Methods of generating adipocytes

Abstract

The present invention relates to methods for generating adipocytes using direct reprogramming.

Description

Technical Field

[0001] This invention relates to a method for generating adipocytes by overexpressing one or more polypeptides with transcription factor combination activity and / or the transcription factor combination itself, i.e., by positive reprogramming. Background Technology

[0002] Adipocytes, also known as fat cells or fat cells, are the main components of adipose tissue and play a crucial role in energy metabolism in vertebrates. They have various uses in medical research and treatment, such as tissue generation and wound healing. Furthermore, adipocytes are also used in cultured meat production because the mixture of muscle cells and adipocytes produces meat products with better texture.

[0003] Currently, methods for differentiating stem cells into adipocytes typically involve treating stem cells with differentiation-inducing substances (such as insulin, dexamethasone, and IBMX) and culturing them for extended periods. However, these differentiation-inducing substances are expensive and have low cell differentiation efficiency. In contrast, forward programming strategies deliver mature human cell types with unprecedented speed and efficiency. Forward programming involves directly converting pluripotent stem cells (including human pluripotent stem cells (hPSCs)) into specific mature cell types by forcibly expressing peptides with key lineage transcription factor activity and / or the key lineage transcription factors themselves.

[0004] There is a need in the art to provide methods for generating adipocytes suitable for use as potential therapeutic agents, for research, and for tissue engineering. Summary of the Invention

[0005] According to a first aspect of the invention, a method for generating adipocytes is provided, comprising increasing the expression of one or more transcription factors in a cell population, wherein the one or more transcription factors are selected from: peroxisome proliferator-activated receptor (PPAR) protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof, and culturing the cell population to obtain adipocytes.

[0006] According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of one or more transcription factors in a cell population (preferably a pluripotent stem cell population, more preferably a human induced pluripotent stem cell (hiPSC) population), said transcription factors being selected from: one or more PPAR proteins (such as PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CCAAT enhancer-binding proteins (CEB proteins, such as CEBPA and / or CEBPB) and variants thereof, and culturing the cell population to obtain adipocytes.

[0007] In a preferred embodiment, the transcription and translation (expression) of the polypeptide and / or the transcription factor itself, which have transcription factor activity, are controlled intracellularly, preferably by using external stimuli.

[0008] According to another aspect of the present invention, a method for producing adipocytes from source cells (preferably pluripotent stem cells, more preferably hiPSCs) is provided, comprising the following steps: a) Inserting (preferably targeted) the gene encoding a transcriptional regulatory protein into the first genomic safe harbor site of the source cell; and b) Inserting (preferably targeted insertion) at least one nucleotide sequence into a second genomic safe harbor site of a source cell, the nucleotide sequence encoding one or more polypeptides having one or more transcription factor activities and / or encoding one or more transcription factors selected from: one or more PPAR proteins (such as PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (such as CEBPA and / or CEBPB) and variants thereof, the nucleotide sequence being operatively linked to an inducible promoter, wherein the inducible promoter is regulated by a transcriptional regulatory protein; and c) Culture source cells containing the insert to obtain adipocytes.

[0009] According to another aspect of the present invention, a method for producing adipocytes from source cells is provided, comprising the following steps: a) Targeting the insertion of genes encoding transcriptional regulatory proteins into the first genomic safe harbor site of the source cell; and b) Targeting at least one nucleotide sequence into a second genomic safe harbor site in a source cell, said nucleotide sequence encoding one or more transcription factors selected from the group consisting of: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423, and variants thereof, said nucleotide sequence being operatively linked to an inducible promoter, said inducible promoter being regulated by a transcriptional regulatory protein; and c) Culture source cells containing the insert to obtain adipocytes.

[0010] According to another aspect of the invention, there is a use for generating adipocytes with at least two or more transcription factors, wherein the two or more transcription factors are selected from: PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof.

[0011] According to another aspect of the invention, there is provided the use of one or more polypeptides having one or more transcription factor activities and / or one or more transcription factors for generating adipocytes (preferably human adipocytes), wherein the one or more transcription factors are selected from: PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (such as CEBPA and / or CEBPB) and variants thereof.

[0012] According to another aspect of the invention, a cell is provided that can be obtained by any of the methods defined herein.

[0013] According to another aspect of the invention, a cell is provided comprising one or more exogenous expression cassettes, the expression cassettes comprising nucleotide sequences encoding at least one or more transcription factors, wherein the one or more transcription factors are selected from: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof.

[0014] According to another aspect of the invention, a cell (preferably a pluripotent stem cell, more preferably a hiPSC) is provided, comprising one or more exogenous expression cassettes, the expression cassettes comprising encoding one or more polypeptides having one or more transcription factor activities and / or nucleotide sequences encoding at least one or more transcription factors, wherein the one or more transcription factors are selected from: one or more PPAR proteins (such as PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (such as CEBPA and / or CEBPB) and variants thereof.

[0015] According to another aspect of the invention, cells (preferably human cells) as defined herein are provided for therapeutic, in vitro diagnostic, drug screening, or preparation of cultured meat.

[0016] According to another aspect of the present invention, a kit for differentiating cells into adipocytes is provided, comprising: (i) source cells and reagents that activate or increase the expression or amount of at least one or more transcription factors; and / or (ii) One or more expression cassettes containing nucleotide sequences encoding at least one or more transcription factors. The one or more transcription factors mentioned therein are selected from: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and their variants.

[0017] According to another aspect of the present invention, a kit for differentiating cells (preferably pluripotent stem cells, more preferably hiPSCs) into adipocytes is provided, comprising: (i) source cells and reagents that activate or increase the expression or amount of at least one or more transcription factors; and / or (ii) One or more expression cassettes comprising encoding one or more polypeptides having one or more transcription factor activities and / or nucleotide sequences encoding at least one or more transcription factors. The one or more transcription factors mentioned therein are selected from: one or more PPAR proteins (such as PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (such as CEBPA and / or CEBPB) and their variants.

[0018] According to another aspect of the invention, a kit as defined herein is provided for use in differentiating cells into adipocytes.

[0019] According to another aspect of the invention, a drug screening method is provided, the method comprising contacting adipocytes generated using a method defined herein or adipocytes defined herein with a drug, and observing changes in the adipocytes induced by the drug.

[0020] According to another aspect of the invention, a method for treating a subject suffering from a disease or disorder or at risk of a disease or disorder is provided, the method comprising administering to the subject a therapeutically effective amount of adipocytes produced using methods defined herein or as defined herein. Attached Figure Description

[0021] Figure 1 Single-cell gene expression data visualized on UMAP plots, including undifferentiated iPSCs (G10), end-sorted live cells (NG), FABP4-GFP positive cells, PLIN1-mCherry positive cells, and marker-negative cells (FN).

[0022] Figure 2 Distribution of transcription factors (eTFs) detected in the UMAP diagram.

[0023] Figure 3 UMAP cell atlas annotated with CellTypist based on the adipose tissue cell gene expression profile reference dataset. In addition to adipocytes, four other cell types (cell types 1-4) are also shown in the figure.

[0024] Figure 4 The dot plot shows a double-positive cell population of FABP4-GFP and PLIN1-mCherry. Figure 1 The expression levels of adipokines, adipocyte marker genes, and cardiac marker genes (as controls) are shown in the figure. The size of the dot represents the proportion of cells expressing the gene in each cell population, and the gray level represents the average expression level.

[0025] Figure 5 The dot plot shows the different cell types annotated by CellTypist ( Figure 3 The expression levels of marker genes in brown adipose tissue are shown in the figure. The size of the dot represents the percentage of cells expressing the gene in each population, and the gray level represents the average expression level.

[0026] Figure 6 The dot plot shows the different cell types annotated by CellTypist ( Figure 3 The expression levels of marker genes in white adipose tissue are shown in the figure. The size of the dot represents the percentage of cells expressing the gene in each population, and the gray level represents the average expression level.

[0027] Figure 7 Log-likelihood enrichment of each eTF combination in the adipocyte population compared to the control (NG) population. Upward: Adipocytes in FABP4-GFP positive cells in each experimental replicate; Downward: Adipocytes in PLIN1-mCherry positive cells in each experimental replicate.

[0028] Figure 8 On day 10 of reprogramming, RT-qPCR was performed on adipocyte markers encoding PPARG, PPARA, HOXC8, ZNF423, EBF1, and EBF2 in iPSC lines. The expression levels shown were relative to the internal control hydroxymethylcholine synthase (HMBS).

[0029] Figure 9 On day 10 of reprogramming, immunocytochemical staining was performed on adipocyte markers FABP4 and PLIN1 in iPSC lines encoding PPARG, PPARA, HOXC8, ZNF423, EBF1, and EBF2. Lipid accumulation was assessed by LipidTOX staining. Scale bar: 100 µm. Detailed Implementation

[0030] The present invention provides a method for generating adipocytes from source cells by expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of a selected set of transcription factors, which the inventors have determined to induce cell differentiation into adipocytes.

[0031] definition

[0032] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the following terms have the meanings assigned to them as follows.

[0033] As used herein, the term "transcription factor" refers to a protein involved in gene regulation in both prokaryotes and eukaryotes. In one embodiment, a transcription factor can have a positive effect on gene expression and is therefore referred to as an "activator" or "transcriptional activator." In another embodiment, a transcription factor can have a negative effect on gene expression and is therefore referred to as a "repressor" or "transcriptional repressor." Activators and repressors are commonly used terms, and their functions are readily recognized by those skilled in the art.

[0034] Regarding increasing the amount, level, or expression of transcription factors, the terms "increased expression" or "increased amount" refer to increasing the amount of transcription factors in target cells (e.g., source cells). In some embodiments, the amount of transcription factors in cells is increased (e.g., by an expression cassette that directs the expression of one or more polynucleotides encoding a transcription factor) when the amount of the transcription factor is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a control (e.g., source cells without the expression cassette or control cells with baseline expression of zero or negligible). In some embodiments, increased expression involves "overexpressing" the transcription factor, i.e., increasing the expression of the transcription factor to a level higher than the endogenous expression level of the transcription factor in the cells.

[0035] The method of this invention can be used for a "cell population," that is, a collection of cells that can differentiate into a desired cell type. The cell population may contain "source cells," also known as "initial cells," that is, cell types prior to differentiating into the desired cell type.

[0036] The term "pluripotent" as used herein refers to cells with the potential to differentiate into all cell types found in organisms. One form of pluripotent stem cell, called induced pluripotent stem cells (iPSCs), is of particular interest in this invention. Induced pluripotent stem cells (iPSCs) are cells that have been reprogrammed into an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the most typical characteristics of embryonic stem cells. A 2006 study demonstrated that overexpression of four specific transcription factors can convert adult cells into pluripotent stem cells. Certain members of the Oct-3 / 4 and Sox gene families have been identified as potential key transcriptional regulators involved in the induction process. Other genes, including certain members of the Klf, Myc, Nanog, and Lin28 families, can enhance induction efficiency. Examples of genes that can be used as reprogramming factors to generate iPSCs include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Lin28b, Sall4, Esrrb, Tbx3, and Glis1, GATA3, GATA6. These reprogramming factors can be used alone or in combination of two or more. In particular, the reprogramming factors may contain at least Yamanaka factors, namely Oct3 / 4, Sox2, Klf4, and c-Myc. These reprogramming factors can also be used in combination with the target transcription factors of this invention.

[0037] The term "somatic cell" as used herein refers to any type of cell that constitutes an organism, excluding germ cells. Therefore, somatic cells include, for example, skin, heart, muscle, bone, or blood cells and their stem cells. Somatic cells may also be referred to as differentiated cells. In one embodiment, a somatic cell may be an adult cell or a cell derived from an adult that exhibits one or more detectable characteristics of adult or non-embryonic cells.

[0038] The method of the present invention (e.g., cell reprogramming of iPSCs) is used to generate "adipocytes," also referred to as "lipocytes" or "adipocytes." As used herein, the term "adipocyte" refers to a cell associated with vertebrate adipose tissue. This term includes white adipocytes (which are the primary site of triglyceride / energy storage) and brown adipocytes (which play an important role in energy expenditure in the form of thermogenesis). In one embodiment, the adipocyte is a white adipocyte. In another embodiment, the adipocyte is a brown adipocyte. The term "adipocyte" includes adipocyte-like cells exhibiting some, but not all, of the characteristics of adult adipocytes, as well as mature, fully functional, and / or metabolically active adult adipocytes. The term also includes adult and fetal adipocyte progenitor cells (also called preadipocytes) and fetal adipocytes. The term also includes other cells capable of implanting into adipose tissue when transplanted in vivo. Adipocytes generated by this method can be at least as functional as adipocytes generated to date through directed differentiation.

[0039] The term "culture" as used herein includes adding cells (e.g., cell populations, i.e., source cells) to a culture medium containing growth factors and / or essential nutrients. It should be understood that such culture conditions can be adjusted based on the cells or cell populations produced by the methods of the present invention.

[0040] When referring to a polypeptide, the term "variant" can refer to, for example, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. When referring to a nucleic acid sequence, the term "variant" can refer to, for example, a nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% identity to the original full-length nucleic acid sequence. A variant can be a fragment of a full-length polypeptide, particularly a functional fragment of the polypeptide. The fragment can be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of the length of a domain of the full-length wild-type polypeptide or the domain having the intended activity (e.g., the ability to differentiate source cells into adipocytes). Variations that eliminate or significantly reduce protein activity, as known in the art, are preferably avoided. In some embodiments, the variant lacks the N- and / or C-terminal portions of the full-length polypeptide, for example, lacking up to 10, 20, or 50 amino acids from either end. In some embodiments, the functional variant or fragment has at least 50%, 60%, 70%, 80%, 90%, 95%, or more of the activity of the full-length wild-type polypeptide. Using assays known in the art, those skilled in the art will know or be able to readily determine whether a particular polypeptide variant or fragment is functional. For example, the assays described herein can be used to assess the ability of the transcription factor variants listed in Table 1 to generate adipocytes. In particular, variants can be biologically active variants. “Biologically active variant” includes any molecular variant having substantially (at least partially) the same function and / or biological properties (e.g., binding properties) and / or the same structural features (e.g., binding domains) as the molecule described herein. It also refers to molecules exhibiting the same functional features as the transcription factors disclosed herein.

