WO2024068768A9

WO2024068768A9 - Glycoengineering using leishmania cells

Info

Publication number: WO2024068768A9
Application number: PCT/EP2023/076767
Authority: WO
Inventors: Amirreza Faridmoayer; Rainer FOLLADOR; Tina SCHELBERT; Manuela Mally
Original assignee: Glycoera Ag
Priority date: 2022-09-28
Filing date: 2023-09-27
Publication date: 2024-06-13
Also published as: WO2024068768A1; WO2024068753A1

Abstract

The present application relates to Leishmania host cells, methods of engineering Leishmania host cells, methods of culturing Leishmania host cells, methods of making a polypeptide of interest using a Leishmania host cell, and polypeptides of interest produced by the methods. In particular, the Leishmania host cells provided herein are capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2.

Description

GLYCOENGINEERING USING LEISHMANIA CELLS

1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Serial No. 63/410,936, filed September 28, 2022, and U.S. Serial No. 63/410,955, filed September 28, 2022, each of which is incorporated herein by reference in its entirety.

2. SEQUENCE LISTING

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14688-006-228_SEQLISTING.xml”, was created on September 22, 2023 and is 330,759 bytes in size.

3. INTRODUCTION

[0003] The present application relates to Leishmania host cells, methods of engineering Leishmania host cells, methods of culturing Leishmania host cells, methods of making a polypeptide of interest using a Leishmania host cell, and polypeptides of interest produced by the methods. In particular, the Leishmania host cells provided herein are capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2.

4. BACKGROUND

[0004] A glycoprotein is a glycoconjugate in which a protein carries one or more glycans covalently attached to a polypeptide backbone, usually via N- or O-linkages. An N-glycan (N-linked oligosaccharide, N-[Asn]-linked oligosaccharide) is a sugar chain covalently linked to an asparagine residue of a polypeptide chain, commonly involving a GlcNAc residue in eukaryotes, and the consensus peptide sequence: Asn-X-Ser/Thr (Varki, Ajit (2009): Essentials of glycobiology. 2ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).

[0005] Protein glycosylation is a ubiquitous post-translational modification found in all domains of life. There is a significant complexity in animal systems and glycan structures have crucial biological and physiological roles, from contributions in protein folding and quality, control to involvement in a large number of biological events, like recognition, stability, action, and turnover of these molecules (Moremen et al. 2012). Therapeutic Glycoproteins like monoclonal antibodies, enzymes, and hormones are the major products of the biotechnology industry (Lagasse, H A Daniel et al. 2017; Dimitrov 2012) and the impact of glycan heterogeneity has more and more been recognized as “critical quality attribute”. Of the many properties determining product quality, glycosylation is regarded as even one of the most important ones: influencing the biological activity, serum half-life and immunogenicity of the protein. Glycans are relevant for increased serum circulation times and many of the biologies approved or under development suffer from an insufficient half-life necessitating frequent applications in order to maintain a therapeutic concentration over an extended period of time. Half-life extension strategies are key to allow the generation of long-lasting therapeutics with improved pharmacokinetics (Kontermann 2016). Glycosylation also appears to improve protein solubility and stability, for example, through a reduced propensity for aggregation and leads to increased circulatory lifetimes due to the prevention of proteolytic degradation. Additionally, N-glycans with different terminating monosaccharides can be recognized by lectins leading to their degradation (Blasko et al., 2013; Varki, 2017). Consequently, monitoring and control of glycosylation is critical in biopharmaceutical manufacturing and a requirement of regulatory agencies (Costa et al. 2014; Eon-Duval et al. 2012; Reusch and Tejada 2015). For these reasons, glycoengineering of expression platforms is increasingly recognized as an important strategy to improve biopharmaceuticals in many aspects (Dicker and Strasser 2015).

[0006] Endocytic lectins are involved in receptor-mediated endocytosis by capturing glycosylated proteins via specific glycan structures to mediate degradation (Cummings et al., Cold Spring Harbor Laboratory Press, (2017). Endocytic lectins are ubiquitous in humans and can recognize various glycan structures.

[0007] Carbohydrate binding receptors are highly diverse and can be exploited by glycoengineering to develop novel therapeutics with unprecedented effectiveness for different diseases, including but not limited to: inflammatory, blood disorders, autoimmune and cancer. This allows development of novel therapeutics based on the concept of glycan- mediated protein degradation. Leveraging natural protein degradation through the glycosylation of monoclonal antibodies can lead to novel therapeutics. The present invention shows a novel finding of Leishmania host cells are capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2, which mediates protein degradation.

[0008] The compositions and methods provided herein address the unmet medical need of patients suffering from various difficult to treat diseases such as cancer, autoimmune and inflammatory diseases, and infectious diseases, treated with glycosylated proteins, such as monoclonal antibodies, and provide related advantages.

5. SUMMARY OF THE INVENTION

[0009] Provided herein are Leishmania host cells, methods of engineering Leishmania host cells, methods of culturing Leishmania host cells, methods of making a polypeptide of interest using a Leishmania host cell, and polypeptides of interest produced by the methods. [0010] Provided herein is a Leishmania host cell comprising: (a) a recombinant nucleic acid encoding a polypeptide of interest; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases, or functionally active variants thereof. In certain embodiments, the GalNAc transferase is heterologous to the host cell. In certain embodiments, the GalNAc transferase is p4-GalNAcT3, or a functionally active variant thereof. In certain embodiments, the P4-GalNAcT3 is human p4-GalNAcT3, or a functionally active variant thereof. In certain embodiments, the GalNAc transferases are P4-GalNAcT3 and P4-GalNAcT4, or functionally active variants thereof. In certain embodiments, the P4-GalNAcT3 and P4-GalNAcT4 are human p4-GalNAcT3 and P4- GalNAcT4, or functionally active variants thereof.

[0011] In certain embodiments, the host cell further comprises: (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

[0012] In certain embodiments, the host cell further comprises: (a) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

[0013] In certain embodiments, the host cell further comprises: (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc; and (c) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway. [0014] Also provided herein is a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a polypeptide of interest; (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases, or functionally active variants thereof; and (c) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway. In certain embodiments, the host cell further comprises one or more recombinant nucleic acids encoding heterologous UDP-GalNAc biosynthetic pathway proteins capable of generating UDP-GalNAc. In certain embodiments, the host cell further comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc; and/or (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein are co-localized in the secretory pathway. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP- GalNAc transporter protein each independently comprise: (a) a signal peptide localizing the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein in the secretory pathway; and/or (b) a retention sequence retaining the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein in the secretory pathway. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP- GalNAc transporter protein each independently comprise the same signal peptide and/or retention sequence. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein comprise different signal peptides and/or retention sequences. In certain embodiments, the signal peptide of the one or more GalNAc transferases and/or the signal peptide of the heterologous UDP-GalNAc transporter protein are derived from a Leishmania species. In certain embodiments, the retention sequence of the one or more GalNAc transferases and/or the retention sequence of the heterologous UDP- GalNAc transporter protein are derived from a Leishmania species. In certain embodiments, the Leishmania species is Leishmania tarentolae. In certain embodiments, the signal peptide of the one or more GalNAc transferases and/or the signal peptide of the heterologous UDP- GalNAc transporter protein are processed and removed.

[0015] In certain embodiments, the recombinant UDP-GalNAc biosynthetic pathway proteins and/or recombinant UDP-GalNAc transporter protein are heterologous to the host cell. [0016] In certain embodiments, one or more of the recombinant nucleic acids are integrated into the [ssuPolI] locus of the host cell.

