Compositions and methods for regulating cell osmolarity

Abstract

The invention provides compositions and methods for regulating intracellular osmolarity in cells, e.g., in cultured cells, including in cultured cells in bioreactors. The invention provides nucleic acids comprising at least one osmo-responsive transcriptional regulatory element (OR-TRE), and cells, vectors, products of manufacture, artificial organs or implants and the like containing an osmo-responsive transcriptional regulatory element (OR-TRE).

1 . An isolated nucleic acid molecule comprising at least one osmo-responsive transcriptional regulatory element (OR-TRE) comprising at least one TonEBP-responsive transcriptional enhancer or NFATc-responsive transcriptional enhancer operatively linked to a transcriptional regulator and at least one Activator Protein (AP-1)-responsive transcriptional enhancer operatively linked to said TonEBP-responsive transcriptional enhancer or NFATc-responsive transcriptional enhancer. 
     
     
         2 . The nucleic acid molecule of  claim 1  wherein said transcriptional regulator comprises a promoter, an enhancer or a combination thereof. 
     
     
         3 . The nucleic acid molecule of  claim 2  comprising a first TonEBP-responsive transcriptional enhancer or NFATc-responsive transcriptional enhancer positioned 5′ to a promoter, and a second TonEBP-responsive transcriptional enhancer or NFAT-responsive transcriptional enhancer positioned 3′ to said promoter. 
     
     
         4 . The nucleic acid molecule of  claim 1  comprising a first Activator Protein-1 (AP-1)-responsive transcriptional enhancer positioned 5′ of said OR-TRE and a second Activator Protein-1 (AP-1)-responsive transcriptional enhancer positioned 3′ of said OR-TRE. 
     
     
         5 . The nucleic acid molecule of  claim 1  wherein the Activator Protein-1 (AP-1)-responsive transcriptional enhancer is positioned 5′ of said transcriptional regulator wherein said transcriptional regulator is a promoter. 
     
     
         6 . The nucleic acid molecule of  claim 1  wherein the Activator Protein-1 (AP-1)-responsive transcriptional enhancer is positioned 3′ of said transcriptional regulator wherein said transcriptional regulator is a promoter. 
     
     
         7 . The nucleic acid molecule of  claim 1  comprising a first Activator Protein-1 (AP-1)-responsive transcriptional enhancer positioned 5′ to said transcriptional regulator and a second Activator Protein-1 (AP-1)-responsive transcriptional enhancer positioned 3′ to said transcriptional regulator, wherein said first transcriptional regulator and said second transcriptional regulator are promoters. 
     
     
         8 . The nucleic acid molecule of  claim 1  wherein
 (a) at least one TonEBP-responsive transcriptional enhancer comprises the nucleic acid sequence of SEQ ID NO:1; or 
 (b) at least one NFATc-responsive transcriptional enhancer comprises the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:4; or 
 (c) at least one Activator Protein-1 (AP-1)-responsive transcriptional enhancer comprises the nucleic acid sequence of SEQ ID NO:3 
 
       wherein N can be any nucleotide. 
     
     
         9 . The nucleic acid molecule of  claim 1  wherein said OR-TRE comprises the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. 
     
     
         10 . The nucleic acid molecule of  claim 1  wherein the nucleic acid molecule further comprises additional nucleic acid molecule operatively linked to the transcriptional regulator, wherein said transcriptional regulator is a promoter that is transcriptionally active in a eukaryotic cell. 
     
     
         11 . The nucleic acid molecule of  claim 10 , wherein the additional nucleic acid molecule comprises (a) a protein-coding nucleic acid molecule encoding a protein of interest; or (b) a regulatory nucleic molecule. 
     
     
         12 . The nucleic acid molecule of  claim 10  wherein said regulatory nucleic acid molecule is an inhibitory molecule, a stabilizing molecule, an up-regulating nucleic acid molecule, or produces an antisense molecule. 
     
     
         13 . The nucleic acid molecule of  claim 11 , wherein the additional nucleic acid molecule encodes an osmo-protective protein or peptide. 
     
     
         14 . The nucleic molecule of  claim 11 , wherein the additional nucleic acid molecule encodes a protein or peptide that confers a beneficial property to proteins expressed in cells. 
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein the additional nucleic acid molecule encodes a polypeptide selected from the group consisting of an anti-apoptotic protein; a protein that confers resistance to oxidative stress to a cell; a chaperone protein involved in facilitating protein folding; a protein involved in extracellular secretion of proteins; a glycolytic enzyme; a cell cycle regulation protein; a glycosylation enzyme, or any combination thereof. 
     
     
         16 . The nucleic acid molecule of  claim 12 , wherein the inhibitory nucleic acid molecule comprises an antisense sequence, a ribozyme, a short interfering RNA (siRNA), or a microRNA (miRNA). 
     