[0041] In one implementation, a variant is an isoform of the listed transcription factor. Many transcription factors have one or more isoforms, generated for example by transcription initiation via alternative splicing or translocation. Different polypeptides are generated based on different transcript variants (i.e., mRNAs). Different transcript variants may have different translation initiation sites.

[0042] A “promoter” is a nucleotide sequence recognized by proteins involved in initiating and regulating polynucleotide transcription. An “inducible promoter” is a nucleotide sequence in which the expression of a gene sequence operatively linked to the promoter is controlled by analytes, cofactors, regulatory proteins, etc. The terms “promoter” or “control element” are intended to encompass both the full-length promoter region and the functional (e.g., controlling transcription or translation) segments of these regions.

[0043] The term "operably linked" refers to the arrangement of elements in which the components, as described above, are configured to perform their usual functions. Thus, a given promoter operably linked to a gene sequence can influence the expression of that sequence when a regulatory factor is present. The promoter need not be contiguous with the sequence, as long as it plays a role in directing its expression. Therefore, for example, an inserted untranslated but transcribed sequence can exist between the promoter sequence and the gene sequence, and the promoter sequence can still be considered "operably linked" to the gene sequence. Therefore, the term "operably linked" is intended to encompass any spacing or orientation between the promoter element and the gene sequence in the inducible cassette that allows transcription of the inducible cassette to be initiated when the transcription complex recognizes the promoter element.

[0044] As used herein, the term "vector" is intended to refer to a nucleic acid molecule used as a carrier to carry genetic material into a cell. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop or loop in which additional DNA segments can be attached. Another type of vector is an infectious but non-pathogenic viral vector, in which additional DNA segments can be attached to certain viral genetic elements. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian and yeast vectors). After introduction into a host cell, other vectors (e.g., non-free-living mammalian vectors) can integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technologies are in the form of plasmids. However, this invention aims to include other forms of such expression vectors with equivalent functionality, such as viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, Sendai viruses, and adeno-associated viruses), as well as bacteriophage and phage particle systems. Another type of vector contains RNA molecules, such as mRNA and stable RNA, which carry encoded genetic information into the cell. This also includes synthetic self-replicating RNA vectors.

[0045] The terms "subject," "patient," or "individual" refer to a subject to treatment, particularly a mammalian subject. Mammal subjects include humans, non-human primates, farm animals (e.g., cattle), athletic animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human mammal, such as a mouse.

[0046] The term "sufficient amount" refers to an amount sufficient to produce the desired effect. The term "therapeutic effective amount" is an amount that effectively improves the symptoms of a disease or ailment. Therapeutic effective amount can also be "preventive effective amount," since prevention can be considered treatment.

[0047] As used herein, the term “about” as used herein includes being up to 10% and inclusive above a specified value, and up to 10% and inclusive below a specified value, appropriately, up to 5% and inclusive above a specified value, and up to 5% and inclusive below a specified value, particularly the specified value. The term “between” includes values ​​that specify boundaries.

[0048] It should be understood that any method described herein may have one or more steps performed in vitro, ex vivo, or in vivo.

[0049] Transcription factor activity

[0050] The methods described herein may involve increasing the expression (particularly protein expression) of a sufficient amount of transcription factor-active polypeptides (or transcription factors themselves) (e.g., portions listed in Table 1 and their variants and isotypes) to differentiate a cell population into adipocytes, thereby causing the cell population to differentiate into adipocytes. In the context of this invention, these factors may also be referred to as “reprogramming factors.” As described herein, the expression of exogenous or endogenous (particularly exogenous) transcription factors can be increased.

[0051] According to one aspect of the invention, a method for generating adipocytes is provided, the method comprising increasing the expression of one or more transcription factors in a cell population, wherein the one or more transcription factors are selected from: a peroxisome proliferator-activated receptor (PPAR) protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof, and culturing the cell population to obtain adipocytes.

[0052] According to another aspect of the invention, a method for generating adipocytes is provided, the method comprising increasing the expression of one or more polypeptides having one or more transcription factor activities and / or increasing the expression of one or more transcription factors selected from: one or more PPAR proteins (e.g., PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) and variants thereof) in a cell population, and culturing the cell population to obtain adipocytes.

[0053] The term "peroxisome proliferator-activated receptor" (PPAR) or "PPAR protein" mentioned in this article refers to a transcription factor called peroxisome proliferator-activated receptor in the nuclear receptor proteome. There are three types of PPARs in this group: PPARα (PPARA), PPARβ / δ (PPARD), and PPARγ (PPARG). All PPARs heterodimerize with the retinoic acid X receptor (RXR) and bind to peroxisome proliferator hormone response elements (specific regions on the target gene DNA). These specific regions share a common DNA sequence of 5'-AGGTCANAGGTCA-3' (SEQ ID NO: 1), where N is any nucleotide.

[0054] In one embodiment, PPAR is selected from PPARA and PPARG. It should be understood that one or more transcription factors as mentioned herein comprise more than one type of PPAR protein, such as a combination of PPARA and PPARG. In one embodiment, PPAR is PPARA. In other embodiments, PPAR is PPARG. It should be understood that if the expression of one or more peptides having one or more transcription factor activities and / or more than one transcription factor is increased, one or more PPAR proteins may be included. Therefore, in one embodiment, the method comprises increasing the expression of one or more peptides having PPARA and PPARG activities and / or PPARA and PPARG themselves.

[0055] The terms “PPARA”, “PPARα”, or “peroxisome proliferator-activated receptor α” used herein refer to members of the PPAR nuclear hormone receptor subfamily. In one embodiment, PPARA is human PPARA. Wild-type human PPARA is identified by UniProtID: Q07869 and is manufactured by [the relevant authority / organization]. PPARA The gene is encoded by Ensembl Gene ID: ENSG00000186951.

[0056] The terms “PPARG”, “PPARγ”, or “peroxisome proliferator-activated receptor γ” used herein refer to members of the PPAR nuclear hormone receptor subfamily and include all isotypes of PPARG, such as PPARG1 and PPARG2. In one embodiment, PPARG is human PPARG. Wild-type human PPARG is identified by UniProt ID: P37231 and is provided by [the relevant authority / organization]. PPARG The gene is encoded by Ensembl Gene ID: ENSG00000132170.

[0057] The “HOXC8” mentioned in this article refers to the homeobox protein Hox-C8. In one implementation, HOXC8 is human HOXC8. Wild-type human HOXC8 is identified by UniProt ID: P31273 and is... HOXC8 The gene is encoded by EnsemblGene ID: ENSG00000037965. HOXC8 belongs to the homeobox gene family, which encodes a highly conserved family of transcription factors. HOXC8 is involved in the regulation of cartilage differentiation, cell junctions, and the regulation of CDH11 expression and function.

[0058] The “EBF1” mentioned in this article refers to early B cell cytokine 1 (also known as transcription factor COE1). In one implementation, EBF1 is human EBF1. Wild-type human EBF1 is identified by UniProt ID: Q9UH73 and is... EBF1 The gene is encoded by Ensembl Gene ID: ENSG00000164330. EBF1 is involved in olfactory signaling pathways and nervous system development. EBF1 activates B cell-specific genes, such as BCR or CD40, and suppresses genes associated with T cell fate, such as GATA3 and TCF7.

[0059] The “EBF2” mentioned in this article refers to early B cell cytokine 2 (also known as transcription factor COE2). In one implementation, EBF2 is human EBF2. Wild-type human EBF2 is identified by UniProt ID: Q9HAK2 and is... EBF2 The gene is encoded by Ensembl Gene ID: ENSG00000221818. EBF2 regulates osteoclast differentiation by activating the RANKL decoy receptor TNFRSF11B.

[0060] The references to “ZNF467” or “zinc finger protein 467” in this document refer to zinc finger proteins. In one embodiment, ZNF467 is human ZNF467. Wild-type human ZNF467 is identified by UniProt ID: Q7Z7K2 and is... ZNF467 The gene is encoded by Ensembl Gene ID: ENSG00000181444. ZNF467 binds to STAT3 via a common sequence 5'-CTTCTGGGAAGA-3' (SEQ ID NO: 2).

[0061] The “ZNF423” mentioned in this article refers to zinc finger protein 423. In one embodiment, ZNF423 is human ZNF423. Wild-type human ZNF423 is identified by UniProt ID: Q2M1K9 and is... ZNF423The gene is encoded by Ensembl Gene ID: ENSG00000102935. ZNF423 plays a central role in BMP signaling and olfactory neurogenesis. ZNF423 acts as a transcriptional repressor by interacting with EBF1 (a transcription factor involved in the differentiation of terminal olfactory receptor neurons). ZNF423 participates in olfactory neurogenesis by regulating the developmental switch that controls the transition of olfactory receptor neurons from differentiation to maturity.

[0062] The “CEB protein” mentioned in this document refers to a CCAAT enhancer-binding protein. In one embodiment, the CEB protein is the human CEB protein. The CEB protein can be such as CEBPA (also known as CEBPα or C / EBPα) and / or CEBPB (also known as CEBPβ ​​or C / EBPβ). Wild-type human CEBPA is identified by UniProt ID: P49715 and is manufactured by [unclear - likely a company name]. CEBPA The gene is encoded by Ensembl Gene ID: ENSG00000245848. Wild-type human CEBPB is identified by UniProt ID: P17676 and is... CEBPB The gene is encoded by Ensembl Gene ID: ENSG00000172216. As its name suggests, the CEB protein interacts with the CCAAT box motif present in the promoters of several genes. The CEB protein possesses a highly conserved basic leucine zipper domain at its C-terminus. It also has activation and regulatory domains at its N-terminus. The CEB protein recruits coactivators, such as CREB-binding proteins, which further open chromatin structures or recruit basal transcription factors.

[0063] In one embodiment, the CEB protein is CEBPB. In another embodiment, the CEB protein is not CEBPA.

[0064] In one embodiment, the method comprises expressing one or more polypeptides having two or more transcription factor activities, particularly three or more, four or more, or five transcription factors, and / or increasing the expression of two or more transcription factors, particularly three or more, four or more, five or more, and six or more transcription factors, wherein the transcription factors are selected from: PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (such as CEBPA and / or CEBPB); and variants thereof.

[0065] In one embodiment, the method comprises expressing one or more polypeptides having one or more transcription factor activities, particularly two or more, three or more, four or more, or five transcription factors, and / or increasing the expression of one or more transcription factors, particularly the expression of two or more, three or more, four or more, five or more, and six or more transcription factors, wherein the transcription factors are selected from: PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB); and variants thereof.

[0066] Once the activity of transcription factors is understood, not only the transcription factors themselves but also engineered peptides to replicate their effects—such as synthetic or artificial transcription factors—can be used to regulate endogenous transcriptional mechanisms. For example, CRISPR (clustered regularly spaced short palindromic repeats), TALE (transcription activator-like effectors), or zinc finger technology can be used to modulate the expression of endogenous cellular genes to achieve faster and more efficient nuclear reprogramming under conditions suitable for clinical and commercial applications. As described in US2016 / 362705, this patent is incorporated herein by reference.

[0067] Alternatively, with the development of AI tools such as AlphaFold in high-precision protein structure prediction, it is now possible to directly design peptides that have a structure and / or activity very similar to the target transcription factor, while their amino acid sequences are almost dissimilar to those of the target transcription factor. For example, large language models trained on biodiversity have been used to develop proteins that share only about 70% similarity with naturally occurring CRISPR-Cas proteins but possess comparable or superior biological activity and specificity (Ruffolo et al. (2024) bioRxiv, doi:https: / / doi.org / 10.1101 / 2024.04.22.590591). Such peptides are included within the scope of this invention.

[0068] In some embodiments of the invention, a polypeptide (particularly a single polypeptide) is engineered to mimic the activities of more than one target transcription factor. In another embodiment, expression of a polypeptide having one or more transcription factor activities is combined with increased expression of another transcription factor. For example, a polypeptide having PPARA activity can be expressed while simultaneously increasing HOXC8 expression (and vice versa).

[0069] According to another aspect of the invention, a method for generating adipocytes is provided, comprising increasing the expression of three or more transcription factors in a non-adipocyte population, wherein the one or more transcription factors are selected from: PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof, and culturing the cell population to obtain adipocytes.

[0070] According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of one or more transcription factors themselves in a non-adipocyte population, wherein the one or more transcription factors are selected from: PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) and variants thereof, and culturing the cell population to obtain adipocytes.

[0071] In one implementation, (e.g., at least one or more) transcription factors are selected from PPARA, HOXC8, EBF1, EBF2 and their variants.

[0072] In one implementation, (e.g., at least two or more) transcription factors are selected from PPARA, HOXC8, EBF1, EBF2 and their variants.

[0073] In another embodiment, the method comprises expressing one or more peptides having PPARA and HOXC8 activity and / or increasing the expression of PPARA and HOXC8.

[0074] In another embodiment, the method comprises expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves, wherein the transcription factors are PPARA, EBF1, and EBF2. According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves in a cell population, wherein the transcription factors are PPARA, EBF1, and EBF2, and culturing the cell population to obtain adipocytes.

[0075] In another embodiment, the method comprises expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves, wherein the transcription factors are HOXC8, EBF1, and EBF2. According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves in a cell population, wherein the transcription factors are HOXC8, EBF1, and EBF2, and culturing the cell population to obtain adipocytes.

[0076] In one implementation, (e.g., at least two or more) transcription factors are selected from: PPARA, PPARG, HOXC8, ZNF467, ZNF423 and their variants.

[0077] In another embodiment, the method comprises expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves, wherein the transcription factors are PPARA, PPARG, and ZNF467. According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves in a cell population, wherein the transcription factors are PPARA, PPARG, and ZNF467, and culturing the cell population to obtain adipocytes.

[0078] In another embodiment, the method comprises expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves, wherein the transcription factors are PPARA, PPARG, and HOXC8. According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves in a cell population, wherein the transcription factors are PPARA, PPARG, and HOXC8, and culturing the cell population to obtain adipocytes.

[0079] In another embodiment, the method comprises expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves, wherein the transcription factors are PPARA, PPARG, and ZNF423. According to another aspect of the invention, a method for generating adipocytes is provided, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of the transcription factors themselves in a cell population, wherein the transcription factors are PPARA, PPARG, and ZNF423, and culturing the cell population to obtain adipocytes.