[0017] In certain embodiment, the one or more GalNAc transferases are derived from a mammalian source. In certain embodiment, the GalNAc transferase is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens. In certain embodiments, the one or more GalNAc transferases, or functionally active variants thereof, are capable of catalyzing the addition of a GalNAc to a N-acetyl glucosamine- terminated glycan. In certain embodiments, the GalNAc transferase, or a functionally active variant thereof, is capable of catalyzing the addition of a GalNAc to a N-acetyl glucosamine- terminated glycan. In certain embodiments, the one or more GalNAc transferases are selected from the group consisting of p4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or functionally active variants thereof. In certain embodiments, the GalNAc transferase is selected from the group consisting of p4-GalNAcT3, P4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or functionally active variants thereof. In certain embodiments, the one or more GalNAc transferases are selected from the group consisting of P4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the GalNAc transferase is selected from the group consisting of P4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the one or more GalNAc transferases comprise P4-GalNAcT3, or an N- terminally truncated variant thereof. In certain embodiments, the GalNAc transferase is P4- GalNAcT3, or an N-terminally truncated variant thereof. In certain embodiments, the one or more GalNAc transferases comprise p4-GalNAcT3. In certain embodiments, the GalNAc transferase is p4-GalNAcT3. In certain embodiments, the one or more GalNAc transferases comprise an N-terminally truncated variant of P4-GalNAcT3. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of P4-GalNAcT3. In certain embodiments, the N-terminally truncated variant comprises an amino acid sequence of SEQ ID NO: 2. In certain embodiments, the one or more GalNAc transferases comprise a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to p4-GalNAcT3. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT3. In certain embodiments, the one or more GalNAc transferases comprise P4-GalNAcT4, or an N-terminally truncated variant thereof. In certain embodiments, the GalNAc transferase is P4-GalNAcT4, or an N-terminally truncated variant thereof. In certain embodiments, the one or more GalNAc transferases comprise P4- GalNAcT4. In certain embodiments, the GalNAc transferase is P4-GalNAcT4. In certain embodiments, the one or more GalNAc transferases comprise an N-terminally truncated variant of P4-GalNAcT4. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of P4-GalNAcT4. In certain embodiments, the N-terminally truncated variant is comprises an amino acid sequence of SEQ ID NO: 4. In certain embodiments, the one or more GalNAc transferases comprise a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to p4-GalNAcT4. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to p4-GalNAcT4. In certain embodiments, the one or more GalNAc transferases comprise CeP4GalNAcT, or a variant that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the one or more GalNAc transferases comprise CeP4GalNAcT. In certain embodiments, the one or more GalNAc transferases comprise Ptp4GalNAcT, or a variant that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the one or more GalNAc transferases comprise Ptp4GalNAcT.

[0018] In certain embodiments, the recombinant nucleic acid encodes at least two GalNAc transferases. In certain embodiments, the at least two GalNAc transferases are different GalNAc transferases. In certain embodiments, the at least two GalNAc transferases are selected as a combination of GalNAc transferases listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the at least two GalNAc transferases are selected from the group consisting of p4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the at least two GalNAc transferases are human p4-GalNAcT3 and human P4- GalNAcT4; or human p4-GalNAcT3 and CeP4GalNAcT; or human p4-GalNAcT3 and Ptp4GalNAcT; or functionally active variants thereof.

[0019] In certain embodiments, the recombinant nucleic acid encoding one or more GalNAc transferases comprises a first open reading frame (ORF) encoding a first GalNAc transferase and a second ORF encoding a second GalNAc transferase. In certain embodiments, the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in the same genetic module. In certain embodiments, the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in separate genetic modules. In certain embodiments, the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the [ssuPolI] locus of the host cell. In certain embodiments, the first and the second GalNAc transferases are different GalNAc transferases. In certain embodiments, the first and the second GalNAc transferases are selected as a combination of GalNAc transferases listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the first and the second GalNAc transferases are human p4-GalNAcT3 and human p4-GalNAcT4, respectively; or human p4-GalNAcT3 and CeP4GalNAcT, respectively; or human P4- GalNAcT3 and Ptp4GalNAcT, respectively; or functionally active variants thereof.

[0020] In certain embodiments, the host cell comprises a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases. In certain embodiments, the additional recombinant glycosyltransferase is heterologous to the host cell. In certain embodiments, the additional recombinant glycosyltransferase comprises one or more N- acetyl glucosamine transferases. In certain embodiments, the N-acetyl glucosamine transferase is selected from the group consisting of MGAT1 and MGAT2, or functionally active variants thereof. In certain embodiments, the additional recombinant glycosyltransferase comprises MG ATI and MGAT2.

[0021] In certain embodiments, the host cell is capable of producing polypepetides comprising a biantennary, GalNAc-terminated N-glycan. In certain embodiments, the host cell is capable of producing polypeptides comprising an N-glycan of the following structure:

wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the polypeptide of interest. In certain embodiments, the host cell attaches the N-glycan to an N-glycosylation site of the polypeptide. In certain embodiments, the amino acid residue is Asn. In certain embodiments, the N-glycosylation site comprises a consensus sequence of N-X-S/T or N-X- C, wherein X is any amino acid except proline.

[0022] In certain embodiments, one or more endogenous enzymes from the glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated. In certain embodiments, the host cell does not have endogenous N-glycan elongation. In certain embodiments, the host cell has been genetically engineered such that the formation of an O- linked GlcNAc on a polypeptide in the host cell is reduced or eliminated. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania cell prior to genetic engineering is catalyzed by at least one N-acetylglucosamine (GlcNAc)-transferase. In certain embodiments, the gene encoding the at least one GlcNAc-transferase is functionally inactivated, downregulated, deleted, or mutated. In certain embodiments, the formation of the O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a reference Leishmania cell. In certain embodiments, the at least one GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2 and OGNTL, and homologous GlcNAc-transferases thereof. In certain embodiments, the host cell is a OGNT1, OGNT2 and OGNTL triple knockout.

[0023] In certain embodiments, the host cell further comprises a recombinant nucleic acid encoding heterologous UDP-GalNAc biosynthetic pathway proteins capable of generating UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc are derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP- N-acetyl hexosamine pyrophosphorylase (UAP1), or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP- N-acetyl hexosamine pyrophosphorylase (UAP1). In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2), or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP- GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2). In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting UDP-GlcNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting UDP- GlcNAc to UDP-GalNAc are derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens. In certain embodiments, the heterologous UDP- GalNAc biosynthetic pathway proteins comprise hGalE, or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprise hGalE, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise hGalE. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting UDP-GlcNAc to UDP-GalNAc are derived from a bacterial source. In certain embodiments, the bacterial source is Campylobacter jejuni. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne, or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne. [0024] In certain embodiments, the host cell further comprises a recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to the secretory pathway. In certain embodiments, the heterologous UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source, a mammalian source, a brachiopod source, a chordate source, choanoflagellate source, a gyrista source, a fungi source, a mollusk source, or a placozoan source. In certain embodiments, the heterologous UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source. In certain embodiments, the nematode source is C. elegans,' the mammalian source is Homo sapiens,' the brachiopod source is Lingula unguis,' the chordate source is Parambassis ranga, Geotrypetes seraphini, or Scophthalmus maximus,' the choanoflagellate source is Salpingoeca rosetta, the gyrista source is Fragilariopsis cylindrus,' the fungi source is Dentipellis fragilis,' the mollusk source is Octopus bimaculoides; and/or the placozoan source is trichoplax sp. H2. In certain embodiments, the nematode source is C. elegans. In certain embodiments, the heterologous UDP-GalNAc transporter protein is CeC03H5.2, or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc transporter protein is CeC03H5.2, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP- GalNAc transporter protein is CeC03H5.2. In certain embodiments, the heterologous UDP- GalNAc transporter protein is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to CeC03H5.2. In certain embodiments, the heterologous UDP-GalNAc transporter protein is GnF, GnG, GnH, GnI, or GnJ. In certain embodiments, the heterologous UDP-GalNAc transporter protein is UGTREL7, or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc transporter protein is UGTREL7, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc transporter protein is UGTREL7. In certain embodiments, the heterologous UDP-GalNAc transporter protein is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to UGTREL7. In certain embodiments, the heterologous UDP-GalNAc transporter protein is GnL, GnM, GnN, or GnO.

