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WO2023233340A1 - Cellules de levure génétiquement modifiées, protéines recombinées produites à partir de celles-ci et leurs applications - Google Patents

Cellules de levure génétiquement modifiées, protéines recombinées produites à partir de celles-ci et leurs applications Download PDF

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WO2023233340A1
WO2023233340A1 PCT/IB2023/055617 IB2023055617W WO2023233340A1 WO 2023233340 A1 WO2023233340 A1 WO 2023233340A1 IB 2023055617 W IB2023055617 W IB 2023055617W WO 2023233340 A1 WO2023233340 A1 WO 2023233340A1
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Prior art keywords
cells
growth factor
recombinant
cell
igf
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Inventor
Po San Mario CHIN
Kai Yi Carrie CHAN
Chun Hei POON
Lok Hin WONG
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Avant Meats Co Ltd
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Avant Meats Co Ltd
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Priority claimed from US17/831,351 external-priority patent/US20220298480A1/en
Application filed by Avant Meats Co Ltd filed Critical Avant Meats Co Ltd
Priority to EP23815409.0A priority Critical patent/EP4532532A1/fr
Priority to CN202380043076.XA priority patent/CN119278210A/zh
Publication of WO2023233340A1 publication Critical patent/WO2023233340A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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

Definitions

  • Embodiments discussed herein generally relate to genetically engineered strains of yeast and improved methods for producing recombinant proteins, cell products or cultivated meat. Embodiments discussed herein also generally relate to the improved methods for cell growth and protein expression using growth factors.
  • Animal meat is high in protein, and supplies all the amino acids needed to build the protein used to support body functions. Meat for consumption is traditionally obtained from animals or fish that are reared on farms. However, agriculture and aquaculture for producing animal meat require a large amount of energy and resources and have a high carbon footprint. Meat produced by agriculture or aquaculture may pose a public health risk as the production processes may expose the meat to diseases, pollutants, and toxins. A number of concerns such as a growing population, increasing demand for meat, environmental concerns, limited land and water resources, biodiversity loss, and the negative perception associated with animal slaughter have led scientists to develop techniques to produce meat by alternative processes.
  • in vitro meat production is the process by which muscle tissue or organ tissue from animals is grown in laboratories using cell culture techniques to manufacture meat and meat products. Apart from food products such as meat, in vitro cell culture can also be employed to produce active ingredients of various products or the products themselves (including but not limited to dietary supplements, hair care, skincare, wound care, cosmetic products, supplements, drugs and other medicinal applications).
  • in vitro meat and meat products includes animal protein products as well as non-meat products including soluble forms and solid forms. While still in an early stage of development, in vitro meat and meat products may offer a number of advantages over traditional meat products such as health and environmental advantages and benefits to animal welfare. It is a next-generation and emerging technology that operates as part of a wider field of cellular agriculture, or the production of agricultural products from cell cultures.
  • Cells for the production of in vitro meat may be cells (e.g., muscle cells, somatic cells, stem cells, etc.) taken from animal biopsies, which may then be grown separately from the animal in culture media in a bioreactor or other type of sterile environment.
  • the cells may grow into a semi-solid or solid form mimicking an animal organ by attaching to an edible three-dimensional scaffold that is placed in the bioreactor.
  • the starter cells may be primary cells directly obtained from the animal’s tissues, or continuous cell lines. If grown under the right conditions in appropriate culture media, primary cells will grow and proliferate, but only a finite number of times that is related to the telomere length at the end of the cell’s DNA.
  • Continuous cell lines can be cultured in vitro over an extended period.
  • Cell biology research has established procedures on how to convert primary cells into immortal continuous cell lines.
  • Primary cells may be transformed into continuous cell lines using viral oncogenes, chemical treatments, or overexpression of telomerase reverse transcriptase to prevent the telomeres from shortening.
  • the culture media may contain components necessary for cell proliferation such as amino acids, salts, vitamins, growth factors, and buffering systems to control pH.
  • Current methods usually add exogenous animal products such as fetal bovine serum (FBS) or recombinant mammalian growth factors or cytokines to the media prior to use to provide vital macromolecules, growth factors, and immune molecules to maintain cell proliferation and increase cellular yields.