[0080] In one embodiment, the method comprises expressing one or more polypeptides having one or more additional transcription factor activities and / or increasing the expression of one or more additional transcription factors themselves. The additional transcription factors may be one or more transcription factors listed in Table 1.

[0081] In one embodiment, the transcription factor comprises PPARA. PPARA may be used in combination with one or more (e.g., one, two, three, four, or five) transcription factors selected from the list in Table 1. In another embodiment, the method comprises expressing one or more polypeptides having two to seven transcription factor activities and / or increasing the expression of two to seven transcription factors themselves, said transcription factors being selected from combinations of PPARA with PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof. In yet another embodiment, the method comprises expressing one or more polypeptides having two to five transcription factor activities and / or increasing the expression of two to five transcription factors themselves, said transcription factors being selected from combinations of PPARA with PPARG, EBF1, EBF2, ZNF467, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof.

[0082] In one embodiment, the transcription factor comprises PPARG. PPARG may be used in combination with one or more (e.g., one, two, three, four, or five) transcription factors selected from the list in Table 1. In another embodiment, the method comprises expressing one or more polypeptides having two to seven transcription factor activities and / or increasing the expression of two to seven transcription factors themselves, said transcription factors being selected from combinations of PPARG with PPARA, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof. In yet another embodiment, the method comprises expressing one or more polypeptides having two to five transcription factor activities and / or increasing the expression of two to five transcription factors themselves, said transcription factors being selected from combinations of PPARG with PPARA, HOXC8, EBF1, EBF2, ZNF467, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof.

[0083] In one embodiment, the transcription factor comprises ZNF467. ZNF467 may be used in combination with one or more (e.g., one, two, three, four, or five) transcription factors selected from the list in Table 1. In another embodiment, the method comprises expressing one or more polypeptides having two to seven transcription factor activities and / or increasing the expression of two to seven transcription factors themselves, said transcription factors being selected from combinations of ZNF467 with PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof. In yet another embodiment, the method comprises expressing one or more polypeptides having two to three transcription factor activities and / or increasing the expression of two to three transcription factors themselves, said transcription factors being selected from combinations of ZNF467 with PPARA, PPARG, or variants thereof.

[0084] In one embodiment, the transcription factor comprises ZNF423. ZNF423 may be used in combination with one or more (e.g., one, two, three, four, or five) transcription factors selected from the list in Table 1. In yet another embodiment, the method comprises expressing one or more polypeptides having two to seven transcription factor activities and / or increasing the expression of two to seven transcription factors themselves, said transcription factors being selected from combinations of ZNF423 with PPARA, PPARG, HOXC8, EBF1, EBF2, ZNF467, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof. In yet another embodiment, the method comprises expressing one or more polypeptides having two to three transcription factor activities and / or increasing the expression of two to three transcription factors themselves, said transcription factors being selected from combinations of ZNF423 with PPARA, HOXC8, or variants thereof.

[0085] In one embodiment, the transcription factor comprises EBF1. EBF1 may be used in combination with one or more (e.g., one, two, three, four, or five) transcription factors selected from the list in Table 1. In another embodiment, the method comprises expressing one or more polypeptides having two to seven transcription factor activities and / or increasing the expression of two to seven transcription factors themselves, said transcription factors being selected from combinations of EBF1 with PPARA, PPARG, HOXC8, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof. In yet another embodiment, the method comprises expressing one or more polypeptides having two to four transcription factor activities and / or increasing the expression of two to four transcription factors themselves, said transcription factors being selected from combinations of EBF1 with PPARA, HOXC8, EBF2 or variants thereof.

[0086] In one embodiment, the transcription factor comprises EBF2. EBF2 may be used in combination with one or more (e.g., one, two, three, four, or five) transcription factors selected from the list in Table 1. In another embodiment, the method comprises expressing one or more polypeptides having two to seven transcription factor activities and / or increasing the expression of two to seven transcription factors themselves, said transcription factors being selected from combinations of EBF2 with PPARA, PPARG, HOXC8, EBF1, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) or variants thereof. In yet another embodiment, the method comprises expressing one or more polypeptides having two to four transcription factor activities and / or increasing the expression of two to four transcription factors themselves, said transcription factors being selected from combinations of EBF2 with PPARA, HOXC8, EBF1 or variants thereof.

[0087] The method of this invention includes the use of variants of the target transcription factor (i.e., as described in Table 1). The involvement of transcription factors also includes species variants, isotypes, homologs, allele forms, mutant forms, and equivalents thereof, including conserved substitutions, additions, or deletions that do not adversely affect their structure and / or function. Changes in the nucleic acid sequence of a transcription factor gene can lead to conserved changes or substitutions in the amino acid sequence. Therefore, this invention includes polypeptides having conserved changes or substitutions. This invention includes sequences that have undergone conserved substitutions without altering the activity of the target transcription factor protein.

[0088] Table 1. Transcription factors used to produce adipocytes, including accession numbers (accessed July 27, 2023)

[0089] Cell types

[0090] The method can be used for any cell type, including stem cells. In the case of stem cells, the generation of adipocytes using the method can be termed "cell reprogramming," "forward reprogramming," "direct programming," or "direct differentiation," i.e., pluripotent stem cells differentiating into adipocytes. Furthermore, adipocyte reprogramming can be used as a general term to refer to the differentiation of source cells into adipocytes using transcription factors.

[0091] The cell source suitable for the methods of the present invention can include, for example, any stem cell or non-adipocyte. For example, the stem cells can be pluripotent stem cells, such as induced pluripotent stem cells, embryonic stem cells, or pluripotent stem cells derived from nuclear transfer or cell fusion. It is preferable that the embryonic stem cells are derived without destroying the embryo, particularly when the cells are human. In some embodiments, the stem cells are not derived from human or animal embryos; that is, the present invention does not involve any method involving the destruction of human or animal embryos. The stem cells can also include pluripotent stem cells, oligopotent stem cells, or unipotent stem cells. The stem cells can also include fetal stem cells or adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells. In some aspects, the stem cells can be isolated from the umbilical cord, placenta, amniotic fluid, chorion, blastocyst, bone marrow, adipose tissue, brain, peripheral blood, cord blood, menstrual blood, blood vessels, skeletal muscle, skin, and liver.

[0092] In one implementation, the cell population is of human origin, such as non-adipocytes, which may be of human origin. It is well known that genome engineering of human pluripotent stem cells is challenging compared to non-human pluripotent cells, partly due to factors such as low transfection / transduction efficiency and high apoptosis rates under stresses such as low-density plating, drug screening, and sorting (Cerbini). et al. , PLOS ONE , 10(1), e0116032).

[0093] In one implementation, the cell population is animal-derived source cells, such as non-adipocytes, which may be of animal origin. In some respects, the cells are preferably derived from livestock animals. Livestock animals include animals such as pigs, cattle, horses, buffalo, bison, goats, sheep, deer, reindeer, donkeys, Balinese cattle, yaks, chickens, ducks, and turkeys.

[0094] In one embodiment, the cell population comprises stem cells, such as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells, mesenchymal stem cells, or neuronal stem cells. In a further embodiment, the cell population comprises pluripotent stem cells, such as iPSCs or ESCs.

[0095] In one embodiment, the source cell is a stem cell, such as an iPSC, an ESC, a hematopoietic stem cell, a mesenchymal stem cell, or a neuronal stem cell. In a further embodiment, the source cell is a pluripotent stem cell, such as an iPSC or an ESC. In some embodiments, the source cell is an iPSC.

[0096] Methods for preparing induced pluripotent stem cells (iPSCs) are also known in the art. Induction of iPSCs typically requires expression or exposure to at least one member of the Sox family and at least one member of the Oct family. Sox and Oct are considered central to a transcriptional regulatory hierarchy that defines the identity of ES cells. For example, Sox can be Sox-1, Sox-2, Sox-3, Sox-15, or Sox-18; Oct can be Oct-4. Other factors can increase reprogramming efficiency, such as Nanog, Lin28, Klf4, or c-Myc; specific groups of reprogramming factors can be those containing Sox-2, Oct-4, Nanog, and optionally Lin-28; or those containing Sox-2, Oct4, Klf, and optionally c-Myc. In one approach, iPSCs can be generated by transfecting cells with the transcription factors Oct4, Sox2, c-Myc, and Klf4 using viral transduction. In an alternative approach, iPSCs can be generated by transfecting cells with RNA that encodes transcription factors that induce stem cell characteristics (such as transcription factors selected from Oct4, Sox2, c-Myc, and Klf4).

[0097] In one implementation, the adipocytes are human adipocytes.

[0098] In one implementation, induced pluripotent stem cells are derived from the patient's somatic cells or germ cells. The use of such autologous cells eliminates the need for cell-recipient matching. Alternatively, commercially available iPSCs, such as those available from WICELL (WiCell Research Institute, Inc., Wisconsin, US), can be used. Alternatively, the cells can be tissue-specific stem cells, which can also be autologous or donated.

[0099] Delivery of transcription factors

[0100] It should be understood that methods for expressing polypeptides with transcription factor activity and / or increasing transcription factor expression in cells to be reprogrammed into adipocytes can include any methods known in the art, such as inducing the expression of one or more expression cassettes previously introduced into the cells, or introducing nucleic acids (such as DNA or RNA), polypeptides, or small molecules into the cells. Increasing the expression of certain endogenous transcriptional repressor genes can also reverse the silencing or repression of these genes by modulating upstream transcription factor expression or epigenetic regulation. Therefore, the methods of the present invention can include culturing cell populations under conditions that artificially increase the expression levels of one or more transcription factors described herein.

[0101] In one embodiment, the expression of peptides and / or transcription factors themselves, which have transcription factor activity, is increased by contacting a cell population with peptides and / or transcription factors (i.e., proteins encoding transcription factors). Delivery of transcription factors can be performed by directly electroporating the transcription factor protein into the cells.

[0102] In another implementation, transcription factor expression is increased by introducing a promoter (e.g., a strong promoter) before the endogenous gene encoding the transcription factor.

[0103] In another embodiment, the expression of a polypeptide with transcription factor activity and / or the transcription factor itself is increased by contacting a cell population with one or more agents that activate or increase the expression of (exogenous or endogenous) transcription factors. In the case of polypeptides with transcription factor activity or exogenous transcription factors, these agents can still be used after the gene of the polypeptide and / or transcription factor has been inserted into the cell.

[0104] In one embodiment, the reagent is selected from: nucleic acids (i.e., polynucleotides, such as messenger RNA (mRNA), encoding DNA sequences), proteins, aptamers and small molecules, ribosomes, RNAi reagents, guide RNA (gRNA), and peptide-nucleic acid (PNA) and their analogues or variants. In one embodiment, the reagent is a transcriptional activation system (e.g., gRNA used in gene activation systems such as CRISPR / Cas9 or TALEN) for increasing the expression of one or more endogenous transcription factors.

[0105] Methods for inducing differentiation of a cell population (i.e., source cells) may involve delivering to cells a nucleic acid containing one or more open reading frames (OPFs) encoding one or more polypeptides with transcription factor activity, one or more transcription factors themselves (e.g., in an expression cassette), a transcription factor protein, and / or a transcription activator encoding an OPF that encodes a polypeptide and / or an OPF that encodes a transcription factor. This results in an increase in the amount of transcription factors in the cells, and the cells differentiate into adipocytes. The OPF may be part of a recombinant expression cassette.

[0106] In one embodiment, the nucleic acid comprises a recombinant or exogenous expression cassette containing a sufficient number of one or more transcription factor sequences (or genes) to induce cellular reprogramming of the source cell into an adipocyte. The exogenous expression cassette may contain an external inducible transcriptional regulatory element for inducing the expression of one or more transcription factors, such as an inducible promoter, for example, containing a tetracycline response element or a variant thereof.

[0107] If the expression of a transcription factor is increased by introducing a foreign sequence encoding the transcription factor (e.g., a transcription factor gene), then it is understandable that any suitable system can be used to deliver the sequence. Gene delivery systems can be transposon systems; viral gene delivery systems; free gene delivery systems; or homologous recombination systems, such as those utilizing zinc finger nucleases, transcription activator-like effector nucleases (TALENs), or large-scale nucleases, or CRISPR / Cas9, etc.

[0108] Alternatively, as described herein or known to those skilled in the art, the introduction of nucleic acids (e.g., DNA or RNA) into cells can be achieved using any suitable method for nucleic acid delivery used to transform cells. Such methods include, but are not limited to, direct delivery of DNA, such as by in vitro transfection, by injection (including microinjection), by electroporation, by calcium phosphate precipitation, by using DEAE-glucan followed by polyethylene glycol, by direct sonic loading, by liposome-mediated transfection, by receptor-mediated transfection, by microparticle bombardment, by stirring with silicon carbide fibers, by Agrobacterium-mediated transformation, and any combination of these methods. By applying these techniques, cells can be stably or transiently transformed.

[0109] Furthermore, the expression cassette (e.g., an inducible recombinant expression cassette) may contain cleavable sequences. Such sequences are those recognized by entities capable of specifically cleaving DNA and contain restriction sites that are target sequences of restriction enzymes or sequences recognized by other DNA-cleaving entities such as nucleases, recombinases, ribozymes, or artificial constructs. At least one cleavable sequence may be contained, but two or more are preferred. These cleavable sequences can be located at any suitable point within the cassette, allowing for the selective removal of selected portions or the entire cassette if necessary. Thus, cleavable sites may be located flanking portions / all of the gene sequence that may need to be removed. Therefore, the method may also include the removal of the expression cassette and / or genetic material.

[0110] In another embodiment, a cell population is exposed to one or more agents having the same effect as activating or increasing the expression or amount of transcription factors (i.e., an indirect method of increasing transcription factor expression). In this respect, the invention includes introducing exogenous agents that mimic the effect of increasing the expression of transcription factors described herein. For example, such methods may include introducing proteins with DNA-binding activities similar to transcription factors (e.g., engineered zinc finger nucleases). For example, PPAR proteins bind to peroxisome proliferator-response elements, so the activity of these transcription factors can be reproduced by engineering zinc finger nucleases to bind the same domains.

