of individuals include humans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cattle, horses, dogs, and cats.

[0051] As used herein, the term "needs treatment" refers to a judgment made by a caregiver (e.g., a physician, nurse, nursing practitioner, or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that an individual needs treatment or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's area of ​​expertise, including the understanding that the individual is ill or will become ill due to a condition that can be treated with the compounds disclosed herein.

[0052] Approximately 23% of the adult population is currently affected by metabolic syndrome, and its prevalence is increasing worldwide. Therefore, there is a significant need for therapeutic targets that can alleviate obesity and insulin resistance (two hallmarks of metabolic syndrome). Adaptive thermogenesis is a physiological process that generates heat by releasing stored chemical energy in response to environmental cues. This process is crucial because even slight differences in energy expenditure caused by adaptive thermogenesis can significantly affect systemic metabolism over time. Therefore, targeting adaptive thermogenesis is a promising strategy for the prevention and treatment of metabolic diseases. Thermogenesis primarily occurs in thermogenic adipose tissue, specifically brown and beige / light adipose tissue (BAT), making it an attractive intervention target.

[0053] As illustrated and broadly described herein, one aspect of this disclosure relates to a population of genetically engineered mesenchymal stem cells (MSCs). In some embodiments of this disclosure, MSCs contain genetic variations and / or exogenous genes that enhance the expression of genes involved in adipogenic differentiation, mitochondrial biosynthesis, and brown adipocyte markers. In some embodiments of this disclosure, MSCs contain exogenous genes involved in adipogenic differentiation, mitochondrial biosynthesis, and brown adipocyte markers, wherein the expression of these genes is enhanced.

[0054] For example, the MSCs described herein may constitute at least 70% of the total number of cells in the cell population, with a more preferred percentage, such as at least 85, 90, 95 or 99%.

[0055] In some embodiments disclosed herein, genetically engineered mesenchymal stem cells possess brown-like functions and are referred to as BAT-like mesenchymal stem cells (BATMen). Brown adipose tissue (BAT) and its associated beige adipose tissue are specifically used for energy expenditure and fuel utilization. Furthermore, BAT exhibits endocrine function by communicating with various cells / organs to regulate systemic metabolism through secreted factors. Increasing evidence suggests that BAT activation can enhance glucose homeostasis, insulin sensitivity, and inflammation reduction, thus combating the onset of type 2 diabetes and cardiovascular disease in a clinical context. Significantly, BATmen administration substantially enhances glucose homeostasis by improving insulin sensitivity and promoting efficient energy utilization. Additionally, BATmen effectively alleviate MASLD-related inflammation by remodeling the immune cell population within the liver microenvironment. Overall, these findings highlight the therapeutic potential of BATmen cell therapy in addressing MASLD.

[0056] In some embodiments disclosed herein, gene mutations enhance the expression of peroxisome proliferation factor activating receptor γ (PPARγ). PPARγ is a major regulator of adipogenesis, the process by which preadipocytes differentiate into mature adipocytes. Its activation induces the expression of genes involved in fat storage, lipid metabolism, and insulin sensitivity. PPARγ promotes the transformation of mesenchymal stem cells into adipocytes by regulating the expression of genes such as C / EBPα and FABP4, which are key to lipid uptake and fat storage.

[0057] In some embodiments disclosed herein, gene mutations enhance the gene expression of peroxisome proliferation factor-activated receptor-γ coactivator 1-α (PGC1α). PGC-1α is a transcriptional coactivator that induces mitochondrial biosynthesis and thermogenesis. By coactivating transcription factors such as PPARγ with nuclear respiratory factor (NRF), PGC-1α increases mitochondrial density and uncoupling protein 1 (UCP1) expression, which is a marker of thermogenic cells. Therefore, it plays a key role in the browning of white adipose tissue and the transformation of white adipocytes into beige or brown-like adipocytes. Browning is characterized by the development of mitochondrial-rich thermogenic adipocytes, which can burn energy through non-shivering / adaptive thermogenesis.

[0058] In some embodiments disclosed herein, gene mutations enhance the expression of mitochondrial uncoupling protein 1 (UCP1). High levels of UCP1 are expressed in the mitochondria of thermogenic adipocytes. This uncoupling protein uncouples oxidative phosphorylation, resulting in the dissipation of the majority of the energy generated by proton-driven energy across the inner mitochondrial membrane as heat. However, the benefits of activating thermogenic adipocytes are not limited to energy dissipation. Brown and beige adipocytes can also produce and release factors such as peptides, lipids, and other metabolites that act as endocrine, paracrine, or autocrine factors to regulate metabolism.

