Glycoengineering using
The present application relates to Leishmania cells genetically engineered such that the formation of an O-linked GlcNAc on a polypeptide in the Leishmania cell is reduced or eliminated. The formation of the O-linked GlcNAc may be catalyzed in the Leishmania cell prior to said genetic engineering by at least one N-acetylglucosamine (GlcNAc)-transferase. Also provided herein are methods of making a polypeptide using a Leishmania cell described herein and polypeptides produced by the methods provided herein.
1 . A Leishmania cell genetically engineered such that the formation of an O-linked N-acetylglucosamine (GlcNAc) on a polypeptide in the Leishmania cell is reduced or eliminated, wherein a gene encoding at least one O-linked GlcNAc-transferase of the Leishmania cell is functionally inactivated, downregulated, deleted, mutated, or overexpressed.
2 . The Leishmania cell of claim 1 , wherein the formation of O-linked GlcNAc in the Leishmania cell prior to genetic engineering is catalyzed by the at least one O-linked GlcNAc-transferase.
3 . The Leishmania cell of claim 1 , wherein the formation of the O-linked GlcNAc is reduced by at least 5%, at least 7%, at least 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% from the formation of the O-linked GlcNAc in a reference Leishmania cell.
4 . The Leishmania cell of claim 1 , wherein the at least one O-linked GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2 and OGNTL, and homologous GlcNAc-transferases thereof.
5 . The Leishmania cell of claim 4 , wherein the at least one O-linked GlcNAc-transferase is a GlcNAc-transferase that is homologous to OGNT1, OGNT2 and/or OGNTL.
6 . The Leishmania cell of claim 1 , wherein the number of the at least one O-linked GlcNAc-transferase is one, two or three.
7 . The Leishmania cell of claim 1 , wherein the growth rate of the Leishmania cell is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the growth rate of a reference Leishmania cell.
8 . The Leishmania cell of claim 1 , wherein the Leishmania cell is Leishmania tarentolae.
9 . The Leishmania cell of claim 1 , wherein the polypeptide is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO).
10 . The Leishmania cell of claim 1 , wherein the Leishmania cell comprises a recombinant nucleic acid encoding a heterologous glycosyltransferase.
11 . The Leishmania cell of claim 10 , wherein the Leishmania cell comprises one or more heterologous glycosyltransferases.
12 . The Leishmania cell of claim 10 , wherein the heterologous glycosyltranferase is an N-acetyl glucosamine transferase; and/or a heterologous galactosyltransferase; and/or a heterologous sialyltransferase.
13 . A method of making a polypeptide comprising (a) culturing the Leishmania cell of claim 1 under suitable conditions for polypeptide production; and (b) isolating the polypeptide.
14 . The Leishmania cell of claim 2 , wherein the gene encoding at least one O-linked GlcNAc-transferase of the Leishmania cell is functionally inactivated, downregulated, deleted, or mutated.
15 . The Leishmania cell of claim 14 , wherein the at least one O-linked GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2 and OGNTL.
16 . The Leishmania cell of claim 14 , wherein the at least one O-linked GlcNAc-transferase is a GlcNAc-transferase that is homologous to OGNT1, OGNT2 and/or OGNTL.
17 . The Leishmania cell of claim 15 , wherein the at least one O-linked GlcNAc-transferase is one O-linked GlcNAc-transferase selected from the group consisting of OGNT1, OGNT2 and OGNTL.
18 . The Leishmania cell of claim 15 , wherein the at least one O-linked GlcNAc-transferase is two O-linked GlcNAc-transferases selected from the group consisting of OGNT1, OGNT2 and OGNTL.
19 . The Leishmania cell of claim 15 , wherein the at least one O-linked GlcNAc-transferase is three O-linked GlcNAc-transferases selected from the group consisting of OGNT1, OGNT2 and OGNTL.
20 . The Leishmania cell of claim 15 , wherein the at least one O-linked GlcNAc-transferase is two or three O-linked GlcNAc-transferases, each of which independently is homologous to OGNT1, OGNT2or OGNTL.