[0111] It should be understood that a combination of one or more methods for expressing polypeptides with transcription factor activity or increasing transcription factor expression may be used, wherein the combination generally produces the activity necessary for positive reprogramming to adipocytes.

[0112] carrier

[0113] In one embodiment, a vector is used to introduce a polypeptide with transcription factor activity or the transcription factor itself (e.g., a combination of peptides and / or transcription factors) into a cell population. Those skilled in the art will have the ability to construct vectors using standard recombination techniques. Vectors include, but are not limited to, plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC).

[0114] In one implementation, a nucleic acid sequence encoding one or more transcription factors is introduced into a cell via a transposon system (i.e., involving transposon plasmids). The transposon delivery system consists of two plasmids, one encoding a transposase and the other encoding a transcription factor. The transposase protein mediates the random integration of the transcript encoded in the transposon plasmid into the genome. In one implementation, the transposon system is selected from the PiggyBac or Sleeping Beauty transposon system. The transposon plasmid encodes a payload flanked by two ITRs (internal terminal repeats). The payload may contain a Tet-inducible promoter, a transcription factor, and an optional selection marker, such as an antibiotic selection cassette under a constitutive promoter.

[0115] In one implementation, transposases and transposon plasmids are delivered into cells via nuclear transfection or lipid transfection. The number of integration events, and therefore the copy number of the payload per cell, can be partially controlled by adjusting the total amount and relative ratio of transposase to transposon plasmid DNA. This enables the combined delivery of transcription factors at the single-cell level.

[0116] In one embodiment, the vector is a viral vector. The viral gene delivery system can be an RNA-based or DNA-based viral vector. Viral vectors include retroviral vectors, lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), gamma retroviral vectors, adenovirus (Ad) vectors (including their replicative, replication-defective, and gutless forms), adeno-associated virus-derived (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey's mouse sarcoma virus vectors, mouse mammary tumor virus vectors, Rouss sarcoma virus vectors, and Sendai virus vectors. In a further embodiment, the viral vector is selected from lentiviral vectors, adeno-associated virus vectors, or Sendai virus vectors. In yet another embodiment, the viral vector is a lentiviral vector.

[0117] Lentiviral vectors are well-known in the field. A lentiviral vector is a complex retrovirus capable of randomly integrating into the host cell genome. In addition to common retroviral genes such as gag, pol, and env, it contains other genes with regulatory or structural functions (e.g., helper genes Vif, Nef, Vpu, Vpr). Lentiviral vectors have the advantage of infecting non-dividing cells and can be used for in vivo and in vitro gene delivery and nucleic acid sequence expression. For example, recombinant lentiviral vectors can infect non-dividing cells, where suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, as well as rev and tat.

[0118] In one embodiment, the vector is a self-replicating RNA vector expression system. For example, the system may comprise a self-replicating RNA vector that remains ectopic to the host cell genome and encodes transcription factors that induce reprogramming. Self-replicating RNA vectors are known in the art, and many are based on positive-sense RNA viruses, such as alphaviruses.

[0119] In one implementation, the viral vector is used with a high multiple of infection (MOI). A high MOI helps ensure that more than one transcription factor is introduced into the source cell. In one implementation, the MOI is greater than 0.5, for example, 1.0 or higher.

[0120] In one embodiment, a nucleic acid sequence encoding one or more polypeptides and / or transcription factors with transcription factor activity is introduced into a cell via a plasmid. In another embodiment, at least one nucleic acid sequence encoding a polypeptide and / or transcription factor with transcription factor activity is introduced into the cell on a single plasmid.

[0121] In one implementation, the plasmid is free-floating. Free-floating vectors are capable of introducing large DNA fragments into cells but retaining them outside the chromosome, replicating once per cell cycle, efficiently distributing them to daughter cells, and substantially not triggering an immune response. In alternative implementations, free-floating vectors based on Epstein-Barr virus (EBV), yeast-based vectors, adenovirus-based vectors, simian virus 40 (SV40)-based free-floating vectors, or bovine papillomavirus (BPV)-based vectors may be used.

[0122] Site-specific delivery

[0123] Any suitable technique can be used to insert a nucleic acid sequence into a specific sequence, and several are described in the art. Suitable techniques encompass any method that introduces a break at the desired location and allows the vector to recombine into the gap. Therefore, a key first step in site-specific genome modification is the creation of a double-stranded DNA break (DSB) at the locus of the genome to be modified. Different cellular repair mechanisms can be utilized to repair the DSB and introduce the desired sequence; these include non-homologous end joining repair (NHEJ), which is more prone to error, and homologous recombination repair (HR).

[0124] Several techniques exist that allow for the generation of DSBs at custom sites within the genome. Many of these involve the use of custom-designed endonucleases, such as zinc finger nucleases, TALENs, or clusters of regularly spaced short palindromic repeats / CRISPR-related proteins (CRISPR / Cas, e.g., CRISPR / Cas9) systems.

[0125] Zinc finger nucleases are artificial enzymes created by fusing a zinc finger DNA-binding domain with the nuclease domain of the restriction enzyme FokI. The latter has a non-specific cleavage domain that must dimerize to cleave DNA. This means that two zinc finger nuclease monomers are required for the FokI domain to dimerize and cleave DNA. The DNA-binding domain, which can be designed to target any genomic sequence, is a tandem array of Cys2His2 zinc fingers, each recognizing three consecutive nucleotides in the target sequence. The two binding sites are spaced 5-7 bp apart to achieve optimal dimerization of the FokI domain. Therefore, the enzyme can cleave DNA at specific sites and increases target specificity by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break.

[0126] Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factors / nucleases. They are created by fusing the DNA-binding domain of a TAL effector with a DNA-cutting domain (nuclease). Transcription activator-like effectors (TALENs) can be engineered to bind to virtually any desired DNA sequence, thus allowing for DNA cleavage at a specific site when combined with a nuclease. TAL effectors are produced by bacteria of the genus *Xanthomonas* (…). XanthomonasBacterial secreted proteins contain a DNA-binding domain comprising a highly conserved, repetitive sequence of 33-34 amino acids with distinct 12th and 13th amino acids. These two positions are highly variable and exhibit a strong correlation with the recognition of specific nucleotides. This direct relationship between amino acid sequence and DNA recognition allows for the engineering of specific DNA-binding domains by selecting combinations of repetitive segments containing appropriate residues at the two variable positions. Therefore, TALENs are constructed from arrays of 33 to 35 amino acid modules, each targeting a single nucleotide. By selecting the array of modules, virtually any sequence can be targeted. Similarly, the nuclease used can be FokI or a derivative thereof.

[0127] Three types of CRISPR mechanisms have been identified, with type II being the most studied. The CRISPR / Cas9 system (type II) utilizes the Cas9 nuclease to cause double-strand breaks in DNA at sites determined by short guide RNAs. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR consists of prokaryotic DNA segments containing short, repeating base sequences. Each repeat is followed by a short segment from the "protospacer DNA" previously exposed to a foreign genetic element. CRISPR spacers use RNA interference to recognize and cleave foreign genetic elements. The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules consist of a variable sequence transcribed from protospacer DNA and a CRISPR repeat sequence. Each crRNA molecule then hybridizes with a second RNA, called trans-activating CRISPR RNA (tracrRNA), which ultimately forms a complex with the nuclease Cas9. The protospacer DNA coding portion of the crRNA directs Cas9 to cleave complementary target DNA sequences if they are adjacent to a short sequence called a protospacer adjacent motif (PAM). This natural system has been engineered and developed to introduce DSB breaks at specific sites in genomic DNA, as well as for many other applications. In particular, the CRISPR type II system derived from Streptococcus pyogenes can be used. In short, the CRISPR / Cas9 system comprises two components that are delivered to the cell to provide genome editing: the Cas9 nuclease itself and the gRNA. The gRNA is a fusion of a custom-designed, site-specific crRNA (targeting the target sequence) and a standardized tracrRNA.

[0128] Once a DSB is formed, it provides a donor template homologous to the target locus; the DSB can be repaired via the homology-directed repair (HDR) pathway, thus allowing for precise insertion.

[0129] Derivatives of this system are also possible. Mutant forms of Cas9 are available, such as Cas9D10A, which has only cleavage enzyme activity. This means it cuts only one DNA strand and does not activate NHEJ. Instead, when a homology repair template is provided, DNA repair proceeds only via the high-fidelity (HDR) pathway. Cas9D10A can be used in pairs with Cas9 complexes designed to bind to two sgRNAs (which are complementary to adjacent regions on the opposite strand of the target site) to create adjacent DNA cleavages, which may be particularly advantageous.

[0130] Elements used to break double-stranded DNA can be introduced into one or more vectors, such as plasmids, for expression in cells.

[0131] Therefore, any method for preparing specific, targeted double-strand breaks in the genome to achieve gene / inducible cassette insertion can be used in the method of the present invention. Preferably, the method for inserting the gene / inducible cassette utilizes any one or more of zinc finger nucleases, TALEN and / or CRISPR / Cas9 systems or any derivatives thereof.

[0132] Once the DSB is prepared by any suitable method, a gene / inducible cassette for insertion can be provided in any suitable manner as described below. The gene / inducible cassette and associated genetic material form donor DNA for DNA repair in the DSB and insertion using standard cellular repair mechanisms / pathways. As mentioned above, the initiation mode of the break will alter the pathway used to repair the damage.

[0133] Other methods in the art for site-specific delivery include the use of homologous recombination (HR) and recombinase-mediated cassette exchange (RMCE). DNA damage-mediated site-specific insertion methods (e.g., CRISPR / Cas) can also be used for site-specific integration of DNA recognition sequences ('att' sites), which are then mediated by the activity of tyrosine and serine recombinases or integrases. Once integrated into the genome, these sites (e.g., attP) can be mediated by homologous recombination between homologous attP and attB sites, along with the expression of suitable and homologous recombinases (e.g., Flp, Cre) or integrases (PhiC31, Bxb1). Site-specific insertion of exogenous DNA into transgenes can be achieved using targeting vectors flanked by attB sites as described above.

[0134] Controlled expression

[0135] In one embodiment, transcription factor expression is performed under inducible control. In this aspect of the invention, transcription and translation (expression) of transcription factors can be controlled intracellularly. This allows for overexpression of transcription factors if desired.

[0136] In another embodiment, the expression of the polypeptide and / or the transcription factor itself, which has transcription factor activity, is under inducible control. In this aspect of the invention, the transcription and translation (expression) of the polypeptide and / or transcription factor with transcription factor activity can be controlled intracellularly. This allows for overexpression of the transcription factor, preferably in response to external stimuli, if desired.

[0137] Exogenous expression cassettes carrying polypeptides and / or transcription factors themselves with transcription factor activity can contain external inducible transcriptional regulatory elements (i.e., inducible promoters) for rapidly inducing protein expression, i.e., inducible gene (or transgene) expression, in response to external stimuli. Controlled expression of the genetic sequence within the inducible expression cassette is regulated by the presence or addition of appropriate external stimuli (e.g., proteins, compounds, or chemicals) to cell culture media; and can be applied continuously or transiently as needed to regulate transcription.

[0138] The expression of the transcription factors described herein can be increased using the two-box expression system described in WO2018096343, which is incorporated herein by reference. In this case, inducible transgene overexpression is achieved using components of the Tet-ON system, where transgene expression is controlled by doxycycline. The components are split between two genetic safe harbor sites (GSHs) to reduce the risk of epigenetic silencing. The components are (i) a transcriptional activator protein (reverse tetracycline transactivator (rtTA)) that binds in the presence of doxycycline and (ii) a tetracycline response element (TRE; multiple TetO repeat sequences and a minimal cytomegalovirus (CMV) promoter). Binding of rtTA to TRE transactivates transgene expression. The transactivation coding sequence of the transgene can be human.

[0139] Therefore, in one embodiment, a sequence encoding one or more (e.g., two or more, three or more) polypeptides and / or transcription factors with transcription factor activity is introduced into a cell population (preferably a pluripotent stem cell population, more preferably a hiPSC population) using a method comprising the following: - Insert (preferably targeted) the coding sequence of a transcriptional regulatory protein into the first genomic safe harbor site of the source cell in the cell population; and - Inserting (preferably targeted insertion) one or more inducible cassettes into one or more second genomic safe harbor sites in source cells, wherein the one or more inducible cassettes contain sequences encoding one or more polypeptides and / or transcription factors having one or more transcription factor activities, the sequences being operatively linked to an inducible promoter, and the promoter being regulated by transcriptional regulatory proteins.

[0140] In one implementation, a sequence encoding one or more (e.g., two or more or three or more) transcription factors is introduced into a cell population using a method comprising: - Targeting the insertion of the coding sequence of transcriptional regulatory proteins into the first genomic safe harbor site of the source cells present in the cell population; and - The inducible cassette is targeted and inserted into a second genomic safe harbor site in the source cell, wherein the inducible cassette contains a sequence encoding one or more transcription factors, the sequence being operatively linked to an inducible promoter, and the promoter being regulated by a transcriptional regulatory protein.

[0141] According to another aspect of the present invention, a dual-expression system is provided, comprising: (a) A first expression cassette containing a gene encoding a transcriptional regulatory protein, said gene being flanked by one or more homologous arms that target the first expression cassette to a first genomic safe harbor site; and (b) A second expression cassette comprising a sequence encoding one or more transcription factors, the sequence being operatively linked to an inducible promoter and flanked by one or more homologous arms targeting the second expression cassette to a second genomic safe harbor site, wherein the inducible promoter is regulated by a transcriptional regulatory protein of the first expression cassette, and wherein the one or more transcription factors are selected from: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof.

[0142] According to another aspect of the present invention, a dual-expression system is provided, comprising: (a) A first expression cassette containing a gene encoding a transcriptional regulatory protein, said gene being flanked by one or more homologous arms that target the first expression cassette to a first genomic safe harbor site; and (b) A second expression cassette comprising a sequence encoding one or more polypeptides and / or transcription factors having one or more transcription factor activities, the sequence being operatively linked to an inducible promoter and flanked by one or more homologous arms targeting the second expression cassette to a second genomic safe harbor site, wherein the inducible promoter is regulated by a transcriptional regulatory protein of the first expression cassette, and wherein the one or more transcription factors are selected from: one or more PPAR proteins (e.g., PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) and variants thereof.