  • FBS fetal bovine serum
  • cytokines recombinant mammalian growth factors or cytokines
  • IGF-1 insulin growth factor 1
  • HSA human serum albumin
  • FBS fetal bovine serum
  • mammalian growth factors to enhance the growth of non-mammalian cells are ineffective as the mammalian growth factors may not bind to or act poorly on cellular receptors on non-mammalian cells; product manufactured in this manner may also raise consumer concerns about safety and potential allergic reactions.
  • the present invention provides novel yeast strains that can produce recombinant native or modified growth factors for enhancing cell growth.
  • recombinant growth factors are introduced to the culture medium of the cells to be cultured, thereby increasing the rate of growth or protein expression of the cultured cells.
  • the recombinant growth factor to be supplemented to the cultured cells is of similar species origin as the cultured cell type and/or with its amino acid sequence modified for improved bioactivity and stability profiles as compared to the native sequence.
  • the present invention significantly reduces the amount of growth factors required to be supplemented and risks imposed on consumers.
  • the present invention does not require a complex setup and products produced using the present invention can be produced on a large-scale basis and readily used in most applications without extra cumbersome purification procedures.
  • the present yeast strains could be used to produce both mammalian and non-mammalian growth factors of various kinds and are therefore versatile in their applications.
  • the present invention provides novel yeast strains and improved methods for producing recombinant proteins that promote cell growth in an in vitro culture and enable the production of in vitro meat products and other types of cell products at lowered costs and in a simplified manner.
  • the resulting products are compatible with various applications including cellular agriculture, cosmetics, pharmaceuticals and biotechnology.
  • inventions of the present disclosure provide genetically engineered strains of yeast and improved methods for increasing cell growth and protein expression in an in vitro cell culture.
  • recombinant growth factors are introduced to the culture medium of the cells to be cultured to increase the rate of growth or protein expression of the cultured cells.
  • cell culture users can use the present invention to generate different constructs of growth factors and evaluate their efficacies and choose the constructs that the concerned cell types respond maximally to for a particular application.
  • cell products produced using the present inventions are of a purity that is generally acceptable for most types of applications and can be readily used without complex purification procedures.
  • the resulting in vitro cell products may be further purified using conventional purification techniques.
  • the application of the present invention not only provides flexibility in its application but also significantly reduces production costs for large-scale cultures.
  • a genetically modified yeast cell encoding a recombinant growth factor is provided.
  • the recombinant growth factor can be of a native amino acid sequence or a modified amino acid sequence with improved stability and/or bioactivity as compared to the native form.
  • a method for increasing the rate of growth or protein expression of a plurality of cells in an in vitro cell culture comprises the steps of growing the plurality of cells in a nutrient medium under growth conditions suitable for initiation of cell division and introducing at least one recombinant growth factor to the nutrient medium.
  • the recombinant growth factor is (a) optimized for increasing the rate of growth or protein expression of the plurality of cells or (b) (i) of genetically same or similar species to the plurality of cells and/or (ii) of genetically same genus to the plurality of cells.
  • a method for meat production by in vitro cell culture includes isolating tissue from an animal or plant source and making a cell suspension of cells is provided.
  • the method further includes introducing growth factors that are (a) optimized for increasing the rate of growth or protein expression of the plurality of cells or (b) (i) of genetically same or similar species to the cells and/or (ii) of the genetically same genus to the cells.
  • the method further includes growing the cells on a food-grade scaffold in a culture medium, the cells growing into a solid or semi-solid structure that mimics an animal organ.
  • the growth factor that supports cell growth can be produced from other microorganisms such as bacteria or fungi containing and expressing a transgene of the growth factor.
  • the recombinant growth factor to be introduced to an in vitro culture is produced by a genetically modified yeast cell comprising a gene of the recombinant growth factor and obtained from a cell culture medium growing the genetically modified yeast cell.
  • the recombinant growth factor is purified from the yeast cell culture medium before it is introduced to the in vitro culture.
  • the recombinant growth factor is introduced to the in vitro culture by introducing the yeast cell culture medium containing the growth factor to the in vitro culture.
  • aspects of the invention may genetically engineer a yeast clone that contains a IGF-1 gene of a specific species such as a fish IFGF-1 gene.
  • the recombinant yeasts may be grown in a yeast culture medium to allow protein expression of the IGF-1 gene.
  • the IGF-1 may be secreted into the yeast culture medium.
  • the IGF-1 - containing yeast culture medium may be centrifuged at about 1500 g for 5 minutes to separate the yeast from the medium. The supernatant may be collected and centrifuged again at about 7200 g for about 15 minutes at about 4°C. The clarified supernatant may then be filtered with a 2 pm or smaller pore-size filter.