[0059] According to this disclosure, mesenchymal stem cells can be obtained from various sources, preferably from umbilical cord, adipose tissue, or bone marrow. Depending on the source, mesenchymal stem cells are umbilical cord mesenchymal stem cells (UMSC), adipose-derived mesenchymal stem cells (ADSC), and bone marrow mesenchymal stem cells (BMSC). In some embodiments of this disclosure, MSCs are isolated and purified from the umbilical cord and are referred to as "umbilical cord MSCs" or "UMSC". In some embodiments, the UMSCs of this disclosure have been identified as exhibiting the same selected surface markers and similar activity as MSCs isolated from other organisms.

[0060] In some embodiments disclosed herein, MSCs comprise modified intracellular nucleic acid sequences to generate genetic variations in genetically engineered MSCs. Exemplary genetic variation techniques include (but are not limited to) homologous recombination, gene knock-in, ZFN (zinc finger nucleases), TALEN (transcription activator-like effector nucleases), CRISPR (clustered regularly spaced short palindromic repeats) / Cas9, and other site-directed nuclease techniques. These techniques enable double-stranded DNA breaks at desired gene loci. These controlled double-stranded breaks promote homologous recombination at specific gene loci. This approach focuses on targeting specific sequences of nucleic acid molecules (such as chromosomes) with endonucleases that recognize and bind to these sequences and induce double-stranded breaks in the nucleic acid molecules. Double-stranded breaks are repaired by error-prone non-homologous end joining (NHEJ) or by homologous recombination (HR).

[0061] The terms "CRISPR / Cas" or "clustered regularly spaced short palindromic repeat sequence system" or "CRISPR" interchangeably refer to DNA loci containing short repeats of a base sequence. Each repeat is followed by a short segment of spacer DNA previously exposed to viruses or plastids. Bacteria and archaea have evolved adaptive immune defenses known as CRISPR-CRISPR-associated (Cas) systems, which use short RNAs to guide the degradation of foreign nucleic acids. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. To guide Cas9 to cleave sequences of interest, crRNA-tracrRNA fusion transcripts, hereinafter referred to as "guide RNAs" or "gRNAs," can be designed using the human U6 polymerase III promoter. CRISPR / Cas-mediated genome editing and regulation highlight its transformative potential in fundamental science, cell engineering, and therapeutics. In the type II CRISPR / Cas system, short segments of foreign DNA (called "spacers") are integrated into the CRISPR gene locus and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs bind to trans-activated crRNA (tracrRNA) and guide the Cas protein to perform sequence-specific cleavage and silencing of the invading DNA. Recent studies have shown that Cas9 protein recognition of its target requires the presence of a "seed" sequence within the crRNA and a conserved adjacent motif (PAM) sequence containing a dinucleotide protospacer located upstream of the crRNA binding region.

[0062] In some embodiments disclosed herein, gene mutations and / or exogenous genes are introduced into MSCs using vectors containing genes for adipogenic differentiation, mitochondrial biosynthesis, and brown adipocyte markers. In some embodiments disclosed herein, gene mutations are introduced into MSCs using vectors containing PPARγ, PGC1α, and UCP1 genes. In some embodiments disclosed herein, gene mutation refers to the transfer of exogenous genes or gene fragments into mesenchymal stem cells, enabling them to express the exogenous gene or gene fragment. In another instance, this modification is a stable modification, and the expression can be persistent or inducible.

[0063] In some embodiments disclosed herein, the carrier is a transposable carrier.

[0064] In some embodiments disclosed herein, the vector comprises dCas9-VP64 and MS2-p65-HSF1 fragments.

[0065] In some embodiments disclosed herein, the vector comprises a U6 promoter operatively linked to a foreign gene.

[0066] In some embodiments disclosed herein, the vector is a multi-gene activator plasmid based on integrated CRISPR, which is constructed by the following steps: Provide gRNA selection vectors containing recombination sites and PPARγ, PGC1α, and UCP1 respectively, and The gRNA selection vector was mixed with a transposon backbone vector containing CRISPR / nuclease to generate multi-gene activators based on integrated CRISPR.

[0067] In one embodiment, the genetically modified MSCs described herein contain a promoter that induces constitutive expression of exogenous nucleic acids.

[0068] In one embodiment, the genetically modified MSCs described herein contain a U6 promoter operatively linked to a foreign gene.

[0069] The genetically engineered MSC population described in this article can be prepared as follows: a vector containing a multi-gene activated plastid based on integrated CRISPR is prepared, wherein the plastid is prepared as follows: a gRNA selection vector containing recombination sites and PPARγ, PGC1α and UCP1 are provided, and the gRNA selection vector is mixed with a transposon backbone vector containing CRISPR / nuclease to generate a multi-gene activated plastid based on integrated CRISPR; MSCs are transfected with these vectors to generate genetically engineered MSCs; genetically engineered MSCs are cultured in a medium suitable for the growth and proliferation of genetically engineered MSCs, and the genetically engineered MSC population is harvested.