[0143] This embodiment of the invention provides a dual expression cassette system. Insertion of a gene encoding a transcriptional regulatory protein into a first GSH provides a control mechanism for the expression of an inducible cassette, which is operatively linked to an inducible promoter and inserted into a second GSH site. In one embodiment, the first and second GSHs are distinct (i.e., located at different locations in the genome). It should be understood that if more than one transcription factor is to be introduced into the cell, using a dual expression system, the transcription factor can be introduced into the second GSH site (e.g., within a polycistronic cassette at the same GSH site), or into multiple GSH sites (i.e., as separate cassettes spanning different GSH sites).

[0144] Alternatively, a dual expression cassette system can utilize different alleles of the same GSH locus. In this embodiment, the inducible cassette can be inserted into one allele of the GSH locus, while the system controlling the expression of the inducible cassette is inserted into the other allele of the GSH locus (e.g., DeKelver et al., 2010, Genome Res., 20, 1133-43 and Qian et al., 2014, Stem Cells, 32, 1230-8).

[0145] GSH loci are genomic loci where genes or other genetic material can be inserted without any harmful effect on the cell or the inserted genetic material. Most advantageous are GSH loci, where the expression of the inserted gene sequence is not interfered with by any readthrough expression from neighboring genes, and the expression of the inducible cassette minimizes interference with endogenous transcriptional programs. More formal criteria have been proposed to help determine whether a particular locus will be a future GSH locus (Papapetrou). et al(2011) These criteria include the following sites: (i) 50 kb or more from the 5' end of any gene, (ii) 300 kb or more from any cancer-related gene, (iii) 300 kb or more from any microRNA (miRNA), (iv) outside the transcription unit, and (v) outside the ultraconserved region (UCR). It may not be necessary to meet all of these recommended criteria, as identified GSHs do not meet all of them. A suitable GSH is considered to meet at least 2, 3, 4, or all of these criteria. Any suitable GSH site can be used in the methods of this invention, based on the premise that the site allows insertion of genetic material without harmful effects on the cell and allows the inserted genetic material to be transcribed. Those skilled in the art can use these simplified criteria to identify suitable GSHs, and / or use the more formal criteria listed above.

[0146] Insertion of the coding sequence of transcriptional regulatory proteins and / or inducible cassettes can be performed using the direct delivery methods described above. It should be understood that such direct delivery methods may result in random insertion of genetic material, but screening can be performed to identify clones that do not show detrimental effects, express genetic material, and can be positively programmed or reprogrammed into adipocytes. Doing so confirms that the transcriptional regulatory protein / inducible cassette has been inserted into the GSH site.

[0147] In one embodiment, the insertion of the transcriptional regulatory protein or the induction cassette is targeted. In another embodiment, the insertion of both the transcriptional regulatory protein and the induction cassette is targeted. "Targeted insertion," as with site-specific delivery, can be understood as the insertion of genetic material into a pre-selected GSH site. As described above, this can be performed using techniques known in the art, such as zinc finger nucleases, TALENs, or clustered regularly spaced short palindromic repeats / CRISPR-related protein (CRISPR / Cas, such as CRISPR / Cas9) systems.

[0148] In one implementation, the first and second genetic safe harbor loci (GSHs) are selected from the hROSA26 locus, the AAVS1 locus, and CLYBL Gene, CCR5 Gene or HPRT Genes (especially any two of them). Specific insertions within genetic safe harbor sites are preferred over random genomic integration because this is expected to be a safer modification of the genome and is less likely to cause unwanted side effects such as silencing native gene expression or random insertional mutagenesis.

[0149] The adeno-associated virus integration site 1 (AAVS1) locus is located within the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, and is uniformly and universally expressed in human tissues. AAVS1 has been shown to be a favorable transcriptional environment because it contains open chromatin structures and natural chromosome insulators that enable the induction cassette to resist silencing. There are no known adverse effects of PPP1R12C gene disruption on cells. Furthermore, induction cassettes inserted at this site retain transcriptional activity in many different cell types.

[0150] The hROSA26 locus (ROSA26 – reverse splice acceptor locus #26) has been identified based on sequence similarity to mouse GSH. The hROSA26 locus is located on chromosome 3 (3p25.3) and can be found in the Ensembl database (GenBank: CR624523). The integration site is located within the open reading frame (ORF) of a THUMPD3-length non-coding RNA (reverse strand). Because the hROSA26 locus possesses an endogenous promoter, the inserted genetic material can utilize this endogenous promoter, or alternatively, can be operatively linked to the promoter for insertion.

[0151] Citrate lyase Beta-like intron 2 ( CLYBL The phiC31 integrase gene, located on the long arm of chromosome 13, was identified as a suitable GSH because it is one of the identified phage integration hotspots derived from the phiC31 integrase. Studies have shown that induction cassettes randomly inserted into this locus are stable and expressive. It has been demonstrated that inserting an induction cassette into this GSH does not interfere with local gene expression (Cerbini). et al . (2015) PLOS One, 10(1):e0116032). CLYBL Therefore, a GSH applicable to this invention is provided.

[0152] CCR5 Located on chromosome 3 (position 3p21.31), this locus encodes the major co-receptor of HIV-1. Interest in using this site for GSH arises from null mutations in this gene that appear to have no adverse effects but predispose to HIV-1 resistance. Zinc finger nucleases targeting exon 3 have been developed, allowing for the insertion of genetic material at this locus.

[0153] Inosine-guanine phosphoribosyltransferase ( HPRT The gene encodes a transferase, which plays a central role in the production of purine nucleotides via the purine salvage pathway.

[0154] Other GSH sites, such as Sadelain, have been described in this field. et al.The references described in (2012) Nature Reviews 12:51-58 and WO2021 / 152086 are incorporated herein by reference.

[0155] GSH has been identified in other organisms, including the ROSA26, HRPT, and Hipp11 (H11) loci in mice. Mammalian genomes may contain GSH sites based on pseudo-attP sites. For such sites, the hiC31 integrase (a streptomyces phage-derived recombinase) has been developed as a non-viral insertion tool because it can integrate plasmids containing an induction cassette with an attB site into pseudo-attP sites.

[0156] Technically, the insertion of the first and / or second GSH can occur on one chromosome or on two chromosomes. GSHs are located at the same gene locus on both chromosomes of diploid organisms. Insertion on two chromosomes is advantageous because it allows for increased transcription levels of the genetic material inserted within the induction cassette, resulting in particularly high transcription levels.

[0157] Based on the customized generation of site-specific DNA double-strand breaks at GSH sites, it is possible to specifically insert genetic material into a particular GSH. The genetic material can then be introduced using any suitable mechanism, such as homologous recombination. Any method for preparing a specific DSB in the genome can be used, but preferred systems include CRISPR / Cas9 and its modified versions, zinc finger nucleases, and the TALEN system, or integration or recombination mediated by HR or RCME.

[0158] One or more gene sequences can be controllably transcribed from a second and / or additional GSH. In practice, the induction cassette may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gene sequences (e.g., transcription factor sequences) that are desired to be inserted into the GSH, and their transcription is controllably induced. Therefore, the transcription factors desired in this invention can be contained within the same cassette introduced into the second genetic safe harbor site. For example, three or more transcription factors can be contained in, for example, three monocistronic constructs, one monocistronic construct and one bicistronic construct, or one tricistronic construct. It should be understood that similar combinations of constructs can be used to achieve higher levels of transcription factor expression.

[0159] Alternatively, if a combination of transcription factors is used, individual transcription factors can be introduced into individual GSHs and / or under the control of the same, different, or orthogonal inducible promoters. Thus, in one embodiment, transcription factors are introduced into individual GSHs. This can be achieved by using three or more different GSH sites for three or more transcription factors (i.e., where the transcription factors are introduced as monocistronic boxes). Alternatively, this can be achieved by utilizing the fact that GSHs are present at the same gene loci on two chromosomes of a diploid organism, for example, introducing one transcription factor into a GSH on one chromosome and a different transcription factor into the same GSH on another chromosome. This embodiment is advantageous if different expression levels or expression times of the transcription factors are desired. In one embodiment, the method comprises targeting each transcription factor operatively linked to an inducible promoter to second, third, and fourth genetic safe harbor sites in the source cell. The inducible promoters of each transcription factor can be the same, and therefore all are regulated by transcriptional regulatory proteins.

[0160] Transcription regulatory proteins are proteins that bind to DNA, preferably specifically binding to DNA site sequences located within or near the promoter, and promoting the binding of transcription mechanisms to the promoter, thereby promoting the transcription of DNA sequences (transcription activators) or blocking this process (transcription repressors).

[0161] The DNA sequence to which transcriptional regulatory proteins bind is called a transcription factor binding site or response element, and it is located within or near the promoter of the regulated DNA sequence. Transcriptional activating proteins bind to response elements and promote gene expression. Such proteins are preferred in the method of the present invention for controlling cassette expression. Transcriptional repressor proteins bind to response elements and prevent gene expression.

[0162] Transcriptional regulatory proteins can be activated or inactivated through a variety of mechanisms, including binding to substances, interactions with other transcription factors or co-regulatory proteins (e.g., homodimerization or heterodimerization), phosphorylation, and / or methylation. Activation or inactivation of transcriptional regulatory proteins can control their activity.

[0163] If the transcriptional regulatory protein is a transcriptional activator, then activation of the transcriptional activator is preferred. This activation can occur by any suitable means, but is preferably achieved by adding an exogenous substance to the cell. The supply of the exogenous substance to the cell can be controlled, thereby controlling the activation of the transcriptional regulatory protein. Alternatively, an exogenous substance can be provided to inactivate the transcriptional regulatory protein, and then the supply can be stopped to activate it.

[0164] If the transcriptional regulatory protein is a transcriptional repressor protein, then preferably the transcriptional repressor protein needs to be inactivated. Therefore, a substance is provided to prevent the transcriptional repressor protein from inhibiting transcription, thereby allowing transcription.

[0165] Any suitable transcriptional regulatory protein can be used, preferably one that can be activated or inactivated. It is preferable to provide exogenous substances to control the transcriptional regulatory protein. This transcriptional regulatory protein is also called an inducible transcriptional regulatory protein.

[0166] Tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcriptional activating protein is tetracycline-responsive transcriptional activator (rtTA) or a derivative thereof. The rtTA protein is capable of binding to DNA at a specific TetO operator gene sequence. Several repetitive sequences of this TetO sequence are placed upstream of a minimal promoter (e.g., the CMV promoter), together forming a tetracycline-responsive element (TRE). This system exists in two forms, depending on whether the addition of tetracycline or its derivative activates (Tet-On) or inactivates (Tet-Off) the rtTA protein.

[0167] In the Tet-Off system, tetracycline or its derivatives bind to and inactivate rtTA, preventing it from binding to TRE sequences and thus blocking the transcription of TRE-controlled genes. This system was first demonstrated in Gossen. et al (1992) PNAS 89(12): 5547–5551 Description.

[0168] The Tet-On system consists of two components: (1) a constitutively expressed tetracycline-responsive transcriptional activator protein (rtTa) and an rtTa-sensitive inducible promoter (TRE). This binds to tetracycline or its more stable derivatives (including doxycycline (dox)), leading to rtTa activation, which binds to the TRE sequence and induces the expression of TRE-controlled genes. This is preferably used in the method of the present invention.

[0169] Therefore, the transcriptional regulatory protein can be a reverse tetracycline-controlled transactivator (rtTa) protein, which can be activated or inactivated by an exogenously provided antibiotic, tetracycline or one of its derivatives. If the transcriptional regulatory protein is rtTA, the inducible promoter inserted into the second GSH site contains a tetracycline response element (TRE). The exogenously provided substance is an antibiotic, tetracycline or one of its derivatives.

[0170] Variant and modified rtTa proteins can also be used in the methods of the present invention, including Tet-On Advanced trans activator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0171] Tetracycline response elements (TREs) typically consist of seven repeating 19 bp bacterial TetO sequences (separated by spacer sequences) and a minimal promoter. Variations and modifications of the TRE sequence are possible because the minimal promoter can be any suitable promoter. Preferably, the minimal promoter does not show or shows minimal expression levels in the absence of rtTa binding. The inducible promoter inserted into a second GSH can therefore contain the TRE.

[0172] The tetracycline-controlled modification system is the T-REX system (Thermo-Fisher Scientific), in which the transcriptional regulatory protein is the transcriptional repressor TetR. The components of this system include: (i) an inducible promoter containing a strong human cytomegalovirus immediate early (CMV) promoter and two tetracycline operon 2 (TetO2) sites, and a Tet repressor (TetR). In the absence of tetracycline, the Tet repressor forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the inducible promoter, preventing transcription of the promoter. Upon addition, tetracycline binds with high affinity to each Tet repressor homodimer, preventing it from binding to the Tet operon. The Tet repressor:tetracycline complex then dissociates from the Tet operon, allowing inducible expression. In this case, the transcriptional regulatory protein is TetR, and the inducible promoter contains two TetO2 sites. The exogenously provided substance is tetracycline or a derivative thereof.

[0173] Other inducible expression systems are known and can be used in the methods of this invention. Among these is Agilent Technologies' Complete Control Inducible system. This system is based on the insect hormone ecdysone or its analogue ponasterone A (ponA), which activates transcription in mammalian cells transfected with the gene for the ecdysone receptor (EcR) from the Drosophila melanogaster and an inducible promoter containing the ecdysone receptor binding site. EcR is a member of the nuclear receptor retinol X receptor (RXR) family. In humans, EcR forms a heterodimer with RXR, which binds to the ecdysone response element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

[0174] Therefore, transcriptional regulatory proteins can be repressive proteins, such as the ecdysone receptor or its derivatives. Examples of the latter include the VgEcR synthetic receptor from Agilent Technologies, a fusion of EcR, the DNA-binding domain of the glucocorticoid receptor, and the transcriptional activation domain of herpes simplex virus VP16. The inducible promoter contains the EcRE sequence or a modified form thereof, along with a minimal promoter. The modified version contains Agilent Technologies' E / GRE recognition sequence, in which the sequence has been mutated. The E / GRE recognition sequence contains a reverse half-site recognition element for the retinoid X receptor (RXR) and GR-binding domain. In all permutations, the exogenously provided substance is ecdysone A, which eliminates the repressive effect of EcR or its derivatives on the inducible promoter and allows transcription to occur.