  • the filtered IGF-1 -containing yeast culture medium may be diluted up to 10,000-fold before adding to the cell culture medium to grow fish cells or other types of cells.
  • FIG. 1 is a flowchart of a method for meat production by in vitro cell culture, according to one embodiment of the present disclosure.
  • FIG. 2 is a chart illustrating the respective cell numbers after treating MCF-7 cells with different concentrations (1 pg/ml to 100 ng/ml) of recombinant human IGF-1 (Oryzogen). Cells were harvested on day 10 for direct cell counting.
  • FIG. 3 is a chart illustrating the respective relative fluorescence after the treatment of MCF-7 cells with 15 nM of recombinant human IGF-1 (from 3 different suppliers), recombinant mouse IGF-1 , and recombinant fish (tuna, bream) IGF-1 . Cells were harvested on day 7 and subjected to CyQUANT Cell Proliferation Assay.
  • FIG. 4 is a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells with 10 nM of three different clones of recombinant fish IGF-1 (Fish IGF-1 (A), IGF-1 (B) and IGF-1 (C)). Cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.
  • FIG. 5 is a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells with 1 % of three different recombinant fish IGF-1 -containing yeast culture medium. Cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.
  • FIG. 6 is the Western blot analysis of recombinant native and modified grouper IGF-1 in yeast culture using anti-6X His antibody.
  • FIG. 7 is the characterization of recombinant native and modified grouper IGF-1 purified from yeast culture using immobilized metal affinity chromatography.
  • Panel (a) shows an analysis by total protein staining and panel (b) shows an analysis by Western blotting using anti-6X His antibody.
  • FIG. 8 shows two charts illustrating the results of the resazurin assay on (a) MCF-7 cells and (b) FHM cells treated with purified grouper IGF-1 at 0 nM, 0.1 nM, 1 nM and 10 nM.
  • FIG. 9 shows two charts illustrating the results of the resazurin assay on (a) MCF-7 cells and (b) FHM cells treated with grouper IGF-l-containing yeast culture at 1 : 100 and 1 : 1000 dilutions.
  • in vitro meat production refers to a cell-based meat production process or cell-based agriculture process in which tissues from animals and/or plants are grown in laboratories using cell culture techniques to manufacture meat and meat products.
  • tissue from an animal or a plant is isolated.
  • the tissue is derived from bony fish of the class Osteichthyes including saltwater fish such as a grouper, sea bass, or yellow cocker.
  • other types of animal tissue such as cow tissue, may be isolated.
  • the block 12 may involve collecting organ tissue, such as a swim bladder, from a fish and making a cell suspension.
  • tissue derived from fish sources it will be understood that the concepts may be applied to tissues derived from other types of animal sources and/or plant sources to provide other types of in vitro meat and/or animal protein products, and vegetarian meat and/or protein products.
  • Many of the isolated cells are adult cells and can be made to proliferate continuously using various established methods in medical research (block 14). For example, specific genes, such as Yamanaka factors, may be used to reprogram adult cells into stem cells, such as induced pluripotent stem cells (iPSCs).
  • stem cells such as induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the isolated adult cells may be transformed into continuous cell lines by telomerase reverse transcriptase overexpression.
  • other types of cells may be isolated such as adult stem cells and embryonic stem cells.
  • the methods of the present disclosure include all sources of cell lines.
  • the cells are grown into a solid or semi-solid structure mimicking an animal organ, such as a fish organ, by attaching/adhering to a food-grade biocompatible scaffold in a sterile chamber or container, such as a bioreactor.
  • a sterile chamber or container may be temperature controlled and may have inlets and outlets for introducing and removing substances such as chemicals, nutrients, and cells.
  • the food-grade biocompatible scaffold becomes part of the final edible product, and is made of plant-based or fungi-based materials such as, but not limited to, agarose, alginate, chitosan, mycelium, and konjac glucomannan.
  • Alginate is a biopolymer naturally derived from brown algae and is biocompatible.
  • plant-based chitosan from fungi has antibacterial properties.
  • the block 16 is carried out in the absence of antibiotics or antimicrobial compounds in the sterile container.
  • a block 18 involves supplying the culture medium to the bioreactor to support cell survival and growth.
