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WO2024170702A1 - Maturation de cellules musculaires squelettiques - Google Patents

Maturation de cellules musculaires squelettiques Download PDF

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Publication number
WO2024170702A1
WO2024170702A1 PCT/EP2024/053906 EP2024053906W WO2024170702A1 WO 2024170702 A1 WO2024170702 A1 WO 2024170702A1 EP 2024053906 W EP2024053906 W EP 2024053906W WO 2024170702 A1 WO2024170702 A1 WO 2024170702A1
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pluripotent stem
protein
cells
stem cell
days
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Erik VAN DER WAL
Kyle Bill QUINNEY
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Meatable BV
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Meatable BV
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Priority to AU2024222968A priority Critical patent/AU2024222968A1/en
Priority to CN202480018700.5A priority patent/CN121175409A/zh
Priority to EP24705154.3A priority patent/EP4665842A1/fr
Publication of WO2024170702A1 publication Critical patent/WO2024170702A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2513/00—3D culture

Definitions

  • the present invention relates to modified pluripotent cells and to methods of differentiating said cell into skeletal muscle cells.
  • the invention concerns the expression of the transcriptional regulator proteins, MYOD and PAX7, in a pluripotent stem cell.
  • a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a MYOD protein, wherein the coding sequence for the MYOD protein is operably linked to an inducible promoter; and, iii) an expression construct for expression of a PAX7 protein, wherein the coding sequence for the PAX7 protein is operably linked to an inducible promoter; wherein the expression constructs of ii) and iii) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.
  • the expression constructs of ii) and iii) are both inserted into a second genetic safe harbour site that is different from the first genetic safe harbour site.
  • said first and further genomic safe harbour sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene or the CCR5 gene, preferably wherein the genetic safe harbour site are hROSA26 locus and the AAVS1 locus.
  • the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines.
  • the pluripotent stem cells are of a livestock or poultry species.
  • the livestock species is porcine or bovine, preferably porcine.
  • the expression construct that is inserted into the second genetic safe harbour site encodes a MYOD protein a linker and a PAX7 protein, preferably wherein the linker is a P2A linker, more preferably wherein the linker comprises the sequence of SEQ ID NO: 6.
  • the construct comprises the sequence of SEQ ID NO: 8.
  • the activity of the transcriptional regulator protein is controlled by an exogenously supplied substance derivative.
  • the transcriptional regulator protein is selected from the group consisting of: tetracycline responsive transcriptional activator protein (rtTa), Tetracycline repressor (TetR), VgEcR synthetic receptor or a hybrid transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain from the human progesterone receptor and an activation domain from the human NF-kB, preferably the transcriptional regulator protein is rtTA.
  • the inducible promoter includes a Tet Responsive Element (TRE).
  • TRE Tet Responsive Element
  • the inducible promotor is a tetON promotor.
  • the invention provides for a method for the production of skeletal muscle cells, for example type 1 or type 2 muscle, comprising a ) culturing the pluripotent stem cell according to any one of the preceding claims in a proliferation medium: followed by b) inducing skeletal muscle cell differentiation by adding the exogenous substance as described herein.
  • the differentiation stage of the method as described herein is at most 10 days, at most 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days.
  • the medium used for proliferation and the medium used for differentiation may be the same medium or different media.
  • the produced skeletal muscle cells are for human and non-human dietary consumption.
  • the invention provides for a use of a pluripotent stem cell as described herein or use of the method for producing a skeletal muscle cells as described herein for tissue engineering, optionally for the production of cultured meat.
  • the invention provides for a food product comprising the pluripotent stem cell as described herein or the skeletal muscle cells obtained by the method as described herein.
  • the food product is cultured meat.
  • the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
  • At least a particular value means that particular value or more.
  • “at least 2” is understood to be the same as “2 or more” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.
  • the word “about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.
  • heterologous when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature.
  • Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed.
  • heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein.
  • heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.
  • expression vector or “expression construct” refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame, inducible promoter
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. In the case of the present invention, the control is effected by the transcriptional regulator protein.
  • the inventors have surprisingly found that the time period required for differentiation of pluripotent cells into mature skeletal muscle cells can dramatically be reduced by using a modified pluripotent stem cells that comprise an expression construct for expression of a MYOD protein and an expression construct for expression of a PAX7 protein or, alternatively, an expression construct which expresses both MYOD and PAX7.