[0025] In certain embodiments, the recombinant nucleic acid encodes at least two heterologous UDP-GalNAc transporter proteins. In certain embodiments, the recombinant nucleic acid encodes two copies of the same heterologous UDP-GalNAc transporter protein. In certain embodiments, the recombinant nucleic acid encodes two different heterologous UDP-GalNAc transporter proteins. In certain embodiments, the heterologous UDP-GalNAc transporter proteins are selected as a combination of heterologous UDP-GalNAc transporters listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2, GnF, GnG, GnH, GnI, GnJ, UGTREL7, GnL, GnM, GnN, or GnO, or functionally active variants thereof. In certain embodiments, the heterologous UDP-GalNAc transporter proteins comprise CeC03H5.2. In certain embodiments, the heterologous UDP- GalNAc transporter protein is CeC03H5.2. In certain embodiments, the heterologous UDP- GalNAc transporter proteins are CeC03H5.2 and GnJ.

[0026] In certain embodiments, the recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter comprises a first ORF encoding a first heterologous UDP-GalNAc transporter and a second ORF encoding a second heterologous UDP-GalNAc transporter. In certain embodiments, the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module. In certain embodiments, the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in separate genetic modules. In certain embodiments, the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP- GalNAc transporter are integrated into the [ssuPolI] locus of the host cell. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter are selected as a combination of heterologous UDP-GalNAc transporters listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are the same UDP-GalNAc transporter protein. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are each CeC03H5.2. In certain embodiments, the first and the second heterologous UDP- GalNAc transporter proteins are different UDP-GalNAc transporter proteins. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are CeC03H5.2 and GnJ, respectively. [0027] In certain embodiments, the recombinant nucleic acids encode at least two GalNAc transferases and at least two heterologous UDP-GalNAc transporter proteins. In certain embodiments, the at least two GalNAc transferases are different. In certain embodiments, the recombinant nucleic acids encode two copies of the same heterologous UDP-GalNAc transporter protein. In certain embodiments, the recombinant nucleic acids encode two different heterologous UDP-GalNAc transporter proteins. In certain embodiments, the at least two GalNAc transferases and at least two heterologous UDP- GalNAc transporter proteins are selected as a combination of GalNAc transferases and heterologous UDP-GalNAc transporter proteins listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof In certain embodiments, the recombinant nucleic acids encode human P4-GalNAcT3, CeP4GalNAcT, CeC03H5.2, and GnJ. In certain embodiments, the recombinant nucleic acids encode human p4-GalNAcT3, Ptp4GalNAcT, and CeC03H5.2. [0028] In certain embodiments, the host cell is such that (a) the recombinant nucleic acid encoding one or more GalNAc transferases comprises a first ORF encoding a first GalNAc transferase and a second ORF encoding a second GalNAc transferase; and (b) b. the recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter comprises a third ORF encoding a first heterologous UDP-GalNAc transporter and a fourth ORF encoding a second heterologous UDP-GalNAc transporter. In certain embodiments, the host cell is such that (a) the first open reading frame (ORF) encoding the first GalNAc transferase and and the third ORF encoding the first heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module; and/or (b) the second open reading frame (ORF) encoding the second GalNAc transferase and and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module. In certain embodiments, the host cell is such that (a) the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in separate genetic modules; and/or (b) the third ORF encoding the first heterologous UDP-GalNAc transporter and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in separate genetic modules. In certain embodiments, the first ORF encoding the first GalNAc transferase, the second ORF encoding the second GalNAc transferase, the third ORF encoding the first heterologous UDP-GalNAc transporter, and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module. In certain embodiments, the first ORF encoding the first GalNAc transferase, the second ORF encoding the second GalNAc transferase, the third ORF encoding the first heterologous UDP-GalNAc transporter, and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the [ssuPolI] locus of the host cell. In certain embodiments, the first and the second GalNAc transferases are different GalNAc transferases. In certain embodiments, the first and the second heterologous UDP- GalNAc transporter proteins are the same UDP-GalNAc transporter protein. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are different UDP-GalNAc transporter proteins. In certain embodiments, the first GalNAc transferase, the second GalNAc transferase, the first heterologous UDP-GalNAc transporter protein, and the second heterologous UDP-GalNAc transporter protein are selected as a combination of GalNAc transferases and heterologous UDP-GalNAc transporter proteins listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the first and the second GalNAc transferases are human p4-GalNAcT3 and CeP4GalNAcT, respectively; and the first and the second heterologous UDP-GalNAc transporter proteins are CeC03H5.2, and GnJ, respectively. In certain embodiments, the first and the second GalNAc transferases are human p4-GalNAcT3 and Ptp4GalNAcT, respectively; and the first and the second heterologous UDP-GalNAc transporter proteins are each CeC03H5.2.

[0029] In certain embodiments, the one or more GalNAc transferases and the polypeptide of interest are co-localized in the secretory pathway. In certain embodiments, the GalNAc transferase and the polypeptide of interest are co-localized in the secretory pathway. In certain embodiments, the one or more GalNAc transferases and the additional recombinant glycosyltransferase are co-localized in the secretory pathway. In certain embodiments, the GalNAc transferase and the additional recombinant glycosyltransferase are co-localized in the secretory pathway. In certain embodiments, the one or more GalNAc transferases each independently comprise a signal peptide localizing the one or more GalNAc transferases in the secretory pathway. In certain embodiments, the one or more GalNAc transferases each independently comprise a retention sequence retaining the one or more GalNAc transferases in the secretory pathway. In certain embodiments, the GalNAc transferase comprises a signal peptide localizing the GalNAc transferase in the secretory pathway. In certain embodiments, the additional recombinant glycosyltransferase comprises a signal peptide localizing the additional recombinant glycosyltransferase in the secretory pathway. In certain embodiments, the additional recombinant glycosyltransferase comprises a retention sequence retaining the additional recombinant glycosyltransferase in the secretory pathway. In certain embodiments, the signal peptide is added to an N-terminally truncated variant of the GalNAc transferase and/or the additional recombinant glycosyltransferase. In certain embodiments, the retention sequence is added to an N-terminally truncated variant of the GalNAc transferase and/or the additional recombinant glycosyltransferase. In certain embodiments, the polypeptide of interest comprises a signal peptide localizing the polypeptide of interest to the secretory pathway and/or a retention sequence retaining the polypeptide of interest in the secretory pathway. In certain embodiments, the signal peptide and/or the retention sequence is derived from Leishmania species. In certain embodiments, the signal peptide is derived from Leishmania tarentolae. In certain embodiments, the signal peptide is an invertase signal peptide from derived from Leishmania tarentolae. In certain embodiments, the signal peptide is processed and removed.