  • the culture medium may be a buffered solution containing components such as, but not limited to, inorganic salts (e.g., calcium chloride (CaCl2), potassium chloride (KCI), sodium chloride (NaCI), sodium bicarbonate (NaHCOs), sodium dihydrogen phosphate (NaH2PO4), magnesium sulfate (MgSO4), etc.), amino acids, vitamins (e.g., thiamine, riboflavin, folic acid, etc.), and other components such as glucose, 0- mercaptoethanol, ethylenediaminetetraacetic acid (EDTA), and sodium pyruvate.
  • inorganic salts e.g., calcium chloride (CaCl2), potassium chloride (KCI), sodium chloride (NaCI), sodium bicarbonate (NaHCOs), sodium dihydrogen phosphate (NaH2PO4), magnesium sulfate (MgSO4), etc.
  • amino acids e.g.,
  • Non-limiting examples of growth media include, but are not limited to, Leibovitz’s L-15 medium, Eagle’s Minimum Essential Media (MEM), Medium 199, Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F12 Nutrient Mix, Ham’s F10 Nutrient Mix, MacCoy’s 5A Medium, Glasgow Modified Eagle Medium (GMEM), Iscove’s Modified Dulbecco’s Medium, and RPMI 1640.
  • growth factors and cytokines are introduced into the culture medium in the bioreactor to support cell growth and proliferation.
  • the growth factors and cytokines may include, but are not limited to, insulin growth factor 1 (IGF-1 ), insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11 ), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transferrin.
  • IGF-1 insulin growth factor 1
  • IL-6 interleukin 6
  • IL-6R interleukin 6 receptor
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • transferrin transferrin.
  • growth factors of (i) genetically the same or similar species to the isolated/cultured cells and/or (ii) genetically the same genus to the isolated cells exert higher bioactivities to the isolated cells compared to the use of growth factors and serum of genetically distant species to the isolated cells. Due to the enhanced compatibility, there is no need to supply a megadose of “suboptimal” growth factors when culturing these cells using growth factors of (i) genetically same or similar species and/or (ii) genetically same genus. Higher bioactivity could also help reduce the amount of growth factors needed in the culture medium, shortening the culture period and improving cell quality. The cost of the culture medium could be reduced due to the decrease in the levels of growth factors required in the culture medium for the stimulation of cell growth and differentiation.
  • suboptimal growth factors limits the magnitude of the maximum cellular response (growth, differentiation). In some instances, certain responses can never be reached no matter how much of the suboptimal growth factor is supplied. Species-specific and/or genus-specific growth factors could help overcome these limits.
  • Species-specific or genus-specific growth factors can effectively act on the receptors of the isolated cells. Compared to the conventional growth medium, which is often supplemented by high levels of human protein growth factors and/or FBS irrespective of the species origin of the isolated cell, the use of species-specific or genus-specific growth factors is better optimized. Species-specific or genus-specific variations in amino acid sequence and post-translational modifications of the growth factor(s) and cell receptor(s) may account for this phenomenon.
  • target cells are first seeded in a complete medium (i.e. basal medium + FBS). Upon reaching the target confluence (around 20%-70%), target cells are treated by the growth factor of different species at a range of concentrations (e.g. 1 pM-1 pM). Target cells are kept in the incubator until reaching the desired time point(s) for the studied parameter (e.g. cell growth, differentiation markers, cellular products).
  • a complete medium i.e. basal medium + FBS.
  • target cells are treated by the growth factor of different species at a range of concentrations (e.g. 1 pM-1 pM).
  • Target cells are kept in the incubator until reaching the desired time point(s) for the studied parameter (e.g. cell growth, differentiation markers, cellular products).
  • cell growth can be measured by trypan blue exclusion, the CyQUANT assay, or any other appropriate cell proliferation/death assays.
  • the bioactivities of the growth factors of various species are compared based on their EC50 values (half-maximal effective concentration). For cost-effectiveness, target cells should be cultured using the growth factor with the lowest EC50 value. However, if the aim is to attain the shortest culturing time or the highest cell quality, the growth factor which triggers the highest maximum cellular response is preferably selected.
  • the optimal dose of the growth factor is defined as the lowest concentration required to elicit the maximum cellular response.
  • the block 20 may involve co-culturing bioengineered cells with isolated/cultured cells in the absence of fetal bovine serum (FBS).
  • the bioengineered cells are engineered to secrete the above growth factors and cytokines and supply these biomolecules to the isolated cells as needed for growth and proliferation.
