Engineered cell lines for increased protein production

Abstract

The present disclosure relates to engineered cells that include genetic alterations leading to up- or down-regulation of certain genes in the cells for improved production of a recombinant protein. Also provided are methods of preparing and using such cells.

What is claimed is: 
     
         1 . An isolated engineered mammalian cell comprising one or more genetic alteration resulting in:
 up-regulation of catabolics; and   up-regulation of one or more of protein folding, growth factor production, anti-apoptosis, protein secretion, anabolics, or transcription initiation,   
       wherein the up- or down-regulation is as compared to the cell without the genetic alterations. 
     
     
         2 . The cell of  claim 1 , further comprising a genetic alteration resulting in enhanced cytotoxicity management. 
     
     
         3 . The cell of  claim 1 , wherein the genetic alteration resulting in up-regulation of catabolitcs is selected from the group consisting of:
 up-regulation of PDP;   up-regulation of citrate synthase (CS);   up-regulation of isocitrate dehydrogenase;   up-regulation of hk1;   up-regulation of pfk1;   up-regulation of pkm, and combinations thereof.   
     
     
         4 . The cell of  claim 1 , wherein the genetic alteration resulting in up-regulation of catabolics is selected from the group consisting of:
 up-regulation of PDP;   up-regulation of citrate synthase (CS);   up-regulation of isocitrate dehydrogenase;   up-regulation of hk1;   up-regulation of pfk1;   up-regulation of pkm;   down-regulation of PDK1;   down-regulation of PDK4   up-regulation of PDH;   up-regulation of DLAT;   up-regulation of DLD;   up-regulation of one of the ATP synthase subunits, and combination thereof.   
     
     
         5 . The cell of  claim 4 , wherein the genetic alteration resulting in up-regulation of one or more of protein folding, growth factor production, anti-apoptosis, protein secretion, anabolics, or transcription initiation is selected from modulations of genes A1-A6, S1-S4, F1-F9, G1-G6, P1-P9 or E1-E5 as noted in Table 1 or Table 1. 
     
     
         6 . The cell of  claim 2 , wherein the genetic alteration resulting in enhanced cytotoxicity management is selected from
 down-regulation of LDHA;   up-regulation of carbamoyl phosphate synthetase I;   up-regulation of transcarbamoylase;   up-regulation of CAP, and combination thereof.   
     
     
         7 . The cell of  claim 1 , comprising at least two genetic alterations. 
     
     
         8 . The cell of  claim 1 , comprising at least three genetic alterations. 
     
     
         9 . The cell of  claim 1 , comprising at least four genetic alterations. 
     
     
         10 . The cell of  claim 7 , comprising at least two genetic alterations each selected from a different group of C1-C3, A1-A3, S1-S4, F1, G1-G3, T1-T4, P1-P3, or E1-E3. 
     
     
         11 . The cell of  claim 1 , wherein the cell further comprises an exogenous polynucleotide encoding a polypeptide. 
     
     
         12 . The cell of  claim 11 , wherein the polypeptide is a therapeutic protein. 
     
     
         13 . The cell of  claim 11 , wherein the polypeptide is an antibody or an antibody fragment. 
     
     
         14 . The cell of  claim 1 , wherein the cell is a human cell. 
     
     
         15 . The cell of  claim 1 , wherein the cell is a CHO cell. 
     
     
         16 . A composition comprising the cell of  claim 1  and a cell culture medium. 
     
     
         17 . The composition of  claim 15 , wherein the medium comprises a supplement selected from the group consisting of ubiquinone, aspartic acid, and rapamycin. 
     
     
         18 . A method of producing a protein, comprising culturing the cell in the composition of  claim 16  and isolating the polypeptide.