     
         17 . The nucleic acid molecule of  claim 10 , wherein the additional nucleic acid molecule encodes a NFATc or a TonEBP polypeptide. 
     
     
         18 . The osmo-responsive nucleic acid molecule of  claim 13 , wherein the osmoprotective-protective protein or peptide is a proline or a glycine-betaine, a taurine transporter, a glycine betaine-γ-aminobutyric acid transporter, a sodium-myo-inositol cotransporter, a heat shock protein, an aquaporin, an aldose reductase, or a neuropathy target esterase (NTE). 
     
     
         19 . The osmo-responsive nucleic acid molecule of  claim 15 , wherein the antiapoptotic protein is Bc1-2, Bc1-xL, Mc1-1, BHRF1, X-IAP, IAP1, IAP2 IEX-1L, Bf1-1 or Bc1-w. 
     
     
         20 . The osmo-responsive nucleic acid molecule of  claim 15 , wherein the protein that confers resistance to oxidative stress to a cell is a superoxide dismutase, a catalase, a glutathione peroxidase, a peroxiredoxin, a sulfiredoxin, thioredoxin, thioredoxin reductase, thioredoxin peroxidase, thioltransferase, glutaredoxin or a glutathione reductase. 
     
     
         21 . The osmo-responsive nucleic acid molecule of  claim 15 , wherein the chaperone protein involved in facilitating protein folding is a binding immunoglobulin protein (BiP), calnexin, calreticulin, ERp57 or a protein disulfide isomerase (PDI). 
     
     
         22 . The osmo-responsive nucleic acid molecule of  claim 15 , wherein the glycolytic enzyme is a pyruvate carboxylase or a pyruvate kinase. 
     
     
         23 . The osmo-responsive nucleic acid molecule of  claim 15 , wherein the cell cycle regulation protein is a cyclin or a cyclin-dependent kinase, or an inhibitor of a cyclin or a cyclin-dependent kinase. 
     
     
         24 . The isolated nucleic acid molecule of  claim 1  wherein said OR-TRE is operatively linked to at least one targeting nucleic acid molecule. 
     
     
         25 . The nucleic acid molecule of  claim 24 , wherein the targeting nucleic acid molecule comprises a nucleic acid molecule for targeting a lactogenic gene, a lactogenic message, or a lactogenic protein to decrease expression of said lactogenic gene, lactogenic message, or a lactogenic protein. 
     
     
         26 . The nucleic acid molecule of  claim 25 , wherein the lactogenic gene is a lactate dehydrogenase. 
     
     
         27 . The nucleic acid molecule of  claim 24 , wherein the nucleic acid comprising a nucleic acid molecule targeting a lactogenic gene or lactogenic message comprises a short interfering RNA (siRNA), a microRNA (miRNA), an antisense RNA and/or an RNA with ribozyme activity. 
     
     
         28 . A vector comprising the nucleic acid molecule of  claim 1 . 
     
     
         29 . A cell comprising the vector of  claim 29 . 
     
     
         30 . The nucleic acid molecule of  claim 1  wherein the nucleic acid molecule further comprises (i) additional nucleic acid molecule encoding a polypeptide that confers a beneficial property to proteins expressed in cells operatively linked to the transcriptional regulator, wherein said transcriptional regulator is a promoter that is transcriptionally active in a eukaryotic cell and (ii) a nucleic acid molecule encoding a protein of interest to be expressed in cells wherein the expression of the nucleic acid molecule of (i) imparts said beneficial property to the polypeptide encoded by the nucleic acid molecule of (ii). 
     
     
         31 . The nucleic acid molecule of  claim 30  wherein the additional nucleic acid molecule of (i) encodes a polypeptide selected from the group consisting of an anti-apoptotic protein; a protein that confers resistance to oxidative stress to a cell; a chaperone protein involved in facilitating protein folding; a protein involved in extracellular secretion of proteins; a glycolytic enzyme; a cell cycle regulation protein; a glycosylation enzyme, or any combination thereof. 
     
     
         32 . A method for protecting a cell under conditions of hyperosmolality comprising:
 (a) introducing a polynucleotide into a cell wherein said polynucleotide comprises: (i) the nucleic acid molecule of  claim 13 , and/or an osmo-responsive nucleic acid molecule of  claim 12  and (ii) a polynucleotide encoding a second protein of interest operatively linked to a promoter; and   (b) culturing the cell such that the osmo-protective protein or peptide, or the osmo-protective regulatory nucleic acid and said protein of interest is expressed, thereby protecting the cell under conditions of hyperosmolality and allowing expression of said second protein of interest.   
     
