Compositions and methods for metabolic selection of transfected cells
Inventors
Assignees
Publication Number
US-8076102-B2
Publication Date
2011-12-13
Expiration Date
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Abstract
The present invention relates to novel selection marker vectors, and methods for using these vectors to generate stable gene expression systems in eukaryotic cells utilizing any enzyme useful in the eukaryotic sterol/cholesterol biosynthetic pathway, such as a 3-ketosteroid reductase, as a metabolic selection marker to select transfected cells. In one embodiment, the method comprises transfecting cells that are auxotrophic for cholesterol with a vector encoding 3-ketosteroid reductase and at least one heterologous protein, and selecting cells that have the ability to survive in medium lacking cholesterol and/or producing the heterologous protein in these cells in chemically defined and/or serum-free media.
Core Innovation
The present invention relates to novel metabolic selection marker vectors, and methods for using these vectors to generate stable gene expression systems in eukaryotic cells by utilizing any enzyme useful in the eukaryotic sterol/cholesterol biosynthetic pathway, such as a 3-ketosteroid reductase (3-KSR), as a metabolic selection marker to select transfected cells. The invention includes a vector comprising a polynucleotide encoding an enzyme in the sterol biosynthetic pathway of a eukaryotic cell, a biologically active fragment or variant thereof, and a polynucleotide encoding a heterologous polypeptide, and further includes host cells transformed with such vectors, kits, and methods of selecting and obtaining cells that can survive in medium lacking cholesterol and produce heterologous proteins in chemically defined and/or serum-free media.
The invention addresses the problem that certain eukaryotic cell lines, exemplified by the NS-0 mouse myeloma cell line, are cholesterol auxotrophs and thus require exogenous cholesterol supplementation, which the patent states is intricate, time-consuming, inherently unstable, prone to precipitation in chemically defined, serum-free media, difficult to sterilize by filtration, and increases the possibility of introducing contaminants. The invention provides a means to culture auxotrophic cells without the need for problematic addition of exogenous cholesterol by supplying an enzyme of the sterol biosynthetic pathway (for example 3-KSR) via an expression vector to confer the ability to survive and be cultured on chemically defined and/or serum-free media.
Claims Coverage
Six independent claims were identified, each defining a distinct inventive feature covering transformed host cells, a kit, a composition of supplements, and methods for making, obtaining, or expressing cells/products using a 3-ketosteroid reductase selection marker.
Host cell transformed with 3-ketosteroid reductase and heterologous polypeptide
A host cell transformed with a vector comprising a polynucleotide encoding a 3-ketosteroid reductase and a polynucleotide encoding a heterologous polypeptide, wherein the host cell is a eukaryotic cell and is auxotrophic for cholesterol.
Kit comprising vector, host cells, media, growth supplements, and protocol
A kit comprising: a vector comprising a polynucleotide that encodes a 3-ketosteroid reductase; a plurality of host cells that are auxotrophic for cholesterol; chemically defined, serum-free media; growth supplements that support the growth of said plurality of host cells at low-seeding and clonal densities; and at least one protocol to utilize said kit.
Composition of cell culture supplements
A composition of cell culture supplements comprising fatty acid-free BSA, rhIL-6, recombinant human insulin, sodium selenite, sodium pyruvate, and ethanolamine in final concentrations as claimed in the patent [procedural detail omitted for safety].
Method of making cholesterol-auxotrophic cells able to survive in cholesterol-free medium
A method comprising transfecting a eukaryotic cell that is auxotrophic for cholesterol with a vector comprising a polynucleotide that encodes a 3-ketosteroid reductase and optionally at least one polynucleotide that encodes a heterologous protein, wherein the polynucleotide encoding the 3-ketosteroid reductase is expressed by the transfected cell to confer the ability to survive in cholesterol-free medium.
Method for obtaining cells that survive without cholesterol and express a heterologous protein
A method comprising transfecting eukaryotic cells that are auxotrophic for cholesterol with a vector comprising a polynucleotide encoding a 3-ketosteroid reductase and at least one polynucleotide that encodes a heterologous protein, and selecting the cells that have the ability to survive in medium lacking cholesterol.
Method of expressing a heterologous protein in cholesterol-free medium
A method comprising transfecting a cell that is auxotrophic for cholesterol with a vector comprising a polynucleotide encoding a 3-ketosteroid reductase and a polynucleotide encoding the heterologous protein; and culturing the transfected cell in a cholesterol-free medium under conditions to provide expression of the heterologous protein.
The independent claims collectively cover transformed eukaryotic host cells carrying a 3-ketosteroid reductase selection marker and heterologous polypeptides, a kit containing the vector, auxotrophic host cells, media and supplements, a defined supplement composition, and methods for conferring cholesterol-independent growth and expressing heterologous proteins in cholesterol-free, chemically defined and/or serum-free media.
Stated Advantages
Provides metabolic selection marker vectors using enzymes of the sterol/cholesterol biosynthetic pathway, such as 3-KSR, to select transfected cells.
Enables generation of stable gene expression systems in eukaryotic cells that can survive and be cultured on chemically defined and/or serum-free media without exogenous cholesterol.
Reduces dependence on problematic addition of exogenous cholesterol to growth media, avoiding issues of solubility, precipitation, filtration loss, short shelf-life, and potential introduction of contaminants.
Addresses regulatory concerns about contamination by enabling use of chemically defined, serum-free media for culturing cells used to express biomolecules.
Documented Applications
Production of heterologous molecules such as peptides, polypeptides, and proteins in eukaryotic cells using 3-KSR selection to allow growth in cholesterol-free, chemically defined and/or serum-free media.
Expression and production of monoclonal antibodies (including complete human antibody light and heavy chain genes cloned in tandem) in transformed host cells selected by 3-KSR in chemically defined media.
Use in generating stable cell lines through transfection with expression vectors in chemically defined/serum-free media capable of expressing heterologous molecules.
Commercial kits for transfection and selection workflows comprising the vector encoding 3-KSR, auxotrophic host cells, chemically defined serum-free media, growth supplements, and protocols.
Use of cell culture systems to test new drugs for toxicity or efficacy and as a platform for cell-based therapeutics, as described in the background of the patent.
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