  • a modified pluripotent stem cells that comprise an expression construct for expression of a MYOD protein and an expression construct for expression of a PAX7 protein or, alternatively, an expression construct which expresses both MYOD and PAX7.
  • full differentiation into mature skeletal muscle cells can be achieved in 6 days or less, for example less than 5 days, for example 4 days or 3 days or more quickly .
  • the use of the herein described pluripotent cells also provides for more scalable production of mature skeletal muscle cells with more mature skeletal muscle cells being produced as compared to what has been previously described.
  • the differentiated cells may develop spindle-like elongated morphology, undergo extensive cell fusion and exhibited strong and homogenous myogenic marker expression based on mRNA and protein levels (MHC isoforms/maturity, immunostainings MYOD/MYOG/MYH/TTN/TNNT/PAX3/7), along with high fusion index, length and thickness. Furthermore, spontaneous muscle fiber contractions may be observed, demonstrating the functionality of MYOD/PAX7.
  • the invention relates to a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a MYOD protein, wherein the coding sequence for the MYOD protein operably linked to an inducible promoter; and, iii) an expression construct for expression of a PAX7 protein, wherein the coding sequence for the PAX7 protein operably linked to an inducible promoter; wherein the expression constructs of ii) and iii) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.
  • a pluripotent stem cell of the invention may optionally comprise no expression constructs other than those set out in i) ii) and iii) above.
  • a pluripotent stem cell of the invention may optionally comprise one or more expression constructs in addition to those set out in i), ii) and iii) above, for example into at least one further genetic safe harbour site that is not the first genetic safe harbour site.
  • Such additional expression constructs may provide for the expression a protein which regulate is a regulator or transcription, for example a transcription factor, other than MYOD or PAX7.
  • a pluripotent stem cell of the invention which comprises one or more antibiotic resistance markers, one or more of such resistance markers may be removed.
  • a pluripotent stem cell of the invention may optionally comprise no antibiotic resistance markers.
  • MYOD also known as myoblast determination protein 1
  • myoblast determination protein 1 is a protein in animals that plays a major role in regulating muscle differentiation.
  • MYOD belongs to a family of proteins known as myogenic regulatory factors.
  • MYOD is one of the earliest markers of myogenic commitment. MYOD is expressed at extremely low and essentially undetectable levels in quiescent satellite cells, but expression of MYOD is activated in response to exercise or muscle tissue damage.
  • the effect of MYOD on satellite cells is dose-dependent: high MYOD expression represses cell renewal, promotes terminal differentiation and can induce apoptosis.
  • MYOD marks myoblast commitment, muscle development is not dramatically ablated in mouse mutants lacking the MYOD gene.
  • MYOD cyclin dependent kinases
  • MYOD enhances its own activity in the cell in a feedforward manner. Sustained MYOD expression is necessary for retaining the expression of muscle-related genes. MYOD is also an important effector for the fast-twitch muscle fiber (types HA, IIX, and IIB) phenotype.
  • the coding sequence for MYOD has the coding sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.
  • PAX7 Paired box protein, is a protein that in humans is encoded by the PAX7 gene. Pax- 7 plays a role in neural crest development and gastrulation, and it is an important factor in the expression of neural crest markers such as Slug, Sox9, Sox10 and HNK-1. PAX7 is expressed in the palatal shelf of the maxilla, Meckel's cartilage, mesencephalon, nasal cavity, nasal epithelium, nasal capsule and pons. Pax7 is a transcription factor that plays a role in myogenesis through regulation of muscle precursor cells proliferation. It can bind to DNA as an heterodimer with PAX3.
  • PAX7 functions as a marker for a rare subset of spermatogonial stem cells, specifically a sub set of Asingle spermatogonia. These PAX7+ spermatogonia are rare in adult testis but are much more prevalent in newborns, making up 28% of germ cells in neonate testis. Unlike PAX7+ muscle satellite cells, PAX7+ spermatogonia rapidly proliferate and are not quiescent. PAX7+ spermatogonia are able to give rise to all stages of spermatogenesis and produce motile sperm.