[0030] In certain embodiments, the host cell is a Leishmania tarentolae host cell. In certain embodiments, culturing the Leishmania host cell produces a composition of the polypeptide of interest, wherein said composition of the polypeptide of interest comprises an N-glycan of the following structure:

wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the polypeptide of interest. In certain embodiments, the host cell is stable to passaging and/or continuous fermentation for 100 or more generations.

[0031] In certain embodiments, the host cell is any one of the strains listed in Table 3 or

Table 9 5.1 Definitions

[0032] As used herein and unless otherwise indicated, the term “about,” when used in conjunction with a number, refers to any number within ±1, ±5 or ±10% of the referenced number.

[0033] As used herein and unless otherwise indicated, the term “subject” refers to an animal (e.g., birds, reptiles, and mammals). In another embodiment, a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In certain embodiments, a subject is a non-human animal. In some embodiments, a subject is a farm animal or pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken). In a specific embodiment, a subject is a human. The terms “subject” and “patient” may be used herein interchangeably.

[0034] As used herein and unless otherwise indicated, the term “effective amount,” in the context of administering a therapy (e.g, a composition described herein) to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s). In certain embodiments, an “effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a disease/disorder or symptom associated therewith; (ii) reduce the duration of a disease/disorder or symptom associated therewith; (iii) prevent the progression of a disease/disorder or symptom associated therewith; (iv) cause regression of a disease/disorder or symptom associated therewith; (v) prevent the development or onset of a disease/disorder, or symptom associated therewith; (vi) prevent the recurrence of a disease/disorder or symptom associated therewith; (vii) reduce organ failure associated with a disease/disorder; (viii) reduce hospitalization of a subject having a disease/disorder; (ix) reduce hospitalization length of a subject having a disease/disorder; (x) increase the survival of a subject with a disease/disorder; (xi) eliminate a disease/disorder in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0035] As used herein and unless otherwise indicated, proteins referred to herein as “colocalized in the secretory pathway” have at least partially overlapping subcellular distribution in the secretory pathway, for example in the endoplasmic reticulum or Golgi apparatus. In certain embodiments, the subcellular distribution of one protein referred to herein as “colocalized in the secretory pathway” with another protein is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% overlapping with with the subcellular distribution of the other protein. Without being bound by theory, in certain embodiments, the overlapping subcellular distribution of two or more proteins referred to herein as “co-localized in the secretory pathway” may be attributed to a signal peptide localizing and/or a retention sequence retaining the two or more proteins in the secretory pathway, for example in the endoplasmic reticulum or Golgi apparatus.

5.2 Conventions and Abbreviations

5.3 \-Glycan Nomenclature

' Mx: number (x) of residues within the oligomannose series; Ax: number (x) of antennae; F: core fucose; Gx: number (x) of galactoses; S: number (x) of sialic acids. Note: Linkage information is given in () parentheses if applicable, e.g. A2G1S1(6) - a2-6 linked sialic acid. Brackets [x] before G or GalNAc indicate which arm of the mannosyl core is galactosylated e.g. [3] G1 indicates that the galactose is on the antenna of the al-3 mannose. ²This typically with IgG associated naming system indicates the presence of core fucose (F), the number of galactoses (G) and the presence of biantennary glycans. It is limited in the number of structures and linkages it can describe but is often used for simplicity. ³ black circle represents mannose (Man), white square is N-acetyl glucosamine (GlcNAc), black square is N-acetyl galactosamine (GalNAc), white circle is galactose (Gal), white diamond is sialic acid, N-acetyl neuraminic acid (Neu5Ac) and white triangle is fucose (Fuc).

6. BRIEF DESCRIPTION OF THE FIGURES

[0036] FIG. 1 depicts the general pathways of UDP-GlcNAc, UDP-GalNAc and UDP- Gal biosynthesis. The enzymes involved in each step are shown besides the arrows. Galactokinase (GK), UDP-sugar pyrophosphorylase (USP), UDP -galactose 4-epimerase (GalE), hexokinase (HXK), phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGP) are identified in Leishmania tarentolae genome and presumed biosynthetic steps are indicated with black arrows. Enzymes not being present in L. tarentolae genome are shown in italics with biosynthesis steps shown as dashed arrows: N- acetylglucosamine kinase (NAGK), human UDP -galactose 4-epimerase (hGalE), Campylobacter jejuni UDP-GlcNAc/Glc 4-epimerase (CjGne), N-acetyl galactosamine kinase (GALK2), UDP -N-acetyl hexosamine pyrophosphorylase (UAP1), Beta-1, 4-N- acetylgalactosaminyltransferase T3 (|34-GalNAcT3), Beta-1,4-N- acetylgalactosaminyltransferase T4 (|34-GalNAcT4), and Beta- 1,4-galactosyltransferase 1 (P4GalTl). These enzymes or transferases chosen for recombinant expression are shown in italics, bold and underlined. The resulting biantennary glycan structure attached to a N-X- S/T polypeptide site is shown, with black circle representing mannose (Man), white square representing N-acetyl glucosamine (GlcNAc), black square representing N-acetyl galactosamine (GalNAc), and white circle representing galactose (Gal).

[0037] FIG. 2 depicts UDP-GalNAc biosynthetic pathways and GalNAc-transferases for engineering host cells. The enzymatic activities are shown besides the arrows. Human UDP- galactose 4-epimerase (hGalE), Campylobacter jejuni UDP-GlcNAc/Glc 4-epimerase (CjGne), N-acetyl galactosamine kinase (GALK2), UDP-N-acetyl hexosamine pyrophosphorylase (UAP1), Beta-l,4-N-acetylgalactosaminyltransferase T3 (|34-GalNAcT3), Beta-l,4-N-acetylgalactosaminyltransferase T4 (|34-GalNAcT3), and Beta-1,4- N-acetyl galactosaminyltransferase (CeGalNAcT). The enzymes or transferases chosen for recombinant expression are shown in italics, bold and underlined. The N-glycan is depicted with black circle representing mannose (Man), white square representing N-acetyl glucosamine (GlcNAc), and black square representing N-acetyl galactosamine (GalNAc). [0038] FIG. 3A depicts UDP-GalNAc measurements in Leishmania tarentolae. A standard containing UDP-GalNAc and UDP-GlcNAc is indicated with sold grey line, L. tarentolae wt extract is shown as solid black line, and the same extract spiked with UDP- GalNAc and UDP-GlcNAc standard in a High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) measurement is shown as dotted black line.

[0039] FIG. 3B depicts UDP-GalNAc measurement of engineered Leishmania tarentolae host cells containing recombinant proteins for UDP-GalNAc biosynthetic pathways.

Different cell lines contain different combinations of recombinant enzymes for the different UDP-GalNAc biosynthetic pathways, for example Transporter +Salvage + Epimerase “TSE”; or Transporter + Salvage (“TS”); or Transporter-i- Epimerization (“TE”) with genetic constructs of coding sequences spaced by different intergenic regions derived from Leishmania major (J, AC vs. Y, Z) and different site-specific integration loci (pfr vs. aTub). Only two strains StCGP2805 and St2848, shown with black and black dashed lines respectively, were supplemented with lOmM GalNAc to the growth medium and UDP- GalNAc peaks were detected. This indicates that the salvage pathway is active when supplemented with GalNAc during growth. Additionally, the epimerization pathway alone (StCGP2807) or in combination with salvage pathway such as StCGP2801, 2803, 2847, which were not fed with GalNAc during growth, did not generate measurable amounts of UDP-GalNAc, or if at all at very low levels. [0040] FIG 4A depicts the recombinant pathway engineered in Leishmania tarentolae strain StCGP2879.