  • “bioengineered” cells are not equivalent to genetically-modified cells.
  • the bioengineered cells have a specific gene that overexpresses one or more specific proteins.
  • the bioengineered cells may be fish cells, or other types of animal cells, such as cow cells.
  • the bioengineered cells and the isolated cells may be genetically similar or identical species. Also, the bioengineered cells and the isolated cells may belong to the same genus.
  • bioengineered fish cells may be co-cultured with isolated fish cells, or bioengineered cow cells may be co-cultured with isolated cow cells.
  • the bioengineered cells may be chicken cells or bird cells if chicken cells are used as the isolated cells.
  • the bioengineered cells may be yellow crocker cells or other fish cells if yellow cracker cells are used as the isolated cells. The bioengineered cells are not present in the final meat product.
  • the co-culturing method of the present disclosure eliminates the need for animal-derived fetal bovine serum (FBS) in the culture medium.
  • FBS animal-derived fetal bovine serum
  • the co-culturing method provides a continuous supply of food-grade specific growth factors and cytokines to the growing isolated cells in situ, and simplifies and reduces the cost of the production process, wherein the growth factors are (i) of genetically same or similar species to the isolated cells and/or (ii) of the same genus to the isolated cells.
  • the growth factors are (i) of genetically same or similar species to the isolated cells and/or (ii) of the same genus to the isolated cells.
  • FBS or other serum or other products containing recombinant growth factors may be used to supply growth factors, cytokines, and other nutrients to support cell growth during the block 16.
  • the block 20 contains recombinant growth factors of genetically the same or similar species to the isolated cells.
  • the recombinant growth factors of the same genus to the isolated cells are used.
  • the recombinant growth factors are introduced into the growth medium in purified form or in a crudely purified form such as a yeast culture (or the supernatant of the yeast culture) obtained from a culture of recombinant yeast cells expressing the recombinant growth factors. The use of such recombinant growth factors exerts higher bioactivities on the isolated cells than growth factors and serum of distant species on the isolated cells.
  • Protein purification is performed by (but not limited to) affinity chromatography, ion-exchange chromatography, size exclusion chromatography, or a combination of these strategies.
  • the recombinant growth factors are recombinant fish growth factors that may be used to support the growth of fish cells.
  • recombinant growth factors of Epinephelus akaara fish
  • the recombinant growth factors for culturing Epinephelus awoara used are Epinephelus akaara’s IGF-1 , insulin and/or transferrin.
  • the concentration of such IGF-1 is ranged from 10ng/ml to 100ng/ml.
  • the concentration of such insulin is ranged from 1 pg/ml - 10pg/ml.
  • the concentration of such transferrin is ranged from 0.5pg/ml - 5pg/ml.
  • the recombinant fish growth factors are used to support or promote the growth of non-fish cells such as human cells or other mammalian cells. In yet another embodiments, the recombinant fish growth factors are used to support or promote the growth of non-mammalian cells other than fish cells.
  • the recombinant growth factors are recombinant bovine growth factors that may be used to support the growth of bovine cells, other mammalian cells or non-mammalian cells.
  • the recombinant growth factors are recombinant avian growth factors that may be used to support the growth of avian cells, other non-mammalian cells or mammalian cells.
  • biomass yield refers to the amount of digestible material (e.g., proteins) in the resulting meat product that is available for energy production upon consumption.
  • the block 22 may involve increasing protein expression by altering micro RNA levels in the cells, with the manipulation of the cells being carried out prior to culturing. Micro RNAs are endogenous, short, non-encoding single-stranded RNA sequences involved in regulating post-transcriptional gene expression.
  • the block 22 may optionally involve increasing the amount of up-regulating micro RNAs that increase protein expression by promoting messenger RNA (mRNA) translation, and/or decreasing the amount of down-regulating micro RNAs that decrease protein expression by suppressing mRNA translation.
  • the micro RNA levels may be increased or decreased by introducing micro RNAs, micro RNA mimics, or micro RNA inhibitors into the cells.
  • the micro RNA mimics have the same function as micro RNAs, but may be more stable and efficient in modulating protein expression.
  • electroporation may be used to introduce episomal vectors into the cells that carry instructions to express specific micro RNAs.
  • an adeno-associated virus may be used as a vehicle carrying episomal instructions to express specific micro RNAs.
