Method to engineer mammanlian-type carbohydrate structures

Abstract

The present invention relates to host cells having modified lipid-linked oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells have a GlcNAcMan 3 GlcNAc 2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyl-transferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

1 - 60 . (canceled) 
     
     
         61 . A  Pichia pastoris  host cell wherein the host cell lacks OCH1 activity and has diminished or depleted activity of one or more enzymes selected from the group consisting of:
 (a) an enzyme having dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity;   (b) an enzyme having dolichyl-P-Man:Man6GlcNAc2-PP-dolichyl alpha-1,2 mannosyltransferase activity; and   (c) an enzyme having dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl alpha-1,6 mannosyltransferase activity.   
     
     
         62 . The host cell of  claim 61 , wherein the diminished or depleted enzyme has dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity. 
     
     
         63 . The host cell of  claim 61 , herein the host cell has depleted dolichyl-P-Man:Man5 GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity. 
     
     
         64 . The host cell of  claim 62  or  63 , further expressing:
 (i) an a1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, 
 (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and 
 (iii) a recombinant glycoprotein. 
 
     
     
         65 . The host cell of  claim 64 , further expressing a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc2Man3GlcNAc2 core structures. 
     
     
         66 . The host cell of  claim 65 , further expressing one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI. 
     
     
         67 . The host cell of  claim 64 , further expressing one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter. 
     
     
         68 . The host cell of claim  60 , wherein the host cell has diminished or depleted activity of an enzyme having dolichyl-P-Man:Man6GlcNAc2-PP-dolichyl alpha-1,2 mannosyltransferase activity. 
     
     
         69 . The host cell of  claim 62  or  63 , further expressing one or more nucleic acids encoding:
 (i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, 
 (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and 
 (iii) a recombinant glycoprotein. 
 
     
     
         70 . The host cell of  claim 69 , further expressing a nucleic acid molecule encoding an α-1,3 and/or α-1,2-1,3 mannosidase catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell. 
     
     
         71 . The host cell of  claim 70 , further expressing a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc2Man3GlcNAc2 core structures. 
     
     
         72 . The host cell of  claim 71 , further expressing one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI. 
     
     
         73 . The host cell of  claim 69 , further expressing one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter. 
     
     
         74 . The host cell of claim  60 , wherein the host cell has diminished or depleted activity of an enzyme having dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl alpha-1,6 mannosyltransferase activity. 
     
     
         75 . The host cell of  claim 73 , further expressing one or more nucleic acid molecules encoding:
 (i) an α1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell,   (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and   (iii) a recombinant glycoprotein.   
     
     
         76 . The host cell of  claim 75 , further expressing a nucleic acid molecule 1,2-1,3 mannosidase catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell. 
     
     
         77 . The host cell of  claim 74 , further expressing a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc2Man3GlcNAc2 core structures. 
     
     
         78 . The host cell of  claim 77 , further expressing one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI. 
     
     
         79 . The host cell of  claim 75 , further expressing one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter.