IP Library › Patent Application 19408114
Patent Application
App. No. 19/408,114

MODIFIED MAMMALIAN CELLS

Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US None
App. No.
19/408,114
Abstract

The present disclosure relates to methods, cells, and compositions for producing a product of interest, e.g., a recombinant protein. In particular, the present disclosure provides improved mammalian cells expressing the product of interest, where the cells (e.g., Chinese Hamster Ovary (CHO) cells) have reduced or eliminated activity, e.g., expression, of certain host cell proteins, e.g., enzymes including, but not limited to, certain lipases, esterases, and/or hydrolases.

Claims (50)

1 . A recombinant host cell, wherein the cell is modified to reduce or eliminate the activity of one or more enzyme relative to the activity of the enzyme in an unmodified cell.

2 . The recombinant host cell of claim 1 , wherein the one or more enzyme is: Lipoprotein lipase (LPL); Phospholipase B-domain containing 2 (PLBL2/PLBD2); Lipase A (Lysosomal acid lipase/cholesteryl ester hydrolase, Lipase) (LIPA); Phospholipase A-2-activating protein (PLAA); Phospholipase D3 (PLD3); Phospholipase A2 group XV (LPLA2); Phospholipase C beta 1 (PLCB1); Phospholipase C delta 1 (PLCD1); DDHD domain containing protein 1 (Fragment) (DDHD1); Lysophospholipase-like protein 1 (LYPLAL1); Phospholipase A2 group XIIA (PLA2G12A); Peroxiredoxin 6 (PRDX6); Sphingomyelin phosphodiesterase (SMPD1); Palmitoyl-protein thioesterase 1 (PPT1); Isoamyl acetate hydrolyzing esterase 1 (putative) (IAH1); OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1); Lysophospholipase 2 (Acyl-protein thioesterase 2) (LYPLA2); Acyl-coenzyme A thioesterase 13 (ACOT13); Fatty acid synthase (FASN); Phospholipase A2 group VII (PLA2G7); Ubiquitin specific peptidase 5 (USP5); Nacylsphingosine amidohydrolase 1 (Acid ceramidase) (ASAH1); Lipase Maturation Factor 1 (LMF1); Apolipoprotein C-II (APOC2); Acylcarnitine hydrolase (HACH); Carboxylesterase 1F (CES1F) or Liver carboxylesterase B-1-like (CES-B1L); Lysophospholipase 1 (LYPLA1); Carboxylesterase 1 (CES1); Phospholipase A1 member A (PLA1A); or Sialic acid acetylesterase (SIAE).

3 . The recombinant host cell of claim 2 , wherein the activity of:

a) PPT1;

b) LPLA2; LPL; and LIPA;

c) LPLA2; LPL; LIPA; PPT1; PLBL2; PLD3; and SMPD1;

d) LPLA2; LPL; LIPA; PPT1; PLBL2; PLD3; SMPD1; PLAA; IAH1; OTUB1; LYPLA2; and PLA2G12A;

e) BAX; BAK; LPLA2; LPL; LIPA; PPT1; PLD3; and SMPD1;

f) BAX; BAK; LPLA2; LPL; LIPA; PPT1; PLBL2; PLD3; SMPD1; CLU; PRDX1; PLAA; and ACOT13;

g) LPLA2; LPL; and PPT1

h) LPLA2; LPL; LIPA and PPT1

i) HACH; CES1F/CES-B1L; and LYPLA1

j) LPLA2; LPL; LIPA; PPT1; HACH; CES1F/CES-B1L; and LYPLA1

k) SMPD1; CES1; PLA1A; and SIAE

l) LPLA2; LPL, LIPA; PPT1; HACH; CES1F/CES-B1L; LYPLA1; SMPD1; CES1; PLA1A; and SIAE

m) LPLA2; LMF1; LIPA; PPT1; HACH; CES1F/CES-B1L; and LYPLA1

n) LPLA2; LMF1; APOC2; LIPA; PPT1; HACH; CES1F/CES-B1L; and LYPLA1 or

o) LMF1 and APOC2

is reduced or eliminated.