  • Decreasing the amount of targeted down-regulating micro RNAs may be achieved by introducing inhibitors for the targeted micro RNAs into the cells by transfection. It is noted here that the methods of increasing protein expression/biomass yield according to the present disclosure is carried out without modifying the genome of the cells.
  • Example 1 Identification Of species-specific or genus-specific growth factor
  • This example describes a method of identifying species-specific or genus-specific growth factors. It includes two major steps, which are the cell growth stimulation step and the measuring cell growth step.
  • Step 1 Cell growth stimulation step
  • the MCF-7 human epithelial cell line is cultured in DMEM/F12 complete medium DMEM/F12 , 10% FBS inside a humidified incubator (34°C; 5% CO2; 95% air). Split cells at a ratio of 1 :4 to 1 :8 for routine maintenance.
  • [0053] Prepare growth factors (e.g. IGF-1 ) at 10x working concentrations in serum-free medium for each species/genius, which growth-stimulating effect to be examined (e.g. recombinant human IGF-1 , human IGF-1 -LR3, mouse IGF-1 , bream IGF-1 and tuna IGF-1 ). Add the 10x growth factors into the wells such that cells will be treated by 1x growth factors (e.g. add 50 pl 10x growth factor to a well containing 450 pl serum-free medium) (e.g. 1 pM-1 pM). Return the cells to the incubator.
  • 1x growth factors e.g. add 50 pl 10x growth factor to a well containing 450 pl serum-free medium
  • 1 pM-1 pM e.g. 1 pM-1 pM
  • Step 2 Measuring cell growth step
  • Cells should have been treated in 24-well plates. Aspirate the culture medium and detach cells by trypsin-EDTA.
  • the CyQUANT Cell Proliferation Assay Kit quantifies cell growth by measuring the nucleic acid content in samples. Cells should have been seeded onto 96-well plates, preferably in triplicate wells per treatment group.
  • IGF-1 stimulates the growth of human MCF-7 cells
  • cells were treated with increasing doses of human IGF-1 (0 pg/ml - 100 ng/ml) for 10 days and processed for cell counting.
  • IGF-1 concentration (1-100 ng/ml) promoted cell growth in a dose-dependent manner.
  • MCF-7 cells are suitable for evaluating the growth-stimulating activity of IGF-1 .
  • Example 2 Human IGF-1, but not the mouse or fish IGF-1, promoted the growth of human MCF-7 cells
  • Example 3 Fish IGF-1 expressed by recombinant yeast cells promoted the growth of fish swim bladder cells
  • FIG. 4 shows a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells by 10 nM of three different clones of recombinant fish IGF-1 (Fish IGF-1 (A), Fish IGF-1 (B) and Fish IGF-1 (C)), each of which has a different nucleotide sequence from each other and the native gene of fish IGF-1 .
  • the cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.
  • FIG. 5 is a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells by 1% (v/v) of three different batches of yeast culture medium, each of which contains exclusively one of the clones of recombinant fish IGF-1 as mentioned in the preceding paragraph (i.e., (Fish IGF-1 (A), Fish IGF-1 (B) and Fish IGF-1 (C)).
  • the three batches of IGF-1 -containing yeast medium were collected, centrifuged and filtered, and the resulting supernatants were added directly to the fish swim bladder cells.
  • the cultured cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.
  • FIGS. 4 and 5 suggest that the addition of fish IFG-1 obtained from recombinant yeast culture to the culture medium of fish swim bladder cells enhanced the growth of the swim bladder cells.
  • other growth factors and cytokines can also be used in connection with these embodiments and other embodiments of this invention, including but not limited to insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11 ), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transferrin, and the resulting recombinant growth factors or cytokines, no matter in purified form or in a mixture form with the yeast medium culture, can be used to supplement cell culture medium for enhancing the growth of various types of cells.
  • IL-6 interleukin 6
  • IL-6R interleukin 6 receptor
  • IL-11 interleukin 11
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • transferrin transferrin
  • growth factors of the same or different origin of the cultured cells can be used, and native or modified growth factors can also be used in order to achieve maximal responsiveness in the cell types in question.
  • the cultured cells to be treated with such exogenous growth factors or cytokines are not limited to fish cells and can be non-fish cells including nonmammalian cells and mammalian cells and that the gene of the growth factors or cytokines to be introduced into the recombinant yeast cells as an expression system by way of genetic engineering shall be preferably (i) of genetically the same or similar species to the target cells and/or (ii) of genetically the same genus to the target cells.