[0070] This disclosure allows the use of any culture medium and culture conditions suitable for the growth and proliferation of genetically engineered MSCs.

[0071] As illustrated and broadly described herein, another aspect of this disclosure relates to a pharmaceutical composition comprising genetically engineered MSCs as described herein and, where applicable, a pharmaceutically acceptable carrier. According to this disclosure, in addition to the genetically engineered MSC population, the composition may contain one or more pharmaceutically permissible inactive carriers. Examples of inactive carriers include preservatives, solubilizers, stabilizers, etc. This composition can be used for non-oral administration, such as intravenous, subcutaneous, intraperitoneal, or topical administration. The dosage of the cell population may vary depending on the disease type, disease severity, route of administration, or individual weight, age, and sex.

[0072] In some embodiments, interstitial stem cells according to this disclosure are included in an injectable formulation. The injectable formulation can be prepared by methods known to be disclosed. For example, the injectable formulation can be prepared, for instance, by dissolving, suspending, or emulsifying a pharmaceutical composition in a sterile aqueous or oily medium known for injection. Examples of aqueous media for injection include physiological saline, an isotonic solution containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (an adduct of polyethylene oxide (50 mol) and hydrogenated castor oil)], etc. Examples of oily media include sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzoate, benzyl alcohol, etc. The resulting injection solution is preferably filled into a suitable ampoule.

[0073] In this invention, the route of MSC administration depends on the tissue or organ requiring treatment. In some embodiments involving individuals with myocardial infarction, the route of MSC administration may be intravenous, intra-arterial, or a combination thereof. The cell-containing solution may be prepared in a suitable diluent, such as water, ethanol, glycerol, liquid polyethylene glycol, various oils and / or mixtures thereof, and other diluents known to those skilled in the art.

[0074] As embodied and broadly described herein, another aspect of this disclosure relates to the use of the pharmaceutical composition described herein for the manufacture of an agent for the prevention, relief, and / or treatment of metabolic disorders in an individual in need. Alternatively, another aspect of this disclosure relates to a method for the prevention, relief, and / or treatment of metabolic disorders in an individual in need, comprising administering to the individual the pharmaceutical composition described herein. Still another aspect of this disclosure relates to the pharmaceutical composition described herein for the prevention, relief, and / or treatment of metabolic disorders in an individual in need.

[0075] Examples of metabolic disorders include (but are not limited to) diabetes, obesity, central obesity, insulin resistance, hypertension, insulin resistance syndrome, hepatic steatosis, heart defects, ischemic heart disease, high blood pressure, inflammation, dyslipidemia of triglycerides, dyslipidemia of HDL, dyslipidemia of cholesterol, elevated fasting plasma glucose, dysregulation of leptin, dysregulation of adiponectin, or cancer. In some embodiments disclosed herein, the metabolic disorder is fatty liver disease (MASLD) associated with metabolic dysfunction.

[0076] In some embodiments disclosed herein, the agent is used to improve insulin sensitivity, promote efficient energy utilization, remodel immune cell populations, and / or improve lipid accumulation, inflammation, or fibrosis in the liver.

[0077] The following examples are provided to illustrate certain aspects of the invention and to assist those skilled in the art in implementing it. These examples should not be construed as limiting the scope of the invention in any way. Without further detailed description, it is believed that those skilled in the art can utilize the invention to the fullest extent based on the description herein. All publications cited herein are incorporated herein by reference in their entirety.

[0078] Example

[0079] Materials and Methods

[0080] Preparation, isolation and characterization of human UMSC

[0081] The collected human umbilical cord tissue was washed three times with Ca2+ and Mg2+-free PBS (DPBS, Life Technology). It was mechanically dissected along the midline with scissors, separating the umbilical arteries, veins, and limiting membranes from the umbilical jelly (Wharton's jelly, WJ). The jelly contents were then thoroughly cut into pieces smaller than 0.5 cm³, treated with type 1 collagenase (Sigma, St Louis, USA), and incubated at 37°C in 5% CO2 for 3 hours. The separated pieces were then cultured in NutriStem® MSC XF medium containing NutriStem® XF supplement mixture (Sartorius, Germany), 5% UltraGRO™-Advanced-PURE cell culture supplement (AventaCell Biomedical, USA), and antibiotics at 37°C in 5% CO2. They were then left undisturbed for 5 to 7 days to allow cell migration from the separated pieces. After 4 to 8 passages, the cell morphology of umbilical cord-derived mesenchymal stem cells (UMSCs) became uniformly spindle-shaped during culture. Specific surface molecules from WJ cells were characterized by flow cytometry. Cells were dissected using TrypLE™ Select enzyme (Gibco, USA), washed with PBS, and cultured with specific antibodies including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1d, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC, and HLA-DR, and bound to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) (BD Biosciences, USA). Cells were then analyzed using a Becton Dickinson flow cytometer and FlowJo v.7.6 software to characterize UMBC markers.