[0041] FIG. 4B depicts high levels of A2GalNAc2 N-glycans produced by engineered Leishmania tarentolae StCGP2879 containing UDP-GalNAc biosynthetic pathways and GalNAc-transferases. Strain StCGP2879 secreting adalimumab antibody was analyzed for its N-glycan composition on its Fab and Fc glycosites. Fab glycosites show 80% A2GalNAc2 N-glycans analyzed by HILIC-UPLC-MS (Top) and N297 canonical Fc glycosite show the main N-glycan A2 (bottom). The N-glycan structure is shown with black circle representing mannose (Man), white square representing N-acetyl glucosamine (GlcNAc), and black square representing N-acetyl galactosamine (GalNAc).

[0042] FIG. 5 depicts a flowchart of CGP engineered host cell lines. The left panel indicates the main N-glycan (M3, A2 or A2GalNAc2) for the strains listed on the right. Strains are in bold. Arrows show the genetic modification step with brief description of the genetic module in italics along with the selection marker, and with the genomic integration locus in brackets. Intergenic regions (IRs) are indicated where relevant. Boxes at the bottom show the quantitative content of A2GalNAc2 on adalimumab (“Fab glycosite”) or endogenous surface glycoproteins (“cell surface”), andn.d. means not determined for respective strain.

[0043] FIG. 6 depicts another flowchart of CGP engineered host cell lines. The left panel indicates the main N-glycan (M3, A2 or A2GalNAc2) for the strains listed on the right.

Strains are in bold. Arrows show the genetic modification step with brief description of the genetic module in italics along with the selection marker, and with the genomic integration locus in brackets. Dashed arrows represent the intended integration of polypeptide of interest or mAb expression modules.

[0044] FIG. 7A depicts N-glycan traces of Fab part of Adalimumab (K84N/D86N) secreted from StCGP3127 CGP engineered host cell lines. N-glycans (A2, A2GalNacl and A2GalNAc2) were analyzed by HILIC-UPLC-MS from Adalimumab (K84N/D86N) in strain StCGP3127, which was derived from StCGP3053 (OGNT+). Adalimumab was cleaved with IdeZ, and the resulting subunits to F(ab’)2 and Fc/2 separated by SDS-PAGE, released by PNGaseF and procainamide labelled. The N-glycan structure is shown with black circle representing mannose (Man), white square representing N-acetyl glucosamine (GlcNAc), and black square representing N-acetyl galactosamine (GalNAc).

[0045] FIG. 7B depicts N-glycan traces of Fab part of Adalimumab (K84N/D86N) secreted from StCGP3220 CGP engineered host cell lines. N-glycans (A2, A2GalNacl and A2GalNAc2) were analyzed by HILIC-UPLC-MS from Adalimumab (K84N/D86N) from StCGP3220, which was derived from strain StCGP3169 (OGNT triple deletion mutant). Adalimumab was cleaved with IdeZ, and the resulting subunits to F(ab’)2 and Fc/2 separated by SDS-PAGE, released by PNGaseF and procainamide labelled. The N-glycan structure is shown with black striped circle representing mannose (Man), white square representing N- acetyl glucosamine (GlcNAc), and black square representing N-acetyl galactosamine (GalNAc).

[0046] FIG. 8A depicts N-glycan traces of Fab part of mAb 5C9 (D86N) secreted from CGP engineered host cell lines. FIG. 8B depicts N-glycan traces of Fab part of mAb 1 lk2 (D86N) secreted from the same CGP engineered host cell lines. N-glycans (A2, A2GalNAcl and A2GalNAc2) were analyzed by HILIC-UPLC from two different IgG4 antibodies, containing engineered glycosite at LC, D86N. The same position of the glycosite within the Framework Region (FR) 3 in the light chain variable domain of the different antibodies show varying degrees of A2GalNAc2 content, when expressed and secreted from the same parental cell line, namely StCGP3169. N-glycans were analyzed from reduced LC, which was separated by SDS-PAGE, released by PNGaseF and procainamide labelled. The N-glycan structure is shown with black circle representing mannose (Man), white square representing N-acetyl glucosamine (GlcNAc), and black square representing N-acetyl galactosamine (GalNAc).

[0047] FIG. 9A depicts N-glycan distribution (%) of surface glycoproteins derived from CGP engineered host cell lines encoding different GalNAc transferases. FIG. 9B depicts N- glycan distribution (%) of mAb Adalimumab (K84N/D86N) derived from CGP engineered host cell lines encoding different GalNAc transferases. FIG. 9C depicts N-glycan distribution (%) of mAb 1 lk2 (S84N/D86N) derived from CGP engineered host cell lines encoding different GalNAc transferases. N-glycans (A2, A2GalNAcl and A2GalNAc2) were analyzed by HILIC-UPLC and quantified, from cell surface glycoprotein of the empty parental strains and from two different mAbs, glycan distribution derived from the Fab of Adali (K84N/D86N), and total N-glycans (“in solution”, includes Fc N297 site) of 1 lk2 (S84N/D86N). The different GalNAc transferases from the parental different parental strains are indicated at the bottom of the corresponding graphs, indicating varying efficiency in A2GalNAcl and A2GalNAc2 conversion on different native (cell surfaces glycoproteins) or engineered glycosite on the different mAbs.

[0048] FIG. 10A depicts a flowchart of engineered host cell lines testing different UDP- GalNAc transporter CeC03H5.2 and selected homologs GnF, GnG, GnH, GnI and GnJ (Table 10) in the StCGP4334 parental cell line background. StCGP4334 contains a GalNAc synthesis module with salvage and epimerization enzymes (“S+E”) and the HsGalNAcT3 transferase, integrated into the ssu-PolI genomic locus; and Adalimumab (K84N/D86N). The left panel indicates the main N-glycan (M3, A2 or A2GalNAc2) for the strains listed on the right. Strain designations are in bold. Arrows show the genetic modification step with brief description of the genetic module in italics along with the selection marker, and with the genomic integration locus in brackets.

[0049] FIG. 10B depicts a flowchart of engineered host cell lines testing different UDP- GalNAc transporter UGTREL7 and selected homologs GnL, GnM, GnN and GnO (Table 11) in the StCGP4334 parental cell line background. StCGP4334 contains a GalNAc synthesis module with salvage and epimerization enzymes (“S+E”) and the HsGalNAcT3 transferase, integrated into the ssu-PolI genomic locus; and Adalimumab (K84N/D86N). The left panel indicates the main N-glycan (M3, A2 or A2GalNAc2) for the strains listed on the right. Strain designations are in bold. Arrows show the genetic modification step with brief description of the genetic module in italics along with the selection marker, and with the genomic integration locus in brackets.

[0050] FIG. 11 depicts A2GalNAc2 N-glycans in % produced by engineered Leishmania tarentolae StCGP4334 (parental cell line) and cell lines modified with putative UDP-GalNAc transporters (indicated in parentheses in the following). Left Panel: Strains StCGP5003 (+CeC03H5.2), StCGP5006 (+GnF), StCGP5007 (+GnG), StCGP5010 (+GnH), StCGP5012 (with GnI), and StCGP5013(+GnJ). Right Panel: Strains StCGP5016 (+UGTREL7), StCGP5017 (+GnL), StCGP5049 (+GnM), StCGP5019 (+GnN), and StCGP5021(+GnO). These cell lines, each secreting Adalimumab (K84N/D86N), were analyzed for the N-glycan composition on the Fab glycosites. Bars represent N-glycan composition in % of A2, A2GalNAcl and A2GalNAc2, released from Adalimumab Fab glycosites and analyzed by HILIC-UPLC-MS.