[0175] Alternatively, the induction system can be based on the synthetic steroid mifepristone as an exogenously provided substance. In this case, a hybrid transcriptional regulator protein is inserted, which is based on the DNA-binding domain of the yeast GAL4 protein, the truncated ligand-binding domain (LBD) of the human progesterone receptor, and the activation domain (AD) of human NF-κB. This hybrid transcriptional regulator protein is available from Thermo-Fisher Scientific (Gene Switch). TM Mifepristone activates the heterozygous protein and allows transcription from an inducible promoter containing the GAL4 upstream activation sequence (UAS) and an adenovirus E1b TATA box. This system is described in Wang et al. (PNAS 91: 8180-8184, 1994).

[0176] Therefore, transcriptional regulatory proteins can be any suitable regulatory protein, either an activator or a repressor. Suitable transcriptional activators are tetracycline-responsive transcriptional activators or gene-switch hybrid transcriptional regulators. Suitable repressors include Tet-Off versions of rtTA, TetR, or EcR. Transcriptional regulatory proteins can be modified or derived as needed.

[0177] Inducible promoters may contain elements suitable for binding to or interacting with transcriptional regulatory proteins. The interaction between transcriptional regulatory proteins and inducible promoters is preferably controlled by exogenously provided substances.

[0178] The exogenous substance can be any suitable substance that binds to or interacts with transcriptional regulatory proteins. Suitable substances include tetracycline (or its derivatives, such as doxycycline), ecdysone A, and mifepristone.

[0179] Genes encoding transcriptional regulatory proteins are preferably operatively linked to constitutive promoters. Alternatively, a first GSH can be selected that already possesses a constitutive promoter, which can also drive the expression of transcriptional regulatory protein genes and any associated genetic material. Constitutive promoters ensure sustained and high levels of gene expression. Commonly used constitutive promoters include the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α (EF1α), phosphoglycerate kinase (PGK), and ubiquitin C (UbC). The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression and is constructed from the following sequences: (C) an early enhancer element of cytomegalovirus (CMV), (A) the promoter, first exon, and first intron of the chicken β-actin gene, and (G) the splice acceptor of the rabbit β-globin gene.

[0180] According to another aspect of the present invention, a method for producing adipocytes from source cells (preferably pluripotent stem cells, more preferably hiPSCs) is provided, the method comprising the following steps: a) Inserting (preferably targeted) the gene encoding a transcriptional regulatory protein into the first genomic safe harbor site of the source cell; and b) Inserting (preferably targeted insertion) at least one nucleotide sequence into a second genomic safe harbor site of a source cell, the nucleotide sequence encoding one or more polypeptides having one or more transcription factor activities and / or one or more transcription factors selected from: one or more PPAR proteins (e.g., PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) and variants thereof, the nucleotide sequence being operatively linked to an inducible promoter, wherein the inducible promoter is regulated by the transcriptional regulatory protein; and c) Culture source cells containing the insert to obtain adipocytes.

[0181] According to another aspect of the present invention, a method for producing adipocytes from source cells is provided, the method comprising the following steps: a) Targeting the insertion of genes encoding transcriptional regulatory proteins into the first genomic safe harbor site of the source cell; and b) Targeting at least one nucleotide sequence into a second genomic safe harbor site in a source cell, said nucleotide sequence encoding one or more transcription factors selected from: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423, and variants thereof, said nucleotide sequence being operatively linked to an inducible promoter, said inducible promoter being regulated by said transcriptional regulatory protein; and c) Culture source cells containing the insert to obtain adipocytes.

[0182] It should be understood that this aspect of the invention can be used in combination with any transcription factor described herein.

[0183] Obtaining fat cells

[0184] In one embodiment, the method further includes monitoring whether the cell population possesses at least one adipocyte characteristic. Cells can be monitored throughout the culture process to identify the expression of key lineage markers.

[0185] For example, monitoring can be performed using engineered “reporter” cell lines (i.e., endogenous marker proteins or positive selection markers controlled by adipocyte-specific promoters) or immunostaining, and detected using fluorescence microscopy or flow cytometry. Such substances contain genes for markers or reporter molecules, such as genes that induce visually recognizable features (containing fluorescent and luminescent proteins). Examples include genes encoding the jellyfish green fluorescent protein (GFP), which causes cells expressing it to emit green light under blue / ultraviolet light; luciferase, which catalyzes the reaction with luciferin to produce light; and a red fluorescent protein derived from the dsRed gene.

[0186] The cell may also contain a screenable and / or selectable reporter gene expression cassette, for example, containing an adipocyte-specific promoter operatively linked to a reporter gene.

[0187] The optional markers may include antibiotic or other drug resistance genes. Examples of drug resistance genes may include: puromycin resistance genes, ampicillin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, or chloramphenicol resistance genes. Cells can be cultured on a medium containing the appropriate drug (i.e., a selective medium), and only those cells incorporating and expressing the drug resistance gene will survive. Therefore, by culturing cells in a selective medium, cells containing and expressing drug resistance genes can be easily selected, thereby positively enriching a target cell population.

[0188] Examples of fluorescent protein genes that can be used as biomarkers include: green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene, or jellyfish bioluminescent protein gene. Cells expressing fluorescent proteins can be detected using fluorescence microscopy, and cell populations can be identified and selected based on the expression of fluorescent proteins using fluorescence activated cell sorting (FACS).

[0189] Fluorescent protein genes can be labeled with nuclear localization signal peptides to restrict the expression of fluorescent proteins to the cell nucleus. This can be helpful in cell types with high lipid content that may not be suitable for FACS. This allows for endpoint fluorescence-activated cell sorting of entire cell populations that retain intact fluorescence signals or purified cell nuclei.

[0190] Examples of chromogenic enzyme genes that can be used as biomarkers, and are known in the art, include, but are not limited to: β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene, or secretory alkaline phosphatase SEAP gene. Cells expressing these chromogenic enzyme genes can be detected by applying appropriate chromogenic substrates (e.g., X-gal for β-galactosidase), so that cells expressing biomarker genes will produce a detectable color (e.g., blue in a blue-white screening test).

[0191] Therefore, this method may include the step of selecting or enriching adipocytes provided by the methods described herein. In one embodiment, the method includes the step of sorting adipocytes using fluorescence-activated cell sorting (FACS) or immunomagnetic bead sorting based on adipocyte marker expression and / or non-adipocyte marker absence. Labeled conjugates targeting target cell surface proteins may be used. Any conjugate capable of specifically binding to a particular epitope may be used for this purpose, such as antibodies or fragments thereof, peptides or synthetic conjugates (e.g., plastic antibodies), or aptamers or oligonucleotides capable of specifically binding to epitopes. The conjugate may be labeled with detectable markers, such as luminescent, fluorescent (e.g., fluorophores), enzymes, or radiolabeled markers; or alternatively, with affinity tags, such as biotin, avidin, streptavidin, or His (e.g., 6x His) tags. In one embodiment, fluorophore-conjugated antibodies targeting cell surface proteins (e.g., adipocyte markers) may be used to sort target cells.

[0192] In another embodiment, adipocytes are enriched by drug resistance selection in genetically engineered source cells that express antibiotic resistance genes under the control of adipocyte-specific promoters.

[0193] This method can generate cells exhibiting at least one adipocyte characteristic (i.e., differentiated cells). One or more characteristics can be used to select adipocytes generated by the method of this invention.

[0194] Features include, but are not limited to, the detection or quantification of cellular markers, enzyme activity, and morphological characteristics and features of intercellular signaling. Biological functions of adipocytes can also be assessed, for example using functional assays, such as the secretion of adipokines (e.g., adiponectin and leptin) and responses to insulin (i.e., insulin sensitivity and / or the production of insulin sensitizers and anti-inflammatory agents).

[0195] In one implementation, the feature (i.e., the feature of adipocytes, particularly human adipocytes) is selected from one or more of the following: (i) Expression of one or more cell markers, such as FABP4, PLIN1, or a combination thereof; (ii) expression of adipokines or response to insulin; or (iii) Morphological characteristics of adipocytes.

[0196] In one embodiment, cells are sorted based on the acquisition of expression of mature adipocyte markers (such as FABP4 and PLIN1). In another embodiment, cells are sorted based on the acquisition of expression of adipocyte markers (such as CEBPA, CEBPB, and CD36). In yet another embodiment, cells are sorted based on the acquisition of expression of brown adipocyte markers (such as UCP1).

[0197] Adipocyte biomarkers can be those obtained through transcriptome analysis. For example, single-cell RNA sequencing has been used to provide detailed transcriptomic profiles of human adipocytes derived from primary human tissue. This information can be used to identify adipocytes produced using the methods described herein. Other resources, such as the Human Cell Atlas and CellTypist, can also be used to identify adipocyte biomarkers.

[0198] This method may include measuring differentiated cells obtained by the method described herein and identifying a set of transcribed genes; comparing the transcribed genome of the differentiated cells with one or more reference transcribed genomes from one or more reference adipocytes; and identifying a match between the differentiated cells and the reference adipocytes.

[0199] In one embodiment, the method includes the step of identifying differentiated cells as adipocyte types by measuring the morphological characteristics of the differentiated cells and matching the morphological characteristics with those of a reference tissue or cell.

[0200] In one embodiment, the method includes the step of identifying differentiated cells as adipocyte types by measuring the expression of protein markers in the differentiated cells and matching the expression of said protein markers with the expression of reference adipocyte protein markers.

[0201] In one implementation, the method includes the step of identifying differentiated cells as adipocyte types by measuring function and matching said function with the function of reference adipocytes.

[0202] In one embodiment, cells obtained by the method of the present invention express an adipocyte phenotype. This phenotype can be defined by the expression (+) or non-expression (-) of one or more of the following markers: FABP4+, PLIN1+, CEBPA+, CEBPB+, CD36+, and UCP1+. Target cells may also be negative for pluripotency markers.

[0203] Alternatively, certain differentiated cells can be sorted from other differentiated cells and cells based on the expression of their lineage-specific cell surface antigens. Another approach is to assess expression at the RNA level, for example, via RT-qPCR or single-cell RNA sequencing, without any sorting or pre-selection steps. This technique is known in the art.

[0204] Cell culture

[0205] In one embodiment, the method involves culturing a cell population for a sufficient time under conditions that allow differentiation into adipocytes. Typically, the cells of the present invention are cultured in a culture medium that is a nutrient-rich buffer solution capable of sustaining cell growth.

[0206] Cell culture media may contain any of the following in appropriate combinations: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Cell culture media commonly used for specific cell types are known to those skilled in the art. For example, the medium may contain a basal medium (e.g., DMEM / F12 or STEMPRO-34) supplemented with GLUTAMAX, antibiotics (such as penicillin or streptomycin), B27 supplementation, and / or N2 supplementation (both available from Thermo Fisher Scientific). The medium can then be further supplemented at different time points during the culture process. For example, one or more peptide hormones and / or cytokines may be added on days 2, 4, and / or 10 of the culture process.

[0207] In one embodiment, the culture medium comprises one or more components selected from: bone morphogenetic protein 4 (BMP4), activin A, fibroblast growth factor 2 (FGF2), insulin, ascorbic acid, and dexamethasone. In one embodiment, the culture medium comprises one or more peptide hormones and / or cytokines selected from: BMP4, activin A, FGF2, and insulin.

[0208] The method of the present invention can be used to obtain adipocytes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after culture. In one embodiment, the method includes culturing the cells under suitable conditions for at least 4 days, such as at least 7 days or at least 10 days. In a further embodiment, the method includes culturing the cells for a period of time sufficient to generate adipocytes (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days or longer, such as from 5 to 40 days, from 7 to 35 days, from 14 to 28 days, or about 21 days). In some embodiments, cells are cultured for several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days). In one embodiment, the method includes culturing cells for at least about 5, 10, 15, or 20 days to generate adipocytes. In one embodiment, the cell culture period is between 4 and 25 days, for example, between 7 and 14 days.

[0209] After culture, the cell population may contain two cell types. For example, such a cell population may contain two cell types, including stem cells and adipocytes. In one embodiment, the cell population contains up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% (or any intermediate range) of adipocytes in the resulting cell population.

[0210] Cell culture can help induce cells to be directed toward a more mature phenotype, preferentially promote the survival of mature cells, or a combination of both effects.

[0211] According to another aspect of the invention, cells that can be obtained by any of the methods defined herein are provided.

[0212] According to another aspect of the invention, a cell is provided comprising one or more exogenous expression cassettes, the expression cassettes comprising nucleotide sequences encoding at least one or more transcription factors, wherein the one or more transcription factors are selected from: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and variants thereof.

[0213] According to another aspect of the invention, a cell is provided comprising one or more exogenous expression cassettes, the expression cassettes comprising nucleotide sequences encoding at least one or more polypeptides and / or transcription factors having one or more transcription factor activities, wherein the one or more transcription factors are selected from: one or more PPAR proteins (e.g., PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) and variants thereof.

[0214] As described herein, exogenous expression cassettes encoding one, two, three, or more polypeptides and / or transcription factors with transcription factor activity can be integrated into the cell's genome. In another embodiment, exogenous expression cassettes encoding two or more polypeptides and / or transcription factors with transcription factor activity are integrated into a (specific) target site of the cell's genome. Alternatively, exogenous expression cassettes encoding two or more polypeptides and / or transcription factors with transcription factor activity are integrated into a non-specific target site of the cell's genome.

[0215] Cellular components

[0216] According to another aspect, a pharmaceutical composition is provided comprising adipocytes generated by the methods described herein and a pharmaceutically acceptable carrier.

[0217] Pharmaceutical compositions may comprise adipocytes as described herein combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions that can be used with the pharmaceutical compositions of the present invention comprise, for example, DMSO.

[0218] For purposes of manufacture, distribution and use, the adipocytes described herein may be provided in the form of cell cultures or suspensions in isotonic excipients or culture media, optionally frozen to facilitate transport or storage.

[0219] Uses of fat cells

[0220] Cells produced by any method according to the present invention have applications in basic and medical research, diagnostic and therapeutic methods. Cells can be used to study cell development in vitro, provide new drug testing systems, develop screening methods, review treatment regimens, provide diagnostic tests, etc. These uses constitute a part of the present invention. Alternatively, cells can be transplanted into human or animal patients for diagnostic or therapeutic purposes. The therapeutic use of cells is also included in the present invention.