[0082] Production of genetically modified UMSC

[0083] To deliver the CRISPR-MAP system to UMSCs, cells were electroporated using a Nucleofector™ 2b device (catalog number AAB-1001; Lonza Inc.) with program Y-001 (HMEC, High Efficiency). In short, 5 × 10⁵ UMSCs per light tube were resuspended in a Nucleofector™ human mesenchymal stem cell kit (catalog number VPE-1001; Lonza Inc.) along with 2.5 µg of transposon-based integrated CRISPR-MAP plastids and 2.5 µg of Super PiggyBac transposase express plastids. Following electroporation, cells were cultured in NutriStem® MSC XF medium containing a NutriStem® supplement mixture and 5% UltraGRO™-Advanced-PURE cell culture supplement in T25 flasks. After 3 days of cultivation, blasticidine (5 µg / mL) was added to the culture medium to select cells with transposons encoding the target gene.

[0084] Adipogenic differentiation of UMSC

[0085] For adipocyte differentiation, cells were grown in DMEM containing 10% FBS for 3 days until confluence, and then treated with adipogenic induction medium for another 12 days, which contained 10% FBS, 0.5 mM isobutylmethylxanthine, 0.1 µM dexamethasone, 0.5 µM human insulin, 2 nM T3, 30 µM indomethacin, 17 µM pantothenate, and 33 µM biotin (Sigma-Aldrich, Dallas, TX).

[0086] Gene expression

[0087] Total RNA was extracted from cells or tissues using Trizol and purified using a rotating column kit (Zymo Research). RNA (1 µg) was reverse transcribed using a high-capacity complementary DNA (cDNA) reverse transcription kit (Applied Biosystems). Real-time PCR was performed in a solution containing cDNA (8 ng), forward and reverse oligonucleotide primers (300 nM each), and SYBR green PCR premix (Roche). Fluorescence was measured and analyzed using an ABI 7900 sequence detection system (Applied Biosystems). Gene expression in human and mouse cells was normalized using 18S rRNA expression, respectively.

[0088] Protein expression

[0089] Cells were scraped from tissue culture trays using RIPA buffer. After centrifugation (12,000 g, 15 min), the protein lysate was harvested and stored at -80°C until further use. Protein concentration was determined according to the manufacturer's instructions using a Pierce BCA kit (Life Technologies). For immunoblotting, the lysate was diluted in Laemmli buffer, boiled, and loaded onto a 4–15% Tris gel for SDS-PAGE. After complete protein separation, it was transferred to a PVDF membrane (Amersham Biosciences) and blocked with Roche blocking buffer. Primary antibody was applied to blocking buffer at 4°C overnight. After washing three times with TBS-T for 10 min, secondary antibody was applied to blocking buffer and maintained for 1 hour. The membrane was washed three more times with TBS-T for 10 min and developed using ThermoFisher chemiluminescence. To quantify the scanned immunoblots, the integrated pixel density of the band of interest was measured using ImageJ software. Total actin was used as an endogenous control for standardization.

[0090] animal

[0091] All animal experiments were conducted in accordance with relevant ethical guidelines for the use of small rodents. C57BL6J mice (stock number 000664) were obtained from Jackson Laboratories. Mice had free access to food and water and were fed a normal diet (Mouse Diet 9F 5020, PharmaServ), which contained 22% calories from fat, 23% from protein, and 55% from carbohydrates. For the MASLD mouse model, mice were fed a high-fat diet containing 60 kcal% fat (catalog number D12492, Research Diets) and 30% fructose water. Mice were transferred to a hopper or a clean cage with no food or feces at the bottom before euthanasia and underwent a 6-hour fasting period. Mice were anesthetized by inhalation of isoflurane (catalog number NDC 66794-017-25, Piramal Critical Care), and blood was collected from the tail for fasting glucose measurement using an Infinity glucometer (US Diagnostics). Additional blood samples were obtained via cardiac puncture, and the plasma was then separated by centrifugation at 4°C and stored at -80°C until future analysis of ALT activity. BAT, pgWAT, scWAT, liver and quadriceps muscle were collected, weighed, and then flash-frozen in liquid nitrogen and stored at -80°C until further analysis.