  • the concentration of recombinant growth factors or cytokines to be added to the culture medium of the target isolated cells are in the range of 0.1 %-1 % (v/v) or 1 nM-10nM.
  • the present invention shows that it is more effective to apply growth factors of (i) genetically the same or similar species or (ii) genetically the same genus as the cultured cell type. The usage of these growth factors or protein factors may be decreased while achieving the same growth rate.
  • Species-specific growth factors and/or genus-specific growth factors represent a promising direction to reduce media cost especially during large-scale cell production for cultivated meat and other applications using the cultivated cell mass. Using more bioactive growth factors can also decrease processing times and improve the quality (e.g. texture, taste, nutritional value) of cultivated meat or cell mass.
  • Grouper IGF-1 genes having the nucleotide sequences set forth in SEQ ID NOs. 1 -5 encoding the amino acid sequences set forth in SEQ ID NOs. 6-10 were cloned into a PichiaPinkTM vector (ThermoFisher) respectively according to the prescribed protocol, resulting in a clone of grouper IGF-1 gene having the native grouper IGF-1 gene sequence and four different clones of modified grouper IGF-1 gene. Details of the clones are summarized in the following Table 1 .
  • the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant IGF-1 selected from the group consisting of SEQ ID NOs. 1 -5.
  • the resulting recombinant IGF-1 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.
  • polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 6-10.
  • polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 6-10.
  • polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 6-10.
  • Example 5 Expression of recombinant fish IGF-1 protein from PichiaPinkTM strains
  • Recombinant yeast cells for expressing recombinant fish IGF-1 protein and the pilot expression of the recombinant fish IGF-1 protein in the resulting recombinant yeast cells were carried out and analyzed as follows:
  • the resulting fish IGF- 1 protein was collected together with the yeast culture medium by the following steps to obtain a IGF-1 containing yeast culture medium:
  • grouper IGF-1 in yeast culture was confirmed by Western blotting.
  • 20 uL of yeast culture medium was harvested and analyzed by Western blotting.
  • expression of recombinant IGF-1 proteins at ⁇ 10 kDa in the present recombinant yeast strain was detected and confirmed by Western blotting using anti-6X His antibody.
  • a band between 35 kDa and 55 kDa was likely to be the IGF-1 multimer as it could be detected with anti-6X His antibody, suggesting the presence of His-tag in the protein.
  • No IGF-1 could be detected in the BMMY only (medium control) and the yeast culture only (uninduced control).
  • Purified grouper IGF-1 was characterized by total protein staining and Western blotting. To characterize native and modified grouper IGF-1 after immobilized metal affinity chromatography (IMAC), total protein staining and Western blotting were performed. As seen in FIG. 7, expression of recombinant IGF-1 proteins in the present recombinant yeast strain was detected and confirmed at ⁇ 10 kDa in total protein staining (panel a) and Western blotting using anti-6X His antibody (panel b).
  • IMAC immobilized metal affinity chromatography
  • the MCF-7 human epithelial cell line was obtained from ATCC and treated with the recombinant fish IGF-1 obtained from the recombinant yeast cells described in the preceding examples following the below steps:
  • Cells are cultured in DMEM/F12 complete medium [DMEM/F12, 10% FBS] inside a humidified incubator (34°C; 5% CO2; 95% air). Split cells at a ratio of 1 : 4 to 1 :8 for routine maintenance.
  • growth factors e.g. IGF-1
  • 2x working concentrations in serum-free medium [DMEM/F12, 0.1 % human serum albumin].
  • 1x growth factors e.g. add 50 uL 2x growth factor to well containing 50 uL serum-free medium. Return the cells to the incubator.
  • the FHM fish epithelial cell line was obtained from ATCC and treated following the below steps: 1. Upon cell line arrival, culture cells in DMEM/F12 complete medium inside a humidified incubator (34°C; 5% CO2; 95% air) for passages 1 -3 (P1 -P3). Split cells at a ratio of 1 :4 to 1 :8. From the 4th passage onwards, sequentially adapt FHM cells to reduced serum conditions and finally to serum-free medium. Culture cells in our in-house serum-free medium formulation inside a humidified incubator (34°C; 5% CO2; 95% air). Split cells at a ratio of 1 :4 to 1 :8 for routine maintenance.
  • growth factors e.g. IGF-1
  • 2x working concentrations in treatment medium [DMEM/F12, 0.1 % human serum albumin, 32 uM ethanolamine].