  • PAX7 is not required for spermatogenesis, as mice without PAX7+ spermatogonia show no deficits in fertility. PAX7 may also function in the recovery in spermatogenesis. Unlike other spermatogonia, PAX7+ spermatogonia are resistant to radiation and chemotherapy. The surviving PAX7+ spermatogonia are able to increase in number following these therapies and differentiate into the other forms of spermatogonia that did not survive. Additionally, mice lacking PAX7 had delayed recovery of spermatogenesis following exposure to busulfan when compared to control mice.
  • the coding sequence for PAX7 has the sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4.
  • the pluripotent stem cell as described herein comprises iv) a further expression construct for expression of a MYOG protein, wherein the coding sequence for the MYOG protein operably linked to an inducible promoter, wherein the expression constructs of iv) is inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site.
  • MYOG or Myogenin
  • Myogenin is a transcriptional activator encoded by the MYOG gene.
  • Myogenin is a muscle-specific basic-helix-loop-helix (bHLH) transcription factor involved in the coordination of skeletal muscle development or myogenesis and repair.
  • MYOG is a member of the MYOD family of transcription factors.
  • MYOG is essential for the development of functional skeletal muscle. MYOG is required for the proper differentiation of most myogenic precursor cells during the process of myogenesis.
  • DNA coding for myogenin was knocked out of the mouse genome, severe skeletal muscle defects were observed.
  • Mice lacking both copies of myogenin suffer from perinatal lethality due to the lack of mature secondary skeletal muscle fibers throughout the body.
  • myogenin can induce myogenesis in a variety of non-muscle cell types.
  • the pluripotent stem cell can comprise expression constructs that provide for the expression of proteins MYOD, PAX7, and MYOG.
  • the nucleic acid molecules encoding the proteins according to the invention may be codon-optimized for expression in mammalian cells. Methods of codonoptimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild-type sequence is replaced by a codon that is more preferred.
  • a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon.
  • the frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon.
  • a transcriptional regulator protein is a protein that bind to DNA, preferably sequence- specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor). Such entities are also known as transcription factors.
  • the DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or nearthe promoter of the regulated DNA sequence.
  • Transcriptional activator proteins bind to a response element and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression.
  • a genetic safe harbour (GSH) site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighboring genes and expression of the inducible cassette minimizes interference with the endogenous transcription program. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al, 201 1 , Nature Biotechnology, 29(1), 73-8. doi: 1 0. 1 038/nbt.
  • These criteria include a site that is (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA(miRNA), (iv) located outside a transcription unit and (v) located outside ultra-conserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of the criteria. It is thought that a suitable GSH will satisfy at least 2, 3, 4 or all of these criteria.
  • the first and further genomic safe harbour sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene or the CCR5 gene.
  • the first and further genomic safe harbour sites are located on chr1 : 152,360,840-152,360,859, chr1 : 175,942,362 -175,942,381 , chr1 :231 ,999,396-231 ,999,415, chr2: 45,708,354 - 45, 708, 373; chr8: 68,720,172 - 68,720,191 of the human genome.
  • the first and further genomic safe harbour sites are selected from any two of the safe harbour sites ROSA26, AAVS1 , the CLYBL gene or the CCR5 gene.
  • the genetic safe harbour sites are hROSA26 locus and the AAVS1 locus.
  • the expression construct for expression of MYOD protein as described herein and the expression construct for expression of a PAX7 protein as described herein are both inserted into a second genetic safe harbour site that is different from the first genetic safe harbour site.
  • the expression construct that is inserted into the second genetic safe harbour site is capable of expressing both the MYOD protein and the PAX7 protein simultaneously.
  • pluripotent stem cells includes embryonic stem cells, embryo- derived stem cells, induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC).
  • EpiSC epiblast-derived stem cells
  • pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.
  • Exemplary pluripotent stem cells can be generated using, methods known in the art.
  • “Induced pluripotent stem cells” iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.
  • the pluripotent stem cell according to the invention can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo- derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.
  • iPS cells can be from any species.
  • the pluripotent stem cell according to the invention, or for use in the invention is an animal cell. In certain embodiments the pluripotent stem cell according to the invention, or for use in the invention if from an edible animal species.
  • the pluripotent stem cell according to the invention is from a livestock or poultry animal or a seafood animal.
  • Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits.
  • the pluripotent stem cell according to the invention is a porcine or a bovine pluripotent stem cell. Most preferably, a porcine pluripotent stem cell.
  • the stem cell according to the invention is a porcine epiblast stem cell (pEpiSCs).
  • Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons.
  • the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare.
  • a pluripotent stem cell according to the invention, or for use in the invention is not a human cell.
  • Seafood species include, but are not limited to, fish and shellfish.
  • Transcriptional repressor proteins bind to a response element and prevent gene expression.
  • Transcriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory proteins, phosphorylation, and/or methylation.
  • the transcriptional regulator may be controlled by activation or deactivation.
  • the transcriptional regulator protein is a transcriptional activator protein
  • the transcriptional activator protein requires activation. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated through the addition to the cell of an exogenous substance.
  • the supply of an exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulator protein can be controlled.
  • an exogenous substance can be supplied in order to deactivate a transcriptional regulator protein, and then supply withdrawn in orderto activate the transcriptional regulator protein.
  • the transcriptional regulator protein is a transcriptional repressor protein
  • it is preferred that the transcriptional repressor protein requires deactivation.
  • a substance is supplied to prevent the transcriptional repressor protein repressing transcription, and thus transcription is permitted.
  • transcriptional regulator protein Any suitable transcriptional regulator protein may be used, preferably one that is activatable or deactivatable. It is preferred that an exogenous substance may be supplied to control the transcriptional regulator protein. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.
  • the pluripotent stem cell according to the invention is controlled by an exogenously supplied substance.
  • the exogenously supplied substance is selected from the group consisting of peptides (such as described by Klotzsche, et al; Journal of Biological Chemistry 280.26 (2005): 24591-24599 or Schlicht et al.; Applied and environmental microbiology 72.8 (2006): 5637- 5642) or the inducers described in Goeke, et al. Journal of molecular biology 416.1 (2012): 33-45; incorporated herein by reference), an aptamer (such as the RNA aptamer described in Hunsicker et al. “Chemistry & biology 16.2 (2009): 173-180; incorporated herein by reference), tetracycline, and anhydroteracyclin or a derivative thereof.
  • the exogenously supplied substance is doxycycline.
  • the transcriptional regulator protein as described herein is selected from the group consisting of tetracycline responsive transcriptional activator protein (rtTa), Tetracycline repressor (TetR), VgEcR synthetic receptor or a hybrid transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain from the human progesterone receptor or an activation domain from the human NF-kB.
  • rtTa tetracycline responsive transcriptional activator protein
  • TetR Tetracycline repressor
  • VgEcR synthetic receptor or a hybrid transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain from the human progesterone receptor or an activation domain from the human NF-kB.
  • Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression well known in the art where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline which is more stable).
  • the transcriptional activator protein is tetracycline - responsive transcriptional activator protein (rtTa) ora derivative thereof.
  • the rtTA protein is able to bind to DNA at specific TetO operator sequences.
  • Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response element (TRE).
  • TRE tetracycline response element
  • Tet-Off tetracycline or a derivative thereof binds rTA and deactivates the rTA, rendering it incapable of binding to TRE sequences, thereby preventing transcription of TRE- controlled genes.
  • the Tet-On system is composed of two components; (1) the constitutively expressed tetracycline - responsive transcriptional activator protein (rtTa) and the rtTa sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes.
  • the transcriptional regulator protein is rtTA.
  • the inducible promoter inserted into the at least one further GSH that is not the first GSH site includes the tetracycline response element (TRE).
  • the inducible promoter includes a Tet Responsive Element (TRE).
  • the expression construct that is inserted into the second genetic safe harbour site is a fusion protein that encodes both the MYOD protein and the PAX7 protein as described herein.
  • expression construct that is inserted into the second genetic safe harbour site encodes a MYOD protein a linker and a PAX7 protein, in preferred embodiments, the construct comprises or consists of SEQ ID NO: 8.
  • the MYOD and PAX7 may be arranged in either order, i.e. MYOD positioned 3’ to PAX7 or vice versa.
  • the linker sequence may be a cleavable linker. That is, the linker sequence may comprise a sequence of amino acids which is capable of being cleaved.
  • the linker sequence may comprise a sequence capable of acting as a substrate for an enzyme capable of cleaving peptide bonds--i.e. a cleavage site. Many such cleavage sites are known to and can be employed by the person skilled in the art of molecular biology.
  • the cleavable linker may comprise an autocleavage site. Autocleavage sites are automatically cleaved without the need for treatment with enzymes.
  • 2A self-cleaving peptides or 2A peptides have been described, which includes 2A peptides P2A, E2A, F2A, and T2A.