[0092] Intravenous transplantation of CRISPR-engineered UMSC

[0093] Male mice were fed a high-fat diet and high-fructose water for 6 weeks, then anesthetized with 0.4 g / kg acetaldehyde hydrate (IP injection) and treated intravenously with 5 × 10⁵ cells. For animal randomization, a computer-generated numbering / sampling method was used to randomize the treatment groups. Control animals were given only PBS. Because mesenchymal stem cells have immunosuppressive properties, the rat host did not receive any immunosuppressive drugs.

[0094] Biodistribution of UMSCs in mouse models

[0095] To assess the homing effect of CRISPR-engineered UMSCs, the biodistribution of cells after intravenous implantation was evaluated using an IVIS imaging system. Cells were labeled with 1 mg / mL 1,1'-di(octadecyl)-3,3,3',3'-hexamethylindole tricarbonylcyanine iodide (DiR) (a near-infrared fluorescent lipophilic carbonylcyanine dye) (Thermo Fisher Scientific, USA) for 60 minutes at room temperature. After labeling, cells were washed with PBS and centrifuged at 3,000 × g for 10 minutes at 4 °C. The DiR-labeled UMSCs were then resuspended in sterile 1×PBS and intravenously injected into mice. One day after injection, mice were sacrificed, and various tissues were immediately collected for imaging using the IVIS system.

[0096] Measurement of lean body mass and body fat

[0097] Body composition, including lean body mass and fat mass, is measured using a dual-energy X-ray absorptiometry (DEXA) scanner. The machine is calibrated according to the manufacturer's instructions prior to the scan. A full-body scan is performed using the body composition mode of the DEXA system, which uses two low-energy X-ray beams to differentiate between bone, lean tissue, and fat tissue based on their attenuation characteristics. The scan duration ranges from 5 to 15 minutes. The data is analyzed using the scanner software, providing detailed measurements of total fat percentage, lean body mass, and localized fat distribution.

[0098] Indirect calorimetry

[0099] Mice were individually housed at room temperature in metabolic cages equipped with a Comprehensive Lab Animal Monitoring System (CLAMS). After a 12-hour acclimatization period, the animals were monitored for 24 hours to obtain the following measurements: oxygen consumption volume (VO2), carbon dioxide production volume (VCO2), respiratory exchange rate (RER) (calculated as the ratio of total VCO2 produced to total VO2 consumed), thermogenesis, activity level, and accumulated food intake.

[0100] Glucose and insulin tolerance test

[0101] For the glucose tolerance test (GTT), animals were fasted for 6 hours (7 AM to 1 PM) and given free access to drinking water. Baseline blood samples were collected from the tail of fully conscious mice, followed by intraperitoneal injection of glucose (2.0 g / kg body weight), with blood samples collected from the tail at 15, 30, 60, and 120 minutes post-injection. For the insulin tolerance test (GTT), animals were fasted for 6 hours (7 AM to 1 PM) and given free access to drinking water. Baseline blood samples were collected from the tail of fully conscious mice. Insulin (1 U / kg body weight) (Humulin O; Eli Lilly) was administered via intraperitoneal injection, with blood samples collected from the tail at 15, 30, 60, and 90 minutes post-injection. Blood glucose concentrations were measured using an InfinityO blood glucose meter (US Diagnostics).

[0102] Histological analysis

[0103] Tissue was fixed in 10% formalin for 24 hours and embedded in paraffin. Paraffin blocks were cut into 5 µm sections and stained with hematoxylin and eosin (H&E). Sections were dewaxed and rehydrated. Antigen retrieval was performed using regulated citrate buffer (Dako targeted retrieval solution, Agilent). Blocking was performed using blocking serum, followed by overnight incubation with primary antibody at 4°C. The next day, slides were washed with PBS and incubated with secondary antibody at a 1:200 dilution for one hour. After hematoxylin contrast staining (S-3309, Agilent), slides were fixed and imaged.

[0104] Oil Red O staining

[0105] The slides were washed twice with PBS and then fixed with 10% buffered formalin for 15 minutes at room temperature. After fixation, the cells were stained with filtered Oil Red O working solution. This working solution was prepared by combining 3 parts isopropanol containing 0.5% Oil Red O with 2 parts water. The staining process was carried out at room temperature for 1 hour. After staining, the cells were washed several times with distilled water and then visualized.