4 . The recombinant host cell of claim 1 , wherein the activity of the one or more enzyme is reduced or eliminated by:

(a) knocking down expression of the enzyme;

(b) or knocking out expression of the enzyme; or

(c) altering the nucleic acid sequence encoding the enzyme.

5 . The recombinant host cell of claim 1 , wherein the cell comprises one or more altered enzyme genes.

6 . The recombinant host cell of claim 5 , wherein the one or more altered enzyme genes have no detectable enzymatic activity.

7 . The recombinant host cell of claim 1 , wherein the cell is a mammalian cell.

8 . The recombinant host cell of claim 7 , wherein the cell is a CHO cell.

9 . The recombinant host cell of claim 1 , comprising a nucleic acid sequence encoding a product of interest.

10 . The recombinant host cell of claim 9 , wherein the product of interest comprises a protein, a viral particle or a viral vector.

11 . The recombinant host cell of claim 9 , wherein the product of interest comprises a recombinant protein.

12 . The recombinant host cell of claim 1 , wherein the product of interest comprises an antibody or an antigen-binding fragment thereof.

13 .- 16 . (canceled)

17 . The recombinant host cell of claim 9 , wherein the nucleic acid sequence is integrated in the cellular genome of the mammalian cell at a targeted location.

18 . The recombinant host cell of claim 17 , further comprising a nucleic acid encoding the product of interest that is randomly integrated in the cellular genome of the mammalian cell.

19 . The recombinant host cell of claim 1 , wherein the modified cell does not express any detectable LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLAL1; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CES-B1L; LYPLA1; CES1; PLA1A; and/or SIAE.

20 . A composition comprising a recombinant host cell of claim 1 .

21 . A method for reducing enzymatic activity in a cell, comprising:

(a knocking down or knocking out the expression of LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLAL1; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CESB1L; LYPLA1; CES1; PLA1A; and/or SIAE;

(b) modulating a cell culture process and/or media composition, wherein modulating a cell culture process and/or media composition results in reduced activity of LPL PLBL2/PLBD2 LIPA PLAA PLD3 LPLA2 PLCB1; PLCD1; DDHD1; LYPLAL1; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CES-B1L; LYPLA1; CES1; PLA1A; and/or SIAE;

(c) selecting cells with reduced activity of LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLA11; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CES-B1L; LYPLA1; CES1; PLA1A; and/or SIAE or

(d) altering the gene encoding one or more of LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLA11; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CES-B1L; LYPLA1; CES1; PLA1A; and/or SIAE.

22 .- 25 . (canceled)

26 . A method for reducing or eliminating enzymatic activity in a cell, comprising administering to the cell a genetic engineering system, wherein the genetic engineering system alters the nucleic acid sequence encoding one or more of LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLA11; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; and ASAH1 so that the one or more of LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLAL1; PLA2G12A; PRDX6; SPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CES-B1L; LYPLA1; CES1; PLA1A; and/or SIAE have reduced or eliminated enzymatic activity.

27 .- 44 . (canceled)

45 . A method of producing a product of interest comprising culturing mammalian cells expressing the product of interest, wherein the mammalian cells express the product of interest and have reduced or eliminated activity of one or more of the following LPL; PLBL2/PLBD2; LIPA; PLAA; PLD3; LPLA2; PLCB1; PLCD1; DDHD1; LYPLA11; PLA2G12A; PRDX6; SMPD1; PPT1; IAH1; OTUB1; LYPLA2; ACOT13; FASN; PLA2G7; USP5; ASAH1; LMF1; APOC2; HACH; CES1F/CES-B1L; LYPLA1; CES1; PLA1A; and/or SIAE.

46 .- 59 . (canceled)

60 . The method of claim 45 , wherein the degradation of polyoxyethylene sorbitan monolaurate is reduced.

61 . The method of claim 45 , wherein the degradation of polysorbate 20 is reduced.

62 . The method of claim 45 , wherein the degradation of polysorbate 80 is reduced.

63 .- 117 . (canceled)