  • F2A is derived from foot-and-mouth disease virus
  • E2A is derived from equine rhinitis A virus
  • P2A is derived from porcine teschovirus-1 2A
  • T2A is derived from thosea asigna virus 2A.
  • the cleavable linker is thus selected from the group consisting of P2A, E2A, F2A, and T2A.
  • the expression construct comprises a Picornavirus 2A (P2A) linker.
  • the expression construct comprises a linker comprising or consisting of the sequence of SEQ ID NO: 6.
  • the expression construct that is inserted into the second genetic safe harbour site encoding a MYOD protein a linker and a PAX7 protein as described herein comprises or consists of the sequence of SEQ ID NO: 8.
  • the inducible promotor that is operably linked to the MYOD protein is different than the inducible promotor that is linked to the PAX7 protein. In certain embodiments, the inducible promotor that is operably linked to the MYOD protein is the same at the inducible promotor that is linked to the PAX7 protein.
  • Inducible promotors are well-known in the art, examples include but are not limited to CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
  • the inducible promotor used in the present invention a tetOn promotor.
  • a tetOn promotor Preferably a 3 rd generation TetOn promotor.
  • the invention relates to a method for the production of skeletal muscle cells, comprising a ) culturing the pluripotent stem cell as described herein in a proliferation medium: followed by b) inducing skeletal muscle cell differentiation by adding an exogenous substance as described herein.
  • the method of the invention is an ex vivo method.
  • the method is for the production of skeletal muscle cells.
  • Mature skeletal muscle cells are herein defined as skeletal muscle cells which show one or more of multinucleation, formation of proteins that are involved in sarcomere formation (eg. Titin and/or one or more myosin heavy chain isoforms) and ultimately contractile functionality.
  • the method of the invention relates to a method for production of type 1 or type 2 muscle.
  • the proliferation medium and the differentiation medium may have the same composition.
  • the same medium may be used for proliferation and differentiation. That is to say, the same culture system can be used for both proliferation and differentiation phases, for example via a medium change or by only adding an inducer of the opti-ox system within the bioreactor.
  • the addition of an inducer for differentiation to the bioreactor is very attractive, as it minimizes capital investment in equipment, processing times and cell manipulation.
  • proliferation and/or differentiation may be carried out in the absence of insulin and/or retinoic acid.
  • proliferation and/or differentiation may be carried out as a single cell suspension.
  • proliferation and/or differentiation may comprise aggregates.
  • the inventors have surprisingly found that use of the pluripotent cell as described herein obviates the need to culture the cells with a commitment induction step.
  • the commitment or determination phase involves the formation of myoblasts, which have lost the potential to differentiate into other cell types.
  • Differentiation of myoblasts to skeletal muscle cells is promoted by a highly regulated network of transcription factors chronologically expressed to promote skeletal muscle morphologic and biochemical features such as the fusion of single myoblasts into multinucleated skeletal muscle cells and the formation of the sarcomeres that require expression of muscle specific proteins that are highly abundant and can count up to 55% of protein content of a skeletal muscle cell ,.
  • the differentiation phase is also divided into four stages: growth arrest, mitotic clonal expansion, early differentiation, and terminal differentiation.
  • Use of the pluripotent stem cells as described herein in the method allows the differentiation of the pluripotent stem cells to mature skeletal muscle cells with a very short (2-4 days) myoblast like cell state and can efficiently differentiate into mature skeletal muscle cells .
  • This short state is particularly advantageous as it reduces the amount of compounds and small molecules that are normally required to be present in the myoblast proliferation medium.
  • the cells as described herein may be able to differentiate without the presence of fetal bovine serum (FBS), dexamethasone and EGF or FGF2 which are normally required for satisfactory differentiation.
  • FBS fetal bovine serum
  • dexamethasone dexamethasone
  • EGF or FGF2 which are normally required for satisfactory differentiation.
  • the ability to leave out these compounds from the differentiation medium reduces the medium costs and eases the way to regulatory acceptance.
  • the method as described herein reduces the differentiation time of the pluripotent cells as described herein to mature skeletal muscle cells dramatically.
  • the time to produce skeletal muscle cells using the method as claimed is at most 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days.
  • the inventors have observed a conversion rate of at least 95% by day 4 of culture, meaning that at least 95% of the cells are mature after 4 days of culture. Accordingly, in certain embodiments the time to produce at least 95% mature skeletal muscle cells is at most 4 days.