[0106] NPC separated from liver

[0107] Hepatic nonparenchymal cells (NPCs) were isolated using a two-step digestion protocol involving streptoprotein and collagenase. In short, the liver was perfused in situ with calcium-free Hank's Balanced Salt Solution (HBSS) containing 0.2 mg / mL EDTA. This was followed by sequential perfusion with 0.4 mg / mL streptoprotein (Sigma, P5147) and 0.2% collagenase type II (Worthington, LS004196). The liver was then minced and further digested in HBSS containing 0.2% collagenase type II, 0.4 mg / mL streptoprotein, and 0.1 mg / mL DNase I (Roche, R104159001) in a shaking water bath at 37°C for 20 minutes. To terminate digestion, DMEM supplemented with 10% serum was added. The resulting hepatocyte suspension was centrifuged at 50 × g for 3 minutes to remove hepatocytes and then passed through a 30 µm Nylon cell filter. Dissolve the remaining red blood cells using 0.8% NH4Cl.

[0108] Phenotyping of liver lymphocytes was performed using blood cell counting and based on time-of-flight (CyTOF).

[0109] After isolating NPCs from mouse livers, hepatic lymphocytes were phenotypically classified. Custom-designed metal-binding antibodies were used to stain cell surface markers for lymphocyte subtyping. Maxpar cytoplasmic / secretive antigen staining was performed using the Fresh Fix kit (Fluidigm, USA) according to the manufacturer's instructions. Briefly, cells were washed with PBS and stained with cisplatin to assess viability. NPCs were then labeled with the six surface markers included in the Maxpar mouse spleen / lymph node basic phenotypic classification kit (Fluidigm, USA). After surface marker staining, cells were fixed with 1.6% formaldehyde and stained with Cell-ID Intercalator-Ir. Before data collection, cells were washed and resuspended in cell collection solution (Fluidigm, USA) at a concentration of 1 × 10⁶ cells. Calibration beads were added at a 1:10 volume ratio for normalization. The stained cells were filtered in tubes with a mesh cap and analyzed using the Helios mass cytometry platform.

[0110] Statistical data

[0111] All statistical data were calculated using Microsoft Excel and GraphPad Prism. Unpaired Student's t-tests were performed for comparisons between only two groups. One-way and two-way ANOVAs were performed, followed by Tukey's post-hoc tests for multiple comparisons. Spearman's correlation tests were used to determine correlations, and Spearman's correlation coefficients are provided. ANCOVA analysis was performed directly from the MMPC energy expenditure analysis webpage to determine significance. All experiments involving mice were compared and statistically analyzed within littermates. A p-value less than 0.05 was considered statistically significant.

[0112] [Example] [1] [Based on] [CRISPR] [Multi-Gene Activation Platform] (CRISPR-MAP)

[0113] To achieve multi-gene activation in MSCs for engineering into multifunctional MSCs, we established a CRISPR-based multi-gene activation platform (CRISPR-MAP), which can simultaneously activate multiple genes after delivery of multiple gRNAs. First, we generated three individual gRNA selection vectors, each with a specific LR sequence for subsequent multi-site gateway selection. After selecting the specific gRNAs into the three vectors, all three vectors were mixed with a transposon backbone vector containing CRISPR-based activation systems (dCas9-VP64 and MS2-p65-HSF1). By adding a multiple-gateway selection enzyme, the three gRNAs were selected into the transposon backbone to generate an integrated CRISPR-based multi-gene activation plastid. [picture] [1a]). After the monolithic plastid containing transposase is delivered into the cell, the CRISPR-SAM system containing three different gRNAs is integrated into the DNA and constitutively activates three different genes ( [picture] [1b]).

[0114] [Example] [2] [use] [CRISPR-MAP] [Method Generation] [BAT] [Mesenchymal stem cells]

[0115] Given that BAT has the potential therapeutic effect in treating MASLD, our aim is to engineer MSCs to acquire BAT function via CRISPR-MAP, and we name the engineered MSCs as... [BAT]-like mesenchymal stem cells (BATMen). To establish BATMen, we targeted three different genes involved in adipogenic differentiation (PPARγ), mitochondrial biosynthesis (PGC1α), and brown marker (UCP1). After co-transfection with transposase-expressing vectors and plastids with or without one of the three gRNAs, we obtained BATMen and CTL-hMSCs ( [picture] [2a]). The results showed that both cell lines exhibited the CRISPR-SAM system (MS2) compared to the parental hMSCs. Importantly, based on the delivery of three gRNAs, BATMEmen showed high levels of PPARγ, PGC1α, and UCP1 ( [picture] [2b]).

[0116] [Example] [3 BATMen] [Therapy Relief] [MASLD] [Abnormal metabolic regulation in mouse models]

[0117] To establish a MASLD disease model, C57BL / 6 mice were fed a high-fat diet (HFD; 20% protein, 60% fat, and 20% carbohydrates, Research diets D12492) and drinking water (HFW) containing 30% (wt / v) fructose for 10 to 14 weeks using a previously established protocol (Softic, S. et al., Divergent effects of glucose and fructose on hepatic lipogenesis and insulin signaling. J Clin Invest, 2017).

