COMPOSITIONS AND METHODS FOR REGULATING CELL OSMOLARITY

• US 20150307888A1
• Filed: 11/21/2014
• Published: 10/29/2015
• Est. Priority Date: 09/15/2008
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: MALPHETTES, Laetitia, SNOWDEN, Andrew, YUK, Inn H.
• Granted Patent: US 10,100,319 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

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).

Claims

1-36. -36. (canceled)

41-45. -45. (canceled)

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) a 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 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.

37. The method of claim 46 wherein the osmolality is increased by spiking with a compound that increases said osmolality.

38. The method of claim 46 wherein said second protein of interest is toxic to said cell.

39. The method of claim 46 wherein said second protein of interest is unstable.

40. The method of claim 46 wherein said second protein of interest is difficult to express under normal culture conditions.

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.

52-54. -54. (canceled)