  • the invention provides for skeletal muscle cells, preferably mature skeletal muscle cells, as obtained by the method as described herein.
  • Culturing the cells as described herein can be performed under so called 2D culturing conditions, which is considered the conventional approach to culturing cells.
  • the method as described can also easily be adapted to allow culturing under 3D conditions as shown in the examples below.
  • 3D cell culture is an artificially-created environment which enables cells to grow or interact with their surroundings in three dimensions.
  • cells typically form 3D colonies, which may be referred to as "spheroids".
  • the 3D culture approach may more accurately model the cells' in vivo growth and behaviour.
  • the skilled person is readily able to carry out 3D cell culture, for example by taking advantage of any of a number of commercially-available culturing tools.
  • the 3D culture may be carried out using scaffold or scaffold-free techniques. Scaffoldbased techniques make use of supports such as solid scaffolds and hydrogels to enable the cells to form a 3D culture.
  • Such scaffolds may aim to mimic the natural extracellular matrix (ECM), which is present in vivo.
  • ECM extracellular matrix
  • 3D spheroids may be established through the use of, for example, low-adhesion plates, hanging-drop plates, micro-patterned surfaces, rotating bioreactors, magnetic levitation and magnetic 3D bioprinting.
  • the skeletal muscle cells that have been transduced with lentiviral vectors are not considered food safe or not safe for human and non-human dietary consumption.
  • the pluripotent cells as described herein of the method as described herein obviate the need to use lentivirally transduced cells.
  • the skeletal muscle cells that are produced according to the method as disclosed herein are suitable for human and non-human dietary consumption.
  • the produced skeletal muscle cells can be used in the production of cultured meat for human consumption.
  • Meat according to the invention may be derived from any non-human animal, including any species mentioned herein.
  • the invention provides for a use of a pluripotent stem cell as described herein or the skeletal muscle cells obtained by the method as described herein for tissue engineering.
  • the use is for the production of cultured meat.
  • the invention provides for a use of a pluripotent stem cell as described herein or the skeletal muscle cells obtained by the method as described herein for tissue engineering, for example in a method of treatment.
  • the invention provides for a food product (also referred to as “foodstuff’) comprising the pluripotent stem cells as described herein or the skeletal muscle cells produced and/or obtained by the method as described.
  • the food product further comprises an edible composition for human or non-human consumption.
  • the edible composition for human or non-human consumption for example comprises at least one of skeletal muscle cells, mature muscle cells, minerals, synthetic substances, flavoring substances (such as for examples herbs and spices), plant based proteins or proteins from microbial origin such as yeast proteins. Plant based proteins and yeast proteins suitable for the use in food products are known to the skilled person in the art.
  • the food product is cultured meat.
  • the invention provides for a method of producing a food product, the method comprising combining the pluripotent stem cells as described herein or the produced and/or obtained skeletal muscle cells with an edible composition for human consumption or non-human consumption as described herein.
  • the food product is cultured meat.
  • Figure 1 EpiSCs-MYOD1 (Left) and EplSCs-PAX7-MYOD1 (right) differentiated for 8 days in 2D monolayer culture. Cultures were stained with Titin and nuclei were visualized with DAPI.
  • Figure 2 Monolayer differentiation of EpiSCs-PAX7-MyoD1 and zoom in on skeletal muscle cell showing extensive pattern of striation indicating full differentiation into functional skeletal muscle cells. Cells were labeled after 8 days of differentiation with anti-titin antibody and nuclei were visualized with DAPI.
  • Figure 3 Comparison of doxycycline duration on differentiation capacity of EpiSCs-PAX7-MYOD1 with 4 days (left) and 2 days (right) with a total culture time of 6 days . Cultures were stained for Titin and nuclei visualized with DAPI.
  • Figure 4 3D suspension differentiation after 4 days of doxycycline addition. Aggregates were stained for Pax7 and nuclei were visualized with DAPI.
  • Figure 5 Comparison between EpiSCs-MyoD1 (left) and EpiSCs-Pax7-MyoD1 (right) after 8 days of 3D suspension differentiation. Aggregates were fixated and labeled with anti-Titin antibody and with DAPI nucleus staining.