[0127] (11): Pages 4059-4074). Following the allogeneic cell therapy protocol, mice were administered PBS, CTL-hMSCs, or BATME via tail vein injection after 6 weeks of HFD and HFW. Cell injections (5 x 10⁵ cells) were given once a week for a total of 10 weeks. [picture] [3a]). After four rounds of cell therapy, the mice were metabolically characterized using the following method. To determine the biodistribution of BATME after tail vein injection, we pre-stained BATME with DiR. One day after injection, red signals were mainly detected in the liver, and some signals were detected in the spleen and lungs. [picture] [3b]) This indicates that BATMEmen can target the liver and modulate the MASLD microenvironment.

[0118] Interestingly, four rounds of BATMEm cell therapy reduced the weight gain following HFD treatment. [picture] [3c]). The results also showed that MASLD mice treated with BATMEen had increased lean body mass and decreased body fat ( [picture] [3d] and [picture] [3e]). Furthermore, according to GTT and ITT monitoring, BATMEn improved glucose tolerance and insulin sensitivity ( [picture] [3f] and [picture] [3g]). Furthermore, BATMEn activation energy consumption leads to the consumption of more energy ( [picture] [3h]). In summary, BATMEm cell therapy can counteract the abnormal regulation of glucose and energy metabolism in MASLD mice.

[0119] [Example] [4 BATMen] [make] [MASLD] [Reduction of lipid accumulation, inflammation, and fibrosis in mouse liver]

[0120] To investigate whether BATMEn alleviates the pathogenesis of MASLD, we examined the histological, biochemical, and molecular changes in the liver. Oil Red O and Masson's trichrome staining methods were used to assess lipid accumulation and fibrosis in the liver. The livers of MASLD mice showed increased lipid droplets and collagen fibers. Interestingly, BATMEn treatment alleviated these abnormalities in the livers of MASLD mice. [picture] [4a] Furthermore, at the molecular level, the upregulation of fibrosis and inflammatory markers (such as collagen type III α1 chain (Col3a1) and tumor necrosis factor α (TNFα)) observed in MASLD mice was reversed by BATMEm therapy, resulting in a decrease in these markers to levels similar to those in mice fed a normal diet. [picture] [4b]).

[0121] [Example] [5 BATMen] [adjust] [MASLD] [Mouse liver] [TME] [Internal Immune Overview]

[0122] Chronic inflammation is a hallmark of MASLD development. It has been shown that the immunosuppressive tissue microenvironment (TME) of MASLD and HCC actually promotes cellular evasion of host immune surveillance, leading to treatment difficulties. Mouse basal autoimmune cells (BAT) can reduce inflammation in the hepatic TME through secreted factors; therefore, we examined whether the BAT™ remodels the immune regulation within the MASLD hepatic TME. After isolating non-parenchymal cell populations (NPCs) from the liver, we used CyTOF analysis on individual cells to elucidate the immune profile in detail. The results revealed that the MASLD liver was infiltrated with more monocytes / macrophages and contained fewer CD8+ T cells (…). [picture] [5a] and [picture] [5b]). Interestingly, BATMen therapy reversed this immune profile within the hepatic TME of MASLD. BATMen downregulated the monocyte / macrophage population and upregulated CD8+ T cells ( [picture] [5a] and [picture] [5b] ) ​​Using tSEN analysis, results showed a significant increase in specific subtypes of monocytes / macrophages in the liver of patients with MASLD, but a decrease after BATMEm treatment (left-bottom). [picture] [5c]). After evaluating MASLD-associated macrophage (NAM) markers (such as GPNMB, TREM2, and APOE) isolated from non-parenchymal cells (NPCs) of mouse liver, we noted that NAM markers in the liver increased after MASLD dietary treatment. [picture] [5d]). Notably, the addition of BATMEmen caused a decrease in the content of NAM markers ( [picture] [5d]). These findings indicate that BATMEM therapy effectively reduces inflammation in the liver of MASLD patients.

Claims

1. A genetically engineered mesenchymal stem cell (MSC) comprising exogenous genes for peroxisome proliferator-activated receptor γ (PPARγ), peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α), and mitochondrial uncoupling protein 1 (UCP1), wherein the gene expression of PPARγ, PGC1α, and UCP1 is increased.

2. The genetically engineered MSCs as requested in item 1, wherein the MSCs are umbilical cord mesenchymal stem cells (UMSC), adipose-derived mesenchymal stem cells (ADSC), or bone marrow mesenchymal stem cells (BMSC).