[0221] According to one aspect of the invention, adipocytes as defined herein are provided for in vitro diagnostics or drug screening.

[0222] Adipocytes generated by the method of the present invention are particularly useful in drug screening. Therefore, in one embodiment, the method further comprises contacting the adipocytes with a test substance and observing changes (e.g., effects) induced by the test substance in the adipocytes. Methods known in the art, such as pharmacological or toxicological assays, can be used to observe the changes or effects. In one aspect, a method for using cells to evaluate a test substance (e.g., a drug, such as a compound) includes determining the pharmacological or toxicological properties of the test substance on adipocytes provided by the method described herein. The method may include: a) contacting the adipocytes described herein with the test substance; and b) determining the effect of the test substance on the adipocytes.

[0223] The evaluation of the activity of candidate molecules may involve combining adipocytes as described herein with candidate molecules, identifying any changes in adipocyte morphology, phenotype, or metabolic activity attributed to the molecule (i.e., compared to controls, such as untreated cells or cells treated with an inert compound), and then correlating the molecule's effects with the observed changes. Screening may be performed because candidate molecules are designed to have pharmacological effects on adipocytes, or because the molecule is designed to have effects in other areas, but it is necessary to determine whether it has any unintended side effects.

[0224] Cytotoxicity can initially be determined by its effects on cell viability, survival, morphology, and enzyme leakage into the culture medium. More detailed analyses can be performed to determine whether the test substance affects cell function without causing toxicity.

[0225] Alternatively, these cells can be used to assess changes in gene expression patterns induced by a potential drug candidate. In this embodiment, changes in gene expression patterns induced by the addition of a drug candidate can be compared to gene expression patterns induced by a control drug with known effects on adipocytes.

[0226] Therefore, according to another aspect, a drug screening method (e.g., assessing drug responsiveness) is provided, the method comprising the step of using adipocytes generated by the methods described herein. According to another aspect of the invention, a drug screening method is provided, the method comprising contacting adipocytes generated using the methods defined herein or adipocytes defined herein with a drug, and observing changes in the adipocytes induced by the drug.

[0227] According to another aspect of the invention, adipocytes as defined herein are provided for therapeutic purposes.

[0228] In one embodiment, the method further includes transplanting adipocytes into a patient. In this aspect of the invention, the cells used to generate adipocytes can be autologous (i.e., adult cells are taken, modified, and returned to the same individual) or derived from a donor (i.e., allogeneic, comprising stem cell lines). Directly reprogramming cells into adipocytes is suitable for generating autologous or allogeneic adipocytes.

[0229] Adipocytes can be used in conjunction with a variety of materials to form compositions for purposes such as reconstructive surgery. Cells can be combined with biological matrices to form two-dimensional or three-dimensional materials as needed. In particular, adipocytes can be used to prepare fat pads and adipose tissue for reconstruction in areas where tissue has been removed.

[0230] In one implementation, the treatment is a cosmetic procedure. Similar to its use in reconstructive surgery, the fat cells generated using the method of this invention can be used in essentially the same way for selective cosmetic procedures, namely, to construct a base tissue under the skin using a composite material of autologous cells and biocompatible materials.

[0231] Therefore, according to another aspect of the invention, a method for treating a subject who has or is at risk of having a disease or disorder is provided, comprising administering to the subject a therapeutically effective amount of adipocytes generated by a method as defined herein or as defined herein.

[0232] The adipocytes of the present invention can be used to develop therapies and treatments for disorders of fat development and function (e.g., lipodystrophy or obesity) and secondary diseases of fat dysfunction (e.g., diabetes, hyperlipidemia, hypertension or cardiovascular disease).

[0233] In a different aspect, cells can be used in tissue engineering. Tissue engineering requires the generation of tissues that can be used to replace human or animal tissues, or even entire organs. Tissue engineering methods are known to those skilled in the art, but involve using a scaffold (extracellular matrix) on which cells are applied to generate tissue / organ. Such methods can be used to produce “artificial” tissues or organs. Methods for generating tissues can include additive manufacturing, also known as three-dimensional (3D) printing, which may involve directly printing cells to create tissue. Therefore, the present invention provides a method for generating tissues using cells produced by the methods described in any aspect of the invention.

[0234] Cell-derived tissues manufactured using the method according to the invention can be used for in vitro / cultured meat. Therefore, according to one aspect of the invention, adipocytes as defined herein are provided for the preparation of cultured meat. According to another aspect of the invention, food products (e.g., cultured meat) comprising adipocytes as defined herein are provided. The primary cell type in cultured meat is myocytes; however, to reproduce the texture and / or flavor of natural meat, it is desirable to use a combination of multiple cell types comprising adipocytes (i.e., adipocytes as described in this invention) to prepare cultured meat. If the engineered tissue is intended for use in cultured meat, then the source cells may be derived from livestock.

[0235] The term "cultured meat" (also known as in vitro meat, laboratory-grown meat, cell-based meat, farmed meat, or synthetic meat) used in this article refers to meat grown from cell or tissue cultures, rather than meat obtained through the slaughter of animals.

[0236] Reprogramming Kit

[0237] According to another aspect, a kit for differentiating cells into adipocytes is provided, comprising: (i) source cells and reagents that activate or increase the expression or amount of at least one or more transcription factors; and / or (ii) One or more expression cassettes containing nucleotide sequences encoding at least one or more transcription factors. The one or more transcription factors mentioned therein are selected from: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 and their variants.

[0238] According to yet another aspect, a kit for differentiating cells into adipocytes is provided, comprising: (i) source cells (preferably pluripotent stem cells, more preferably hiPSCs) and agents that activate or increase the expression or amount of at least one or more transcription factors; and / or (ii) One or more expression cassettes comprising nucleotide sequences encoding one or more polypeptides having transcription factor activity and / or at least one or more transcription factors. The one or more transcription factors mentioned therein are selected from: one or more PPAR proteins (e.g., PPARA and / or PPARG), HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins (e.g., CEBPA and / or CEBPB) and their variants.

[0239] In one embodiment, the expression cassette comprises an inducible expression construct containing a sequence encoding one or more polypeptides having transcription factor activity and / or the transcription factor itself.

[0240] As described herein, combinations of peptides with transcription factor activity and / or transcription factors described herein are particularly useful in this invention. If a combination of peptides and / or transcription factors is desired, these can be encoded on the same or different expression cassettes. Therefore, in one embodiment, the kit comprises an expression cassette (preferably an inducible expression cassette) encoding two or more peptides and / or transcription factors (e.g., three, four, five, six, seven, or eight transcription factors). Preferably, the kit comprises an expression cassette encoding three or more, more preferably four or more peptides and / or transcription factors.

[0241] According to another aspect, the kits defined herein are provided for use in differentiating cells into adipocytes.

[0242] The kit may contain one or more items and / or reagents for performing the method. For example, one or more transcription factor genes, their derivatives, variants, or fragments used in the method described herein may be provided in a separate form and may be part of the kit, for example, in a suitable container, such as a vial, in which the contents are protected from the external environment.

[0243] In one embodiment, the kit further comprises at least one source cell, such as pluripotent stem cells (e.g., induced pluripotent stem cells) or non-pluripotent, non-adipocytes.

[0244] In one embodiment, the kit further comprises a culture medium for culturing cells and instructions for preparing enhanced pluripotent cells or reprogrammed pluripotent cells according to the methods defined herein.

[0245] It should be understood that all embodiments described herein can be applied to all aspects of the present invention.

[0246] Other features and advantages of the invention will be apparent from the description provided herein. However, it should be understood that while the description and specific examples indicate preferred embodiments of the invention, they are given by way of example only, as various changes and modifications will be apparent to those skilled in the art. The invention will now be described using the following non-limiting embodiments: Example Example 1 – Prioritized Filtering method: Plasmid libraries encoding TF, reporter cell lines, and transposase-mediated delivery Based on hierarchical adipocyte marker transcriptomics analysis and using a set of computational tools, a large-scale candidate library of transcription factors (TFs) was initially selected from a chosen multi-omics dataset through scientific screening.

[0247] Each TF and a small set of negative controls were cloned separately into a vector for genome integration mediated by the PiggyBac transposase. TF expression was controlled by a tetracycline-inducible promoter. The vector encoded a puromycin resistance cassette for selecting cells that successfully integrated the genome. Each TF expression cassette encoded a unique DNA tag sequence, allowing for the identification of TFs and the quantification of their frequency in the cell pool.

[0248] To identify cells successfully reprogrammed into adipocytes, the hiPSC reporter cell line was generated. Genomic loci of two mature adipocyte markers, FABP4 and PLIN1, were engineered to generate polycistronic cassettes encoding the fluorescent proteins GFP and mCherry, respectively. Therefore, the fluorescent markers are expressed upon activation of the endogenous markers without disrupting their function. Since cells with high lipid content may not be suitable for flow cytometry or other microfluidic-based protocols, both GFP and mCherry were labeled with nuclear localization signal peptides to restrict the expression of the fluorescent proteins to the nucleus. Therefore, endpoint fluorescence-activated cell sorting can be performed on the entire cell population or purified nuclei that retain intact fluorescence signals.

[0249] Vectors were mixed in equimolar ratios to ensure homogeneous representation of TFs, then retransformed in *E. coli* for large-scale DNA preparation. A mixture of the TF library and PiggyBac transposase was nuclear transfected into hiPSCs. Cell culture scale and nuclear transfection parameters were optimized to ensure adequate coverage of the highly complex combinatorial space.

[0250] Filter Summary

[0251] Two replicates of the priority selection experiment were performed. Prior to nuclear transfection, FABP4-GFP / PLIN1-mCherry hiPSCs were amplified in TesR E8 (Stem Cell Technologies) on standard tissue culture plates coated with polinecin (Life Technologies). After nuclear transfection, cells were cultured as described above, and cells that successfully integrated the genome were selected by adding puromycin to the culture medium. After selection, hiPSCs were plated for reprogramming and cultured for up to 10 days in the presence of doxycycline in a medium that promotes adipocyte maintenance and survival. Sufficient cell numbers were maintained in the culture at all stages to ensure adequate coverage of each TF combination. After reprogramming, cells were harvested, and GFP-positive, mCherry-positive, and negative control populations were sorted by fluorescence-activated cell sorting. In another set of samples, cell nuclei were purified prior to sorting.

[0252] Batch TF tag sequence quantification and analysis

[0253] Genomic DNA was extracted from all biomarker-positive and control samples, and the relative distribution of TF tag sequences in each batch of gDNA samples was quantified by amplicon sequencing.

[0254] Data were analyzed using the Mageck method (Li et al.). Genome Biol (15, 554, 2014). TF tag sequence counts were analyzed using the Mageck count function. Subsequently, the raw count file was used in the test and maximum likelihood estimation (MLE) functions to generate enrichment coefficients and statistics for each TF in FABP4-GFP and PLIN1-mCherry positive samples compared to non-enriched control samples. MLE The function allows for comparisons under multiple conditions and produces a beta value similar to the logFC value in traditional differential expression tests. The analysis considers reproducibility between two replicates. test The function performs pairwise comparisons of the conditions and generates a logFC value and related statistics for each pair.

[0255] result: pass MLE and test The functions generate beta values ​​and logFC measurements to measure the enrichment of TF in the two biomarker-positive populations (FABP4-GFP or PLIN1-mCherry). TF is considered a hypothetical reprogramming factor and is selected for downstream validation if a beta value > 0.3 or a mean logFC > 0.5 in any of the following samples (see Example 2 below): Whole-cell FABP4-GFP positive samples; Whole-cell PLIN1-mCherry positive sample; Purify FABP4-GFP positive samples from cell nuclei; Purify PLIN1-mCherry positive samples from cell nuclei.

[0256] Table 2.

[0257] Table 2 lists 20 hypothetical reprogrammed TFs and displays the beta and logFC values ​​for FABP4-GFP and PLIN1-mCherry positive samples. LogFC values ​​for two replicates are shown, and the beta value includes experimental replicates in the calculation. Values ​​in bold indicate those that meet downstream validation criteria. TFs that do not meet downstream validation criteria are not shown.

[0258]

[0259] Example 2 – Validation Screening method: Plasmid libraries encoding TF and transposase-mediated delivery Following priority screening (see Example 1), the reprogramming potential of TFs was evaluated. Each TF and a negative control were cloned separately into vectors for genome integration mediated by the PiggyBac transposase. TF expression was controlled by a tetracycline-inducible promoter. The vectors encoded puromycin resistance cassettes for selecting cells that successfully integrated the genome. Each TF expression cassette encoded a unique DNA tag sequence. We designed primers to simultaneously amplify the TF and cell tag sequences in a 10x Genomics workflow, allowing for the allocation of TF combinations to single-cell transcriptome atlases.

[0260] Vectors were mixed in equimolar ratios to ensure homogeneous representation of TFs, then retransformed in *E. coli* for large-scale DNA preparation. A mixture of the TF library and PiggyBac transposase was nuclear transfected into iPSCs. Cell culture scale and nuclear transfection parameters were optimized to ensure adequate coverage of the highly complex combinatorial space.

[0261] Filter Summary

[0262] Two replications of the validation screening experiment were performed using the same mixed plasmid library. Prior to nuclear transfection, FABP4-GFP / PLIN1-mCherry hiPSCs were amplified in TesR E8 (Stem Cell Technologies) on standard tissue culture plates coated with Life Technologies. Following nuclear transfection, cells were cultured as described above, and cells that successfully integrated the genome were selected by adding puromycin to the culture medium. After selection, iPSCs were plated for reprogramming and cultured for up to 9 days in the presence of doxycycline in a medium that promotes adipocyte maintenance and survival. Throughout all phases, a sufficient number of cells were maintained in the culture to ensure adequate coverage of each TF combination.

[0263] After reprogramming, cells were harvested and sorted into GFP-positive, mCherry-positive, and negative control populations using fluorescence-activated cell sorting. Unsorted (NS), ungated (NG), and marker-negative (or all-negative, FN) control samples were also collected.