  • Figure 6 Comparison between EpiSCs-MyoD1 (left) and EpiSCs-Pax7-MyoD1 (right) after 8 days of 3D suspension differentiation. Zoom in of Figure 5. Arrows indicate sarcomere formation.
  • FIG. 7 EpiSCs-PAX7 (A) and EplSCs-PAX7-MYOD1 (B) differentiated for 18 and 8 days in 2D monolayer culture. Cultures were stained with Titin and nuclei were visualized with DAPI.
  • Figure 8 Comparison between EpiSCs-PAX7 (left) and EpiSCs-Pax7-MyoD1 (right) after 18 or 8 days of 3D suspension differentiation. Aggregates were fixed and labeled with anti-Titin antibody and with DAPI nucleus staining.
  • Porcine Epiblast-derived Stem Cells (pEplSCs) differentiation to skeletal muscle cells.
  • Undifferentiated pEplSCs (Opti-Ox MyoD1 and Opti-Ox Pax7-MyoD1) were grown in N2B27 proliferation media (50% DMEM Ham’s F-12 (L0093-500, Biowest), 50% Neurobasal media (21103049, Thermofisher), B27 supplement (17504044, Thermofisher), N2 supplement (17502001 , Thermofisher), glutamax (35050061 , Thermofisher), 10 mM 2-Mercaptoethanol (31350010, Thermofisher), 0,02pg/mL Activin A (QK001 , Q-kine), 0,10 pg/mL FGF2 (QK002, Q-kine), 0,625 pg/mL XAV939 (X3004, Sigma Aldrich) on hESC-qualified geltrex (A1413301 .Thermo Scientific) - coated plates.
  • N2B27 proliferation media 50% DMEM Ham’s
  • Single cells were obtained after gentle cell dissociation reagent (07174, STEMCELL technologies) treatment and cell number and viability were assessed.
  • Single pEplSCs were seeded in the corresponding cell culture plates at a density of 100000-200000 cells/cm2.
  • undifferentiated single-pEplSCs (Opti-Ox MyoD1 and Opti-Ox Pax7-MyoD1) were seeded at 3 million cells/mL in 150 mL shaker flasks containing 12.5 mL of proliferation media (containing 50% DMEM Ham’s F-12 (L0093-500, Biowest), 50% Neurobasal media (21103049, Thermofisher), B27 supplement (17504044, Thermofisher), N2 supplement (17502001 , Thermofisher), glutamax (35050061 , Thermofisher), 10 mM 2-Mercaptoethanol (31350010, Thermofisher) , 0,02pg/mL Activin A (QK001 , Q-kine), 0,10 pg/mL FGF2 (QK002, Q-kine), 0,625 pg/mL XAV939 (X3004, Sigma Aldrich), 2X %
  • MYOD and PAX7 were selected for use in a combined cellular reprogramming strategy.
  • 2A peptide linkers are well-characterized short peptide linkers of 18-22 amino acids that result in two separate gene products expressed from a single open reading frame due to ribosome skipping during translation.
  • Stable knock-ins in the AAVS1 GSH were selected by incorporation of a puromycin-resistance cassette and selected with puromycin addition to the cell culture medium.
  • Example 2 EpiSCs-MYOD-PAX72D differentiation into multinucleated skeletal muscle cells pEpiSCs Opti-Ox MYOD and pEpiSCs Opti-Ox PAX7-MYOD were expanded in 3D suspension culture, single celled and seeded in coated plastic wells with 200k cells/cm 2 . One day after attachment doxycycline was added to medium to start the differentiation. Doxycycline was removed on day 4 in the pEpiSCs Opti-Ox PAX7-MYOD culture.
  • Example 3 3D culture of EpiSCs-MYOD and EpiSCs-MYOD-PAX7
  • shakers and/or bioreactors are often used to scale up to large volumes and generate the amounts of cell mass needed for a cultivated meat product at a cost competitive price.
  • primary cells are used derived from muscle biopsies.
  • primary cultures have limited self-renewal, lose their capacity to differentiate during expansion and are variable in quality between biopsies.
  • Other sources such as ESCs and iPSCs have high expansion capacity but are in an undifferentiated state and differentiation is induced either using small molecules to mimic embryonic development (review Chai et al., 2017 Jun 15;144(12):2104-2122.
  • the differentiation of cells into myotubes and subsequently into myofibers is an integral part of the process, which usually occurs in a subsequent separate step.