[0051] FIG. 12 depicts a flowchart of engineered host cell lines testing UDP-GalNAc transporter homologs in the StCGP4106 parental cell line background, that already contains Adalimumab (K84N/D86N) by introducing a combined module that contains the UDP- GalNAc biosynthesis pathway (T+S+E), where T is the designated GalNAc transporter or homolog (either CeC03H5.2, GnF, GnJ or GnM, respectively) along with the PtGalNAc transferase. The left panel indicates the main N-glycan (M3, A2 or A2GalNAc2, respectively) for the strains listed on the right. Strain designations are in bold. Arrows show the genetic modification step with brief description of the genetic module in italics along with the selection marker, and with the genomic integration locus in brackets.

[0052] FIG. 13 depicts A2GalNAc2 N-glycans in %, produced by engineered Leishmania tarentolae StCGP4978, StCGP5351, StCGP5352, and StCGP5694 expressing putative UDP-GalNAc transporters, the PtGalNAcT transferase and each of them secreting Adalimumab (K84N/D86N). Fab glycosites were evaluated after Protein A purification for the N-glycan composition and bars represent N-gly can % of A2, A2GalNAcl and A2GalNAc2, released from Adalimumab Fab glycosites and analyzed by HILIC-UPLC-MS. [0053] FIG. 14 shows the flowchart of engineering entry host cell lines StCGP5359 and StCGP5942. The left panel indicates the main N-glycan (M3, A2 or A2GalNAc2, respectively) for the strains listed on the right. Strain designations are in bold. Arrows show the genetic modification step with brief description of the genetic module in italics along with the selection marker, and with the genomic integration locus in brackets. Dashed arrow indicates that any polypeptide (“target proteins”, mAbs, Ab scaffolds, etc.) containing N- glycosylation consensus site(s) can be introduced in described host cell lines to produce polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2, which mediates protein degradation.

[0054] FIG. 15 shows A2GalNAc2 N-glycans analyzed by HILIC-UPLC-MS (Top Panel) and quantified in % (Bottom Panel), from engineered Leishmania tarentolae StCGP6044, expressing a VHH containing a C-terminal glycotag, which is a peptide stretch harboring a N-glycosylation consensus site N-X-S/T. StCGP6044 is a representative for the performance of entry cell line StCGP5359, which does not express a target polypeptide. StCGP6044 was continuously passaged for 37 and 66 generations, respectively, without addition of any selection pressure. VHH was affinity -purified for N-glycan analysis and bars represent N-glycan % of A2, A2GalNAcl and A2GalNAc2, released from purified protein (Graph at Bottom Panel).

[0055] FIG. 16 shows A2GalNAc2 N-glycans analyzed by HILIC-UPLC-MS (Top Panel) and quantified in % (Bottom Panel), from engineered Leishmania tarentolae StCGP6631, expressing a VHH containing a C-terminal glycotag, which is a peptide stretch harboring a N-glycosylation consensus site N-X-S/T. StCGP663 l is a representative for the performance of entry cell line StCGP5942, which does not express a target polypeptide. StCGP6631 was continuously passaged for 35 and 63 generations, respectively, without addition of any selection pressure. VHH was affinity -purified for N-glycan analysis and bars represent N-glycan % of A2, A2GalNAcl and A2GalNAc2, released from purified protein (Graph at Bottom Panel).

7. DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention relates to Leishmania host cells, methods of engineering Leishmania host cells, methods of culturing Leishmania host cells, methods of making a polypeptide of interest using a Leishmania host cell, and polypeptides of interest produced by the methods described herein.

7.1 Leishmania Host Cells

[0057] Provided herein are Leishmania host cells comprising: (a) a recombinant nucleic acid encoding a polypeptide of interest; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the Leishmania host cells provided herein are capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan. In particular, the Leishmania host cells provided herein are capable of producing polypeptides comprising an N-glycan of the following structure:

wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the polypeptide of interest.

[0058] In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases described in Section 7.1.1. In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases described in Section 7.1.2. In certain embodiments, one or more endogenous enzymes described in Section 7.1.3 from the glycan biosynthesis pathway of the the Leishmania host cells provided herein have been deleted, mutated and/or functionally inactivated. In certain embodiments, the Leishmania host cells provided herein further comprise a recombinant nucleic acid encoding heterologous UDP- GalNAc biosynthetic pathway proteins as described in Section 7.1.4 capable of generating UDP-GalNAc. In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.1.5 capable of transporting UDP-GalNAc to the secretory pathway. In certain embodiments, the strain of the Leishmania host cells provided herein is described in Section 7.1.6. In certain embodiments, the Leishmania host cells provided herein below are genetically engineered using the methods described in Section 7.2. In certain embodiments, the Leishmania host cells provided herein below are cultured according to the methods described in Section 7.3. In certain embodiments, the Leishmania host cells provided herein may be used as an expression system as described in Section 7.4. In certain embodiments, the Leishmania host cells provided herein may be used to make a polypeptide of interest as described in Section 7.5.

7.1.1 N-Acetylgalactosamine (GalNAc) Transferases

[0059] The Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the GalNAc transferase, or a functionally active variant thereof, is capable of catalyzing the addition of a GalNAc to a N-acetyl glucosamine-terminated glycan.

[0060] In certain embodiments, the GalNAc transferase is heterologous to the Leishmania host cell. In certain embodiments, the GalNAc transferase is derived from Homo sapiens, Caenorhabditis elegans, Parasteatoda lepidarionim, Salmo Irulla, or Hucho hucho. In certain embodiments, the GalNAc transferase is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens.

[0061] In certain embodiments, the GalNAc transferase is selected from the group consisting of P4-GalNAcT3, [34-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or functionally active variants thereof. In certain embodiments, the GalNAc transferase is selected from the group consisting of P4-GalNAcT3, P4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. [0062] In certain embodiments, the GalNAc transferase comprises P4-GalNAcT3, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises P4-GalNAcT3. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT3. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of P4-GalNAcT3. In certain embodiments, the P4-GalNAcT3 comprises P4-GalNAcT3 of Homo sapiens, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises P4-GalNAcT3 of Homo sapiens. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 1. In certain embodiments, the GalNAc transferase comprises one that is homologous to P4-GalNAcT3 of Homo sapiens. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT3 of Homo sapiens. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of P4- GalNAcT3 of Homo sapiens comprising an amino acid sequence of SEQ ID NO: 2.

[0063] In certain embodiments, the GalNAc transferase comprises P4-GalNAcT4, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises P4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of P4-GalNAcT4. In certain embodiments, the P4-GalNAcT4 comprises P4-GalNAcT4 of Homo sapiens, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises P4-GalNAcT4 of Homo sapiens. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 3. In certain embodiments, the GalNAc transferase comprises one that is homologous to P4-GalNAcT4 of Homo sapiens. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT4 of Homo sapiens. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of P4- GalNAcT4 of Homo sapiens comprising an amino acid sequence of SEQ ID NO: 4.