[0264] Single-cell transcriptome analysis and TF tag sequence capture

[0265] Cell nuclei were purified and sorted, and analyzed via scRNA-seq using a 10x Genomics Chromium Single Cell 3' Reagent Kits v3 according to the manufacturer's instructions. Up to 10,000 nuclei were targeted for analysis per sample. Following the cDNA amplification step, gene expression libraries were created and sequenced on NovaSeq, aiming for at least 25,000 reads per cell, according to the 10x Genomics 3' v3.1 protocol. In addition to the gene expression library, matched cDNA was used as a template for further targeted amplification of TF-tagged sequences and sequenced on MiSeq.

[0266] Data Analysis

[0267] Single-cell gene expression data generated by the Cell Ranger pipeline were further analyzed in Seurat v3 (Stuart et al., Cell, 2019) and visualized on uniform manifold approximation and projection (UMAP) plots (Becht, Nature Biotech, 2019). Cell identities were assigned using the automated cell classification tool CellTypist (Dominguez Conde et al., Science, 2022) and a selected human adipose tissue reference single-cell atlas (Emont et al., Nature, 2022). Exogenous transcription factor (eTF) tag sequences were assigned to single cells and their corresponding gene expression profiles using 10x cell tag sequences. eTF enrichment was quantified by comparing cells in negative control (NG) and sorted (FABP4-GFP, PLIN1-mCherry) samples with cells in marker-positive populations identified as adipocytes by CellTypist.

[0268] result: Visualize single-cell gene expression data on a UMAP plot that includes undifferentiated hiPSCs (G10), sorted live cells (NG), FABP4-GFP positive cells, PLIN1-mCherry positive cells, and marker-negative cells (all negative, or FN). Figure 1 Predictably, a cluster consisting entirely of undifferentiated hiPSCs separated from all other clusters containing cells collected at the end of reprogramming (NG, FN, FABP4-GFP, PLIN1-mCherry). In the latter, one large cluster almost entirely contained unsorted cells, while more dispersed clusters contained cells from both FABP4-GFP and PLIN1-mCherry positive samples. The partial overlap between FABP4-GFP and PLIN1-mCherry positive cells suggests that both markers enrich cells sharing similar gene expression profiles. A small subset of low-quality nuclei clustered at the right end of the UMAP plot.

[0269] eTF tag sequences were successfully detected and assigned in single cells of all samples. Figure 2 Although negative controls (e.g., NEG1 to NEG5) were randomly distributed in the samples, several eTFs were found in clusters within the sorted cell populations (e.g., PPARA, PPARG, EBF1, EBF2).

[0270] Next, we used CellTypist to assign cell identities to the adipocyte gene expression profile reference dataset (Emont et al., Nature, 2022). In our dataset, we identified cells belonging to at least 5 cell types with high confidence, including adipocytes ( Figure 3 A total of 4,670 cells were identified as adipocytes, most of which were in FABP4-GFP and PLIN1-mCherry positive samples (Table 3 below).

[0271] Table 3. Number of cells identified as adipocytes in each sample

[0272] Expression of FABP4-GFP and PLIN1-mCherry defined two distinct, partially overlapping adipocyte clusters, indicating that each marker identifies a specific adipocyte subset. We evaluated whether these hiPSC-derived adipocytes expressed any functional genes associated with this cell type. For both markers, cells showed high expression levels of several genes commonly associated with adipose tissue in all experimental replicates. Figure 4 Conversely, no cardiac cell markers were found. Of the five cell types identified by CellTypist, adipocytes showed similarities to brown adipose tissue (…). Figure 5) and white adipose tissue ( Figure 6 The highest expression level of common related markers.

[0273] To identify the eTFs that reprogram hiPSCs into adipocytes, we first calculated the log-likelihood of the sets of up to four eTFs in FABP4-GFP and PLIN1-mCherry-positive cells (identified as adipocytes by CellTypist) compared to the control, and then ranked the sets according to enrichment. Figure 7 As shown, several enriched ensembles were identified, with a few exhibiting particularly high enrichment. Table 4 shows the most enriched combinations in FABP4-GFP and PLIN1-mCherry-positive adipocytes. Unsurprisingly, the two datasets identified partially overlapping eTF ensembles that drive reprogramming in different adipocyte subtypes. eTF ensembles containing EBF2, EBF1, PPARA, and / or HOXC8 were identified as reprogramming ensembles in the PLIN1-mCherry-positive dataset. eTF ensembles containing PPARG, PPARA, HOXC8, and / or ZNF467, ZNF423 were identified as reprogramming ensembles in the FABP4-GFP-positive dataset.

[0274] Table 4. Most enriched reprogramming combinations in each adipocyte subpopulation Example 3 – Further Examination of TF Combinations Following the validation screening (see Example 2), the reprogramming potential of some of the highest-scoring TF (PPARG, PPARA, HOXC8, ZNF423, EBF1, and EBF2) combinations was evaluated.

[0275] method: Each TF was cloned separately into a vector for genome integration mediated by PiggyBac transposase. TF expression was controlled by a tetracycline-inducible promoter. The vector encoded a puromycin resistance cassette for selecting cells that successfully integrated the genome. The vectors were mixed in equimolar ratios to ensure homogeneous representation of the TFs. The mixture of the TF library and PiggyBac transposase was nuclear transfected into hiPSCs. Cell culture scale and nuclear transfection parameters were optimized to ensure a sufficient proportion of cells encoding the complete set of six TFs.

[0276] Two replicates of nuclear transfection were performed using the same plasmid library. Prior to nuclear transfection, hiPSCs were amplified in GIBCO ESSENTIAL 8 Medium (Gibco) on standard tissue culture plates coated with LifeTechnologies. After nuclear transfection, cells were cultured as described above, and cells that successfully integrated the genome were selected by adding puromycin to the medium. After selection, hiPSCs were plated for reprogramming and cultured for 10 days in a medium that promotes adipocyte maintenance and survival in the presence of doxycycline. Delivery of TF via transposases can result in a variable number of insertion events, which may bias the analysis of reprogramming efficiency.

[0277] After reprogramming, cells were harvested and RNA extracted, and reverse transcription and quantitative PCR were performed on adipocyte markers and the housekeeping control gene hydroxymethylcholine synthase (HMBS). Alternatively, cells were fixed and immunostained for adipocyte markers, and the results were analyzed using LipidTox™ (…). Thermo Fisher Scientific Staining was used to assess intracellular lipid accumulation.

[0278] result: Transcriptional analysis using qPCR revealed enrichment of key adipocyte markers PLIN1, FABP4, CEBPB, CEBPA, CD36, and UCP1 in cells reprogrammed with six different TF libraries. Figure 8 Among these adipocyte markers, UCP1 is a key regulator of brown adipocyte fate.

[0279] To assess reprogramming efficiency, immunocytochemistry (ICC) was performed on the biomarkers FABP4 and PLIN1. Protein expression of both biomarkers was easily detectable. Figure 9 In addition, intracellular lipid accumulation was detected by LipidTox staining.

[0280] In summary, these data suggest that one or more combinations of TFs, including PPARG, PPARA, HOXC8, ZNF423, EBF1, and EBF2, can drive hiPSC reprogramming into adipocytes.

Claims

1. A method for generating adipocytes, comprising expressing one or more polypeptides having one or more transcription factor activities and / or increasing the expression of one or more transcription factors in a cell population, said transcription factors being selected from the group consisting of one or more PPAR proteins, such as PPARA and / or PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, one or more CEB proteins, such as CEBPA and / or CEBPB and variants thereof, and culturing the cell population to obtain adipocytes.

2. The method of claim 1, wherein the expression of two or more transcription factors selected from the group consisting of: a PPAR protein, HOXC8, EBF1, EBF2, ZNF467, ZNF423 is increased.

3. The method of claim 1 or 2, wherein PPAR is selected from: PPARA, PPARG, and combinations of PPARA and PPARG.

4. The method of any one of claims 1 to 3, wherein the transcription factor is selected from the group consisting of PPARA, EBF1, EBF2, HOXC8 and variants thereof.

5. The method of any one of claims 1 to 3, wherein the transcription factor is selected from the group consisting of: PPARA, PPARG, HOXC8, ZNF467, ZNF423 and variants thereof.

6. The method of claim 1 or 2, wherein the transcription factor comprises one of the following combinations: (i) PPARA, EBF2 and EBF1; (ii) HOXC8, EBF2, and EBF1; (iii) PPARG, ZNF467 and PPARA; (iv) PPARG, HOXC8, and PPARA; (v) HOXC8, ZNF423, and PPARA; (vi) PPARA and CEBPA; (vii) PPARA and CEBPB; (viii) PPARG and CEBPA; or (ix) PPARG and CEBPB.

7. The method of any one of claims 1 to 6, comprising increasing the expression of one or more additional transcription factors as listed in Table 1.

8. The method of any one of claims 1 to 7, wherein the method comprises increasing the expression of three to seven transcription factors.

9. The method of any one of claims 1 to 8, wherein the transcription and translation of the one or more polypeptides having one or more transcription factor activities and / or the transcription factors themselves are controlled within the cell.

10. The method of claim 9, wherein transcription and translation of transcription factors are controlled within the cell.

11. The method of any one of claims 1 to 10, wherein the cell population comprises pluripotent stem cells, particularly induced pluripotent stem cells.

12. The method of any one of claims 1 to 11, wherein the method comprises generating adipocytes by reprogramming the pluripotent stem cells or induced pluripotent stem cells, preferably human induced pluripotent stem cells.

13. The method of any one of claims 1 to 12, wherein the adipocytes are human adipocytes.

14. The method of any one of claims 1 to 13, further comprising monitoring at least one adipocyte characteristic of the cell population.

15. The method of claim 14, wherein the feature is selected from one or more of the following: (i) Expression of one or more cell markers, such as FABP4, PLIN1, CEBPA, CEBPB, CD36, UCP1 or a combination thereof; (ii) expression of adipokines or response to insulin; and (iii) Morphological characteristics of adipocytes.

16. The method of any one of claims 1 to 15, wherein the expression of transcription factors is increased by contacting a cell population with one or more exogenous expression cassettes encoding one or more genes or one or more agents that activate or increase the expression or amount of transcription factors.

17. The method of any one of claims 1 to 16, wherein the expression of the gene is under controlled transcription.

18. The method of any one of claims 1 to 17, wherein a sequence encoding one or more transcription factors is introduced into the cell population using a method comprising: - Insert (preferably targeted) the coding sequence of a transcriptional regulatory protein into the first genomic safe harbor site of the source cell in the cell population; and - An inducible cassette is inserted (preferably targeted) into a second genomic safe harbor site in the source cell, wherein the inducible cassette contains a sequence encoding one or more transcription factors, the sequence being operatively linked to an inducible promoter, and the promoter being regulated by a transcriptional regulatory protein.

19. The method according to any one of claims 1 to 18, comprising culturing under suitable conditions for at least 4 days, for example at least 7 days, particularly about 10 days.

20. The method of claim 19, comprising culturing in a culture medium containing one or more components selected from the group consisting of BMP4, Activin A, FGF2, insulin, ascorbic acid, and dexamethasone.

21. A method for producing adipocytes from source cells, preferably pluripotent stem cells, and more preferably human induced pluripotent stem cells, comprising the following steps: a) Insert (preferably targeted insertion) the gene encoding a transcription regulatory protein into the first genomic safe harbor site of the source cell; and b) Inserting (preferably targeted insertion) at least one nucleotide sequence into a second genomic safe harbor site of a source cell, the nucleotide sequence encoding one or more polypeptides having one or more transcription factor activities and / or encoding one or more transcription factors selected from: one or more PPAR proteins, such as PPARA and / or PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423; one or more CEB proteins, such as CEBPA and / or CEBPB and variants thereof, the nucleotide sequence being operatively linked to an inducible promoter, wherein the inducible promoter is regulated by the transcriptional regulatory protein; and c) Culture source cells containing the insert to obtain adipocytes.

22. Use of one or more transcription factors for the generation of adipocytes, wherein the one or more transcription factors are selected from: one or more PPAR proteins, such as PPARA and / or PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, and one or more CEB proteins, such as CEBPA and / or CEBPB and variants thereof.

23. A cell that can be obtained by the method of any one of claims 1 to 22.

24. A cell, preferably a pluripotent stem cell, more preferably a human induced pluripotent stem cell, comprising one or more exogenous expression cassettes, said expression cassettes comprising encoding one or more polypeptides having one or more transcription factor activities and / or nucleotide sequences encoding at least one or more transcription factors, wherein said one or more transcription factors are selected from the group consisting of one or more PPAR proteins, such as PPARA and / or PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, and one or more CEB proteins, such as CEBPA and / or CEBPB and variants thereof.

25. The cell of claim 23 or 24, wherein a nucleotide sequence encoding one or more transcription factors is integrated into the cell’s genome.

26. The cell of claim 25, wherein a nucleotide sequence encoding one or more transcription factors is integrated into a target site of the cell.

27. The cell according to any one of claims 23 to 26, for therapeutic purposes.

28. The cells of any one of claims 23 to 26, for use in in vitro diagnostics or drug screening.

29. The cell according to any one of claims 23 to 26, for use in the preparation of cultured meat.

30. A kit for differentiating cells, preferably pluripotent stem cells, more preferably human induced pluripotent stem cells, into adipocytes, comprising: (i) source cells and reagents that activate or increase the expression or amount of at least one or more transcription factors; and / or (ii) One or more expression cassettes comprising encoding one or more polypeptides having one or more transcription factor activities and / or nucleotide sequences encoding at least one or more transcription factors. The one or more transcription factors mentioned therein are selected from: one or more PPAR proteins, such as PPARA and / or PPARG, HOXC8, EBF1, EBF2, ZNF467, ZNF423, and one or more CEB proteins, such as CEBPA and / or CEBPB and their variants.

31. Use of the kit as defined in claim 30 for differentiating cells, preferably pluripotent stem cells, more preferably human induced pluripotent stem cells, into adipocytes.

32. A drug screening method comprising contacting adipocytes generated using the method of any one of claims 1 to 21 or adipocytes as defined in any one of claims 23 to 26 with a drug and observing changes in the adipocytes induced by the drug.

33. A method of treating a subject who has or is at risk of having a disease or condition, comprising administering to the subject a therapeutically effective amount of adipocytes generated by the method of any one of claims 1 to 21 or adipocytes as defined in any one of claims 23 to 26.

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