     
         33 . The method of  claim 32  wherein the osmoprotective-protective protein or peptide is a proline or a glycine-betaine, a taurine transporter, a glycine betaine-γ-aminobutyric acid transporter, a sodium-myo-inositol cotransporter, a heat shock protein, an aquaporin, an aldose reductase, or a neuropathy target esterase (NTE). 
     
     
         34 . A method for expressing a protein of interest in a cell under conditions of hyperosmolality comprising:
 (a) introducing a polynucleotide into a cell wherein said polynucleotide comprises: (i) the nucleic acid molecule of  claim 14  and (ii) a polynucleotide encoding a second protein of interest operatively linked to a promoter; and   (b) culturing the cell such that the expression of nucleic acid molecule of (i) imparts said beneficial property to the polypeptide encoded by the polynucleotide of (ii) when said cells are grown under conditions of hyperosmolality.   
     
     
         35 . The method of  claim 34 , wherein the protein or peptide that confers a beneficial property to proteins expressed in cells is an anti-apoptotic protein; a protein that confers resistance to oxidative stress to a cell; a chaperone protein involved in facilitating protein folding; a protein involved in extracellular secretion of proteins; a glycolytic enzyme; a cell cycle regulation protein; a glycosylation enzyme; or any combination thereof. 
     
     
         36 . The method of  claim 32  or  34  wherein in step (b) said cells are initially cultured cell under normal culture conditions and then said osmolality is increased to an amount sufficient to increase expression of said second protein of interest. 
     
     
         37 . The method of  claim 36  wherein the osmolality is increased by spiking said culture with a compound that increases said osmolality. 
     
     
         38 . The method of  claim 36  wherein said second protein of interest is toxic to said cell. 
     
     
         39 . The method of  claim 36  wherein said second protein of interest is unstable. 
     
     
         40 . The method of  claim 36  wherein said second protein of interest is difficult to express under normal culture conditions. 
     
     
         41 . The method of  claim 32  or  34  wherein instep (b) said cells are initially cultured cell under normal culture conditions and allowing culture to become hyperosmotic, thereby increasing expression of said second protein of interest. 
     
     
         42 . The method of  claim 41  wherein said second protein of interest is toxic to said cell. 
     
     
         43 . The method of  claim 41  wherein said second protein of interest is unstable. 
     
     
         44 . The method of  claim 41  wherein said second protein of interest is difficult to express under normal culture conditions. 
     
     
         45 . A method for adding or enhancing a cell's adaptability or resistance to osmotic stress or osmotic shock comprising introducing to a cell a polynucleotide comprising the nucleic acid molecule of  claim 11 , wherein the additional nucleic acid molecule encodes an osmo-protective protein or peptide, or an osmo-protective regulatory nucleic acid. 
     
     
         46 . A method for expressing a protein of interest in a cell under conditions of hyperosmolality comprising introducing a polynucleotide into a cell wherein said polynucleotide comprises:
 (a) the nucleic acid molecule of  claim 11  and   (b) a polynucleotide encoding a second protein of interest operatively linked to a second OR-TRE;   
       wherein said nucleic acid molecule of (a) encodes TonEBP; and wherein under conditions of increased osmolality, said nucleic acid molecule of (a) is expressed, thereby expressing TonEBP protein, and wherein said TonEBP protein positively regulates expression of TonEBP and said second protein of interest. 
     
     
         47 . The method of  claim 46  wherein said second protein of interest is an osmoprotective protein. 
     
     
         48 . The method of  claim 47  wherein said osmoprotective protein is a proline or a glycine-betaine, a taurine transporter, a glycine betaine-γ-aminobutyric acid transporter, a sodium-myo-inositol cotransporter, a heat shock protein, an aquaporin, an aldose reductase, or a neuropathy target esterase (NTE). 
     
     
         49 . The  claim 46  wherein said second protein of interest is a protein that imparts a beneficial property to polypeptides expressed by said cells. 
     
     
         50 . The method of  claim 49  wherein said protein that imparts a beneficial property is an anti-apoptotic protein; a protein that confers resistance to oxidative stress to a cell; a chaperone protein involved in facilitating protein folding; a protein involved in extracellular secretion of proteins; a glycolytic enzyme; a cell cycle regulation protein; a glycosylation enzyme; or any combination thereof. 
     
     
         51 . The method of  claim 49  further comprising a third nucleic acid encoding a third protein of interest operatively linked to a promoter, wherein said protein that imparts a beneficial property acts on said third protein of interest. 
     
     
         52 . The nucleic acid molecule of  claim 11 , wherein the additional nucleic acid molecule encodes TonEBP. 
     
     
         53 . The nucleic acid of  claim 11  further comprising a polynucleotide encoding a second protein of interest operatively linked to a second OR-TRE. 
     
     
         54 . A kit comprising the nucleic acid molecule of  claim 1 .