[0064] In certain embodiments, the GalNAc transferases comprise P4-GalNAcT3 and P- GalNAcT4, or functionally active variants thereof. In certain embodiments, the GalNAc transferases comprise P4-GalNAcT3 and P4-GalNAcT4. In certain embodiments, the GalNAc transferases comprise variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT3 and P-GalNAcT4, respectively. In certain embodiments, the GalNAc transferases comprise N-terminally truncated variants of P4-GalNAcT3 and/or [34-GalNAcT4. In certain embodiments, the GalNAc transferases comprise P4-GalNAcT3 and P4-GalNAcT4 of Homo sapiens, or functionally active variants thereof. In certain embodiments, the GalNAc transferases comprise P4-GalNAcT3 and P4-GalNAcT4 of Homo sapiens. In certain embodiments, the GalNAc transferases comprise amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3. In certain embodiments, the GalNAc transferases comprise ones that are homologous to 4- GalNAcT3 and P4-GalNAcT4 of Homo sapiens. In certain embodiments, the GalNAc transferases comprise variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to P4-GalNAcT3 and P4-GalNAcT4 of Homo sapiens, respectively. In certain embodiments, the GalNAc transferases comprise N- terminally truncated variants of P4-GalNAcT3 and/or [34-GalNAcT4 of Homo sapiens comprising amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. [0065] In certain embodiments, the GalNAc transferase is CeP4GalNAcT, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is CeP4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to CeP4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of CeP4GalNAcT. In certain embodiments, the CeP4GalNAcT is a P4GalNAcT of Caenorhabditis elegans, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is a P4GalNAcT of Caenorhabditis elegans. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 5. In certain embodiments, the GalNAc transferase is one that is homologous to a CeP4GalNAcT of Caenorhabditis elegans. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to a CeP4GalNAcT of Caenorhabditis elegans. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of CeP4GalNAcT of Caenorhabditis elegans comprising an amino acid sequence of SEQ ID NO: 6. [0066] In certain embodiments, the GalNAc transferase is Ptp4GalNAcT, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Ptp4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Ptp4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of Ptp4GalNAcT. In certain embodiments, the Ptp4GalNAcT is a P4GalNAcT of Parasteatoda tepidariorum. or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is a Ptp4GalNAcT of Parasteatoda tepidariorum. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 7. In certain embodiments, the GalNAc transferase is one that is homologous to a Ptp4GalNAcT of Parasteatoda tepidariorum. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to a Ptp4GalNAcT of Parasteatoda tepidariorum. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of Ptp4GalNAcT of Parasteatoda tepidariorum comprising an amino acid sequence of SEQ ID NO: 8.

[0067] In certain embodiments, the GalNAc transferase is Stp4GalNAcT, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Stp4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Stp4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of Stp4GalNAcT. In certain embodiments, the Stp4GalNAcT is a P4GalNAcT of Salmo IruUa. or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is a p4GalNAcT of Salmo trutta. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 9. In certain embodiments, the GalNAc transferase is one that is homologous to a P4GalNAcT of Salmo trutta. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to a p4GalNAcT of Salmo trutta.

[0068] In certain embodiments, the GalNAc transferase is Hhp4GalNAcT, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Hhp4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Hhp4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of Hhp4GalNAcT. In certain embodiments, the Hhp4GalNAcT is a P4GalNAcT of Hucho hucho, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is a P4GalNAcT of Hucho hucho. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the GalNAc transferase is one that is homologous to a p4GalNAcT of Hucho hucho. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to a P4GalNAcT of Hucho hucho.

[0069] Without being bound by theory, combinations of at least two GalNAc transferases can be beneficial for optimizing the glycosylation profile of polypetides of interest, for example by increasing the extent and/or homogeneity of glycoylsation with N-glycan(s) described herein. In certain embodiments, the recombinant nucleic acid encodes at least two GalNAc transferases. In certain embodiments, the recombinant nucleic acid encodes two GalNAc transferases. In certain embodiments, the at least two GalNAc transferases are different GalNAc transferases. In certain embodiments, the at least two GalNAc transferases are selected from the group consisting of p4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the at least two GalNAc transferases are human p4-GalNAcT3 and human P4- GalNAcT4; or human p4-GalNAcT3 and CeP4GalNAcT; or human p4-GalNAcT3 and Ptp4GalNAcT; or functionally active variants thereof.

[0070] In certain embodiments, the GalNAc transferase is localized in the secretory pathway. Without being bound by theory, localization within the secretory pathway includes, but is not limited to, localization to one or more of the following sub-cellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface anchored proteins, and membrane proteins. In certain embodiments, localization in the secretory pathway comprises localization to one or more of said sub-cellular compartments.

[0071] In certain embodiments, the GalNAc transferase comprises a signal peptide localizing the GalNAc transferase in the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the GalNAc transferase (i.e. the signal peptide is not added to the GalNAc transferase, but is one contained in the GalNAc transferase when naturally expressed in the source). In certain embodiments, the GalNAc transferase is localized in the secretory pathway without adding Leishmania signal peptide to the GalNAc transferase. In other embodiments, the signal peptide is added to the GalNAc transferase. In certain embodiments, the signal peptide is fused to the C-terminus of the GalNAc transferase. In certain embodiments, the signal peptide is fused to the N-terminus of the GalNAc transferase. In certain embodiments, the signal peptides is fused to one or more amino acids within the polypeptide of the GalNAc transferase. In certain embodiments, the signal peptide is fused to the N-terminus of an N-terminally truncated variant of the GalNAc transferase. In certain embodiments, the signal peptide is fused to one or more amino acids within the polypeptide of an N-terminally truncated variant of the GalNAc transferase. In certain embodiments, the signal peptide is derived from Leishmania species. In certain embodiments, the signal peptide is a modified version of a signal peptide derived from a Leishmania species. In certain embodiments, the signal peptide is derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of a signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide is an invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of the invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the GalNAc transferase.

[0072] In certain embodiments, the GalNAc transferase comprises a retention sequence retaining the GalNAc transferase in the secretory pathway. In certain embodiments, the retention sequence is derived from the same source as the GalNAc transferase (i.e. the retention sequence is not added to the GalNAc transferase, but is one contained in the GalNAc transferase when naturally expressed in the source). In certain embodiments, the GalNAc transferase is retained in the secretory pathway without adding a Leishmania retention sequence to the GalNAc transferase. In other embodiments, the retention sequence is added to the GalNAc transferase. In certain embodiments, the retention sequence is fused to the C-terminus of the GalNAc transferase. In certain embodiments, the retention sequence is fused to the N-terminus of the GalNAc transferase. In certain embodiments, the retention sequence is fused to one or more amino acids within the polypeptide of the GalNAc transferase. In certain embodiments, the retention sequence is fused to the N-terminus of an N-terminally truncated variant of the GalNAc transferase. In certain embodiments, the retention sequence is fused to one or more amino acids within the polypeptide of an N- terminally truncated variant of the GalNAc transferase. In certain embodiments, the retention sequence is derived from a Leishmania species. In certain embodiments, the retention sequence is a modified version of a retention sequence from derived from a Leishmania species. In certain embodiments, the retention sequence is derived from Leishmania tarentolae. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from Leishmania tarentolae.

[0073] In certain embodiments, the GalNAc transferase and the recombinant UDP- GalNAc transporter protein described in Section 7.1.5 are co-localized in the secretory pathway. In certain embodiments, the GalNAc transferase and the additional recombinant glycosyltransferase described in Section 7.1.2 are co-localized in the secretory pathway. In certain embodiments, the GalNAc transferase and the polypeptide of interest described in Section 7.5 are co-localized in the secretory pathway.

