Incubating cells with a heterologous nucleic acid encoding a recombinant protein in a media containing amphiphilic copolymer having at least one block of poly(oxyethylene) and at least one block of poly(oxypropylene) optionally initiated by ethylenediamine or polyethylenimine; enhanced gene expression
Inventors
Kabanov, Alexander V. • Alakhov, Valery
Assignees
University of Nebraska Medical Center UNMC • University of Nebraska System
Publication Number
US-7422875-B2
Publication Date
2008-09-09
Expiration Date
2024-07-20
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Abstract
Compositions and methods for increasing protein production are provided.
Core Innovation
The invention provides compositions and methods for increasing gene expression and protein yield by incubating cells containing a heterologous nucleic acid encoding a recombinant protein in media containing at least one amphiphilic block copolymer. These amphiphilic block copolymers consist of at least one block of poly(oxyethylene) and at least one block of poly(oxypropylene), such as Pluronic®-type copolymers, or mixtures of different block copolymers, optionally including polycation-conjugated forms.
The problem addressed is that while amphiphilic block copolymers have been known to assist in gene delivery into cells, the prior art did not disclose or suggest their impact on increasing the expression of genes already present in cells. The invention overcomes this limitation by demonstrating that exposing cells containing heterologous nucleic acids to selected amphiphilic block copolymers significantly increases the yield of recombinant proteins and RNA products.
According to the patent, the method enables both short- and long-term incubation (at least three hours, particularly at least nine hours) with these block copolymers to result in increased gene and protein expression. The approach is applicable to a wide range of cell types, including mammalian, bacterial, yeast, and insect cells. Additionally, the invention provides kits for carrying out these methods, which can include the amphiphilic block copolymer and reagents required for transforming and selecting stably transformed cells.
Claims Coverage
There are three independent claims in the patent, each describing a distinct inventive feature regarding methods for increasing protein or RNA production using amphiphilic block copolymers.
Method for producing a protein using amphiphilic block copolymers
A method that includes: 1. Providing cells comprising a heterologous nucleic acid encoding a recombinant protein. 2. Incubating the cells in media containing at least one amphiphilic block copolymer, where the copolymer has at least one block of poly(oxyethylene) and at least one block of poly(oxypropylene). 3. The heterologous nucleic acid encoding the recombinant protein is controlled by a transcription element that includes a binding site for NF-κB or p53. Additional dependent claims detail suitable cell types, promoter elements (e.g., cytomegalovirus promoter), block copolymer features (composition, HLB, concentrations), and selection of block copolymers or their mixtures, including those conjugated to polycations.
Method for producing a protein in a host via pre-incubation with amphiphilic block copolymers
A method that includes: 1. Providing a cell comprising a heterologous nucleic acid encoding a recombinant protein. 2. Incubating the cells in media containing at least one amphiphilic block copolymer that has at least one block of poly(oxyethylene) and at least one block of poly(oxypropylene). 3. Introducing the treated cells into a host. The nucleic acid encoding the recombinant protein is under control of a transcription element comprising a binding site for NF-κB or p53. The claim also covers the case where the cells are obtained from the host and modified in vitro.
Method for enhancing RNA production using amphiphilic block copolymers
A method that includes: 1. Providing cells comprising a heterologous DNA encoding an RNA. 2. Incubating the cells in media containing at least one amphiphilic block copolymer composed of at least one block of poly(oxyethylene) and at least one block of poly(oxypropylene). 3. The heterologous nucleic acid encoding the RNA is controlled by a transcription element comprising a binding site for NF-κB or p53. Dependent claims specify the encoded RNA can be an siRNA, and the promoter may be a cytomegalovirus or polymerase III promoter.
The independent claims broadly cover methods of increasing protein and RNA production using defined amphiphilic block copolymers under specific transcriptional control conditions, including application in cells before introduction into hosts.
Stated Advantages
The disclosed methods increase the production of recombinant proteins from cells in various settings, including laboratory tissue culture and large-scale production.
The increase in protein expression is achieved without the need for metal ions such as copper ions, and in the presence of media containing human or animal-derived protein.
The method is selective for increasing expression driven by foreign or heterologous promoters, without a global increase in total cellular protein.
Increased protein production per cell facilitates purification since the desired protein forms a larger proportion of the total protein produced.
The approach reduces production costs for recombinantly produced proteins intended for commercial or therapeutic use due to the need for fewer cells to achieve the same product yield.
Documented Applications
Recombinant protein expression in tissue culture and large-scale production, particularly for therapeutic proteins.
Production of various recombinant proteins such as cytokines, enzymes, clotting factors (e.g., Factor VIII, Factor IX, tissue plasminogen activator), vaccines, antibodies, growth factors, hormones (e.g., erythropoietin, insulin), hemoglobin, alpha-1-antitrypsin, lactoferrin, cystic fibrosis transmembrane conductase, human protein C, anti-viral agents, and interleukins.
Enhancing RNA production, including siRNA, in cells containing heterologous DNA.
Methods for pre-treating cells ex vivo with amphiphilic block copolymers to enhance gene expression before introducing them into a host, including ex vivo gene therapy settings.
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