3. The genetically engineered MSCs of claim 1, wherein the exogenous genes are introduced into the MSCs by using a vector containing the PPARγ, PGC1α and UCP1 genes.

4. As in request item 3, the genetically engineered MSC, wherein the vector is a transposon vector.

5. The genetically engineered MSCs of claim 1, wherein the foreign genes are introduced into the MSCs by using clustered regularly spaced short palindromic repeats (CRISPR) / nucleases.

6. The genetically engineered MSCs of claim 3, wherein the vector contains dCas9-VP64 and MS2-p65-HSF1 fragments.

7. The genetically engineered MSC of claim 3, wherein the vector contains a U6 promoter operatively linked to the exogenous genes.

8. The genetically engineered MSC of claim 3, wherein the vector is a multi-gene activator plasmid based on integrated CRISPR, which is constructed by the following steps: providing gRNA selection vectors containing recombination sites and PPARγ, PGC1α and UCP1 respectively, and mixing the gRNA selection vectors with a transposon backbone vector containing CRISPR / nuclease to produce a multi-gene activator plasmid based on integrated CRISPR.

9. A population of engineered mesenchymal stem cells as claimed in any one of claims 1 to 8.

10. A vector comprising the exogenous genes PPARγ, PGC1α and UCP1.

11. The carrier of request item 10 contains the dCas9-VP64 and MS2-p65-HSF1 fragments.

12. The vector of claim 10 or 11 contains a U6 promoter operatively linked to the exogenous genes.

13. A method for preparing a population of genetically engineered MSCs as claimed in any one of claims 1 to 8, comprising: preparing a vector containing a multi-gene activated plasmid based on an integrated CRISPR, wherein the plasmid is prepared as follows: providing gRNA selection vectors containing recombination sites and PPARγ, PGC1α and UCP1 respectively, and mixing the gRNA selection vectors with a transposon backbone vector containing CRISPR / nuclease to produce a multi-gene activated plasmid based on an integrated CRISPR; transfecting MSCs with the vectors to produce genetically engineered MSCs; culturing the genetically engineered MSCs in a medium suitable for the growth and proliferation of the genetically engineered MSCs; and harvesting the genetically engineered MSC population.

14. A genetically engineered population of MSCs prepared by the method of claim 13.

15. A pharmaceutical composition comprising a genetically engineered MSC population as claimed in claim 9 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a genetically engineered MSC population as claimed in claim 14 and a pharmaceutically acceptable carrier.

17. Use of a pharmaceutical composition as claimed in claim 15 for manufacturing a medicament for the prevention, relief and / or treatment of metabolic disorders in an individual in need.

18. As claimed in claim 17, wherein the metabolic disorder is diabetes, obesity, central obesity, insulin resistance, hypertension, insulin resistance syndrome, hepatic steatosis, heart defect, ischemic heart disease, high blood pressure, inflammation, dyslipidemia of triglycerides, dyslipidemia of HDL, dyslipidemia of cholesterol, elevated fasting plasma glucose, dysregulation of leptin, dysregulation of adiponectin, or cancer.

19. As claimed in claim 17, wherein the metabolic disorder is fatty liver disease (MASLD) associated with metabolic dysfunction.

20. The use as claimed in claim 17, wherein the agent is used to improve insulin sensitivity, promote efficient energy utilization, remodel immune cell populations, and / or improve lipid accumulation, inflammation, or fibrosis in the liver.

21. As claimed in claim 17, wherein the effective amount ranges from about 1 × 10⁴ cells to about 1 × 10⁸ cells.

22. As claimed in claim 17, wherein the drug is administered by intravenous injection.

23. Use of a pharmaceutical composition as claimed in claim 16 for manufacturing a medicament for the prevention, relief and / or treatment of metabolic disorders in an individual in need.

24. As claimed in claim 23, wherein the metabolic disorder is diabetes, obesity, central obesity, insulin resistance, hypertension, insulin resistance syndrome, hepatic steatosis, heart defect, ischemic heart disease, high blood pressure, inflammation, dyslipidemia of triglycerides, dyslipidemia of HDL, dyslipidemia of cholesterol, elevated fasting plasma glucose, dysregulation of leptin, dysregulation of adiponectin, or cancer.

25. As requested in claim 23, wherein the metabolic disorder is fatty liver disease (MASLD) associated with metabolic dysfunction.

26. The use as claimed in claim 23, wherein the agent is used to improve insulin sensitivity, promote efficient energy utilization, remodel immune cell populations, and / or improve lipid accumulation, inflammation, or fibrosis in the liver.

27. As claimed in claim 23, wherein the effective amount ranges from about 1 × 10⁴ cells to about 1 × 10⁸ cells.

28. As claimed in claim 23, wherein the drug is administered by intravenous injection.

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