  • Differentiation of skeletal muscle cells requires often distinct conditions in terms of nutrients and physical environment. The necessary nutrients can be provided by switching from a nutrient rich proliferation medium to a differentiation medium consisting of low serum, but providing the physical environment that the cells require in order to differentiate is more challenging.
  • the substrate requirements for the proliferation and differentiation phases are typically different in terms of surface chemistry and topography.
  • a particular challenge for skeletal muscle cells in suspension is that skeletal muscle cells need attachment sides to poles, a scaffold, or a surface to form elongated stretched multinucleated myotubes.
  • EpiSCs-MYOD and EpiSCs-PAX7-MYOD were adapted to 3D suspension cell growth and grown as aggregates; EpiSCs expanded in an adherent 6-well cell plate were single celled with Accumax (00-4666-56, ThermoFisher Scientific) according to the manufacturer’s instructions and transferred to a 150mL shaker flask in 12,5mL media and a RHO/ROCK pathway inhibitor and thereafter expanded for at least 3 cycles. Media during expansion in 3D was refreshed daily. 3D adapted EpiSCs were subsequently used in shaker and bioreactor experiments.
  • Example 4 Comparison of differentiation speed of EpiSCs-PAX7 and EpiSCs-PAX7-MYOD pEplSCs
  • Opti-Ox PAX7 were cultured in suspension for 2 days in Pax7 induction medium (DMEM/F12, 1x ITS and 15% KSR) containing 3 uM CHIR99021 , then switched to Pax7 induction medium with 1 ug/ml DOX and 20 ng/ml FGF2 for 10 days. After 10 days cells were either directly differentiated to skeletal muscle cells in aggregates or with single cells plated on geltrex coated dishes.
  • Pax7 induction medium DMEM/F12, 1x ITS and 15% KSR
  • PAX7-MYOD cells were first induced for 4 days with PAX7/MYOD medium containing DMEM/HG, 1x ITS, 15% KSR and 1 ug/ml DOX. On day 4 of culture, DOX was withdrawn and cells were switched to PAX7/MYOD differentiation medium (DMEM HG, 1x ITS and 1 % KSR). Differentiation of cells was measured on for Pax7 cells on day 18 as a shorter differentiation time did not result in visibly differentiated cells and differentiation of the Pax7-MyoD1 cells was measured on day 8, ( Figure 7).
  • Pax7-MyoD1 cells showed the formation of titin positive skeletal muscle cells and even thicker skeletal muscle cells (Figure 7A) than those observed after 18 days of differentiation with the Pax7 cells.
  • Pax7-MyoD1 cells further displayed strong formation of sarcomeric structures as visualized in Figure 7B. Even after 18 days differentiation, the difference in differentiation is still noticeable as the EpiSCs-PAX7 still did not reach the same thickness and sarcomeric structures.
  • aggregates containing EpiSCs-PAX7-MYOD showed much bigger and longer skeletal muscle cells that included many nuclei ( Figure 8).

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Abstract

La présente invention concerne une cellule souche pluripotente comprenant une construction d'expression pour l'expression d'une protéine MYOD et une construction d'expression pour l'expression d'une protéine PAX7. L'invention concerne en outre des procédés de production de cellules musculaires squelettiques comprenant les cellules souches pluripotentes et pour des produits alimentaires comprenant les cellules musculaires squelettiques ou les cellules souches pluripotentes.
PCT/EP2024/053906 2023-02-15 2024-02-15 Maturation de cellules musculaires squelettiques Ceased WO2024170702A1 (fr)

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WO2025133344A1 (fr) 2023-12-21 2025-06-26 Meatable B.V. Procédés de culture de cellules souches pluripotentes par perfusion
WO2025141173A1 (fr) 2023-12-28 2025-07-03 Meatable B.V. Procédés d'augmentation de la différenciation et/ou de l'accumulation de graisse dans des cellules
WO2025141211A1 (fr) 2023-12-29 2025-07-03 Meatable B.V. Cellules pour programmation cellulaire
WO2025141210A1 (fr) 2023-12-29 2025-07-03 Meatable B.V. Procédé de production de cellules grasses cultivées
WO2025181376A1 (fr) 2024-03-01 2025-09-04 Meatable B.V. Produits cellulaires cultivés
WO2025181375A1 (fr) 2024-03-01 2025-09-04 Meatable B.V. Produits cellulaires cultivés

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