  • Add the 2x growth factors into the wells such that cells will be treated by 1x growth factors e.g. add 50 ul 2x growth factor to well containing 50 ul serum-free medium). Return the cells to the incubator.
  • the resazurin assay is commonly used to quantify the number of live cells in a sample and to measure the metabolic capacity of live cells.
  • Purified grouper IGF-I stimulated cell growth in both mammalian and non-mammalian cells.
  • the stimulatory effect of grouper IGF-1 on cell growth was measured by the resazurin assay.
  • Purified native and modified grouper IGF-1 were treated to MCF7 cells (human) and FHM cells (fish) at 0 nM, 0.1 nM, 1 nM and 10 nM respectively.
  • grouper IGF-1 stimulated cell growth in both cell types, indicating their ability to be used after purification. Both cell types also showed different responsiveness to the modified grouper IGF-1.
  • Grouper IGF-1 containing yeast culture could be used directly to stimulate cell growth in both mammalian and non-mammalian cells.
  • a resazurin assay was performed.
  • Grouper IGF-1 yeast cultures after centrifugation and filtering were treated to MCF7 cells (human) and FHM cells (fish) at 1 : 100 and 1 : 1000 dilutions respectively.
  • grouper IGF-1 yeast cultures stimulated cell growth in both cell types, while the BMMY only (medium control) and yeast culture only (uninduced control) did not have significant effects on cell growth.
  • recombinant growth factors or proteins are expressed and used for supporting the growth of in vitro cell culture.
  • embodiments of genetically engineered yeast cells may be used to express recombinant growth factors of various origin and the resulting growth factors may be used to supplement in vitro culture for promoting the growth of cells of identical or similar species or genus.
  • the growth factor is IGF-1 such as IGF-1 having the sequence depicted in Table 2 below.
  • the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant IGF-1 selected from the group consisting of SEQ ID NOs. 11 -55.
  • the resulting recombinant IGF-1 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.
  • polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 56-100.
  • polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 56-100.
  • polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 56-100.
  • the growth factor is fibroblast growth factor 2 (FGF-2) such as FGF2 having the sequence depicted in Table 3 below.
  • FGF-2 fibroblast growth factor 2
  • the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant FGF-2 selected from the group consisting of SEQ ID NOs. 101 -145.
  • the resulting recombinant FGF-2 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.
  • polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 146-190.
  • polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 146-190.
  • polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 146- 190.
  • the growth factor is epidermal growth factor (EGF) such as EGF having the sequence depicted in Table 4 below.
  • EGF epidermal growth factor
  • the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant EGF selected from the group consisting of SEQ ID NOs. 191 -219.
  • the resulting recombinant FGF2 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.
  • polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 220-248
  • polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 220-248.
  • polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 220- 248.

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Abstract

La présente invention concerne un procédé permettant d'augmenter la croissance cellulaire et l'expression des protéines dans une culture cellulaire in vitro à l'aide de facteurs de croissance recombinés produits par une souche de levure génétiquement modifiée. Des facteurs de croissance recombinés sont introduits dans le milieu de culture des cellules à cultiver, augmentant ainsi le taux de croissance ou l'expression des protéines des cellules cultivées. Dans un cas, on utilise un facteur de croissance recombiné (i) génétiquement de la même espèce ou d'une espèce similaire aux cellules cultivées et/ou (ii) génétiquement du même genre que les cellules cultivées. Dans un autre mode de réalisation, on utilise un facteur de croissance recombiné dont la séquence d'acides aminés a été modifiée pour améliorer la stabilité, la bioactivité ou d'autres caractéristiques favorisant la croissance cellulaire et/ou l'expression de la protéine des cellules cultivées.
PCT/IB2023/055617 2022-06-02 2023-06-01 Cellules de levure génétiquement modifiées, protéines recombinées produites à partir de celles-ci et leurs applications Ceased WO2023233340A1 (fr)

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CN103694340A (zh) * 2013-12-09 2014-04-02 重庆大学 重组蛋白igf1-24及其应用
WO2018227016A1 (fr) * 2017-06-07 2018-12-13 Wild Type, Inc. Production de chair ex vivo
CN109320601A (zh) * 2018-10-18 2019-02-12 天津林达生物科技有限公司 重组igf-1蛋白和高效表达及其在促细胞增殖方面的用途
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