7.1.2 Additional Recombinant Glycosyltransferases

[0074] In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases. In certain embodiments, the additional recombinant glycosyltransferase, or a functionally active variant thereof, is capable of catalyzing the addition of a first glycan to a second glycan. In certain embodiments, the additional recombinant glycosyltransferase is a N-acetyl glucosamine transferase, or a functionally active variant thereof, capable of catalyzing the addition of a N-acetyl glucosamine (GlcNAc) to a mannose-terminated glycan, for example, a Man3GlcNAc2 glycan (Man3, see Section 5.3).

[0075] In certain embodiments, the additional recombinant glycosyltransferase comprises one or more N-acetyl glucosamine transferases. In certain embodiments, the N-acetyl glucosamine transferase is heterologous to the host cell. In certain embodiments, the N- acetyl glucosamine transferase is derived from Homo sapiens, Spodoptera frugiperda, Trypanosoma brucei. Pan troglodytes, Macaca mulatto, Mus musculus, Rattus norvegicus, Danio rerio A, Drosophila melanogaster, Anopheles gambiae, Caenorhabditis elegans, Arabidopsis thaliana, Oryza sativa Japonica, Xenopus tropicalis, Canis lupus, Bos taurus, Danio rerio B, or Gekko japonicus. In certain embodiments, the additional recombinant glycosyltransferase is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens.

[0076] In certain embodiments, the N-acetyl glucosamine transferase is selected from the group consisting of MGAT1 (alpha-1, 3-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransf erase) and MGAT2 (alpha- 1,6-mannosylgly coprotein 2-beta-N- acetylglucosaminyltransferase), or functionally active variants thereof. In certain embodiments, the additional recombinant glycosyltransferases comprise MGAT1 and MGAT2.

[0077] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1, or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Homo sapiens (accession number P26572), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferase comprises an amino acid sequence of SEQ ID NO: 13. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MG ATI of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Homo sapiens.

[0078] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2, or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Homo sapiens (accession number: Q10469.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferase comprises an amino acid sequence of SEQ ID NO: 14. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2 of Homo sapiens.

[0079] In certain embodiments, the N-acetyl glucosamine transferases comprise MGAT1 and MGAT2, or functionally active variants thereof. In certain embodiments, the N-acetyl glucosamine transferases comprise MGAT1 and MGAT2. In certain embodiments, the N- acetyl glucosamine transferases are variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 and MGAT2, respectively. In certain embodiments, the N-acetyl glucosamine transferases are MGAT1 and MGAT2 of Homo sapiens, or functionally active variants thereof. In certain embodiments, the N-acetyl glucosamine transferases are MGAT1 and MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferases comprise amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In certain embodiments, the N-acetyl glucosamine transferases are ones that are homologous to MGAT1 and MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferases are variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 and MGAT2 of Homo sapiens, respectively.

[0080] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Spodoptera frugiperda (SfGnT-I, accession number: AEX00082), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Spodoptera frugiperda. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Spodoptera frugiperda. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MG ATI of Spodoptera frugiperda.

[0081] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Trypanosoma brucei (TbGnT-I, accession number: XP 844156), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Trypanosoma brucei. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Trypanosoma brucei. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Trypanosoma brucei.

[0082] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Pan troglodytes (PtMGATl, accession number: XP 001155433.2), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Pan troglodytes . In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Pan troglodytes. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Pan troglodytes.

[0083] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 Macaco. mulatto (MaMGATl, accession number: NP_001244759), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 oiMacaca mulatta. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 oiMacaca mulatta. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1

Macaco mulatta.

[0084] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Mus musculus (MuMGATl, accession number: NP_001103620.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 oi Mus musculus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 oi Mus musculus. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Mus musculus.

[0085] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Rattus norvegicus (RnMGATl, accession number: NP_110488.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Rattus norvegicus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Rattus norvegicus. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Rattus norvegicus.

[0086] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Danio rerio A (DrMGATla, accession number: NP 956970.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Danio rerio A. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Danio rerio A. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Danio rerio A.

[0087] In certain embodiments, the N-acetyl glucosamine transferase comprises an MGAT1 of Caenorhabditis elegans (Cel4MGATl, accession number: NP 497719.1 or Cel3MGATl, accession number: NP 509566.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Caenorhabditis elegans. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Caenorhabditis elegans. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Caenorhabditis elegans.

[0088] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Arabidopsis thaliana (AtMGATl, accession number: NP 195537.2), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Arabidopsis thaliana. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Arabidopsis thaliana. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Arabidopsis thaliana.

[0089] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Oryza sativa Japonica (OsJMGATl, accession number: XP 015624616.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Oryza sativa Japonica. In certain embodiments, the N- acetyl glucosamine transferase comprises one that is homologous to MGAT1 Oryza sativa Japonica. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Oryza sativa Japonica.

[0090] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 oi Xenopus tropicalis (XtMGATl, accession number: NP 001011350.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 oi Xenopus tropicalis. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Xenopus tropicalis. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Xenopus tropicalis.

[0091] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Canis lupus (C1MGAT1, accession number: XP 855658.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Canis lupus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Canis lupus. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Canis lupus.

[0092] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Bos taurus (BtMGATl, accession number: NP 001015653.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Bos taurus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Bos taurus. In certain embodiments, the N- acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Bos taurus.

[0093] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Danio rerio B (DrMGATlb, accession number: NP 001073440.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MG ATI of Danio rerio B. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Danio rerio B. In certain embodiments, the N- acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Danio rerio B. [0094] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Gekko japonicus (GjMGATl, accession number: XP 015280466.1), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Gekko japonicus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Gekko japonicus. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Gekko japonicus.

[0095] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Rattus norvegicus (rMGAT2, accession number: NP 446056), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Rattus norvegicus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Rattus norvegicus. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2 of Rattus norvegicus.

[0096] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Spodoptera frugiperda (SfGnT-II, accession number: AEX00083), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Spodoptera frugiperda. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Spodoptera frugiperda. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2 of Spodoptera frugiperda.

[0097] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Trypanosoma brucei (TbGnT-II, accession number: XP 845654), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Trypanosoma brucei. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Trypanosoma brucei. In certain embodiments, the N-acetyl glucosamine transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2 of Trypanosoma brucei.

[0098] In certain embodiments, the additional recombinant glycosyltransferase is localized in the secretory pathway. Without being bound by theory, localization within the secretory pathway includes, but is not limited to, localization to one or more of the following sub-cellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface anchored proteins, and membrane proteins. In certain embodiments, localization in the secretory pathway comprises localization to one or more of said sub-cellular compartments.

[0099] In certain embodiments, the additional recombinant glycosyltransferase comprises a signal peptide localizing the additional recombinant glycosyltransferase in the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the additional recombinant glycosyltransferase (i.e. the signal peptide is not added to additional recombinant glycosyltransferase, but is one fused to the additional recombinant glycosyltransferase when naturally expressed in the source). In certain embodiments, the additional recombinant glycosyltransferase is localized in the secretory pathway without adding a Leishmania signal peptide to the additional recombinant glycosyltransferase. In other embodiments, the signal peptide is added to the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is fused to the C-terminus of the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is fused to the N-terminus of the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is fused to one or more amino acids within the polypeptide of the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is derived from Leishmania species. In certain embodiments, the signal peptide is a modified version of a signal peptide derived from a Leishmania species. In certain embodiments, the signal peptide is derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of a signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide is an invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of the invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the additional recombinant glycosyltransferase.

[00100] In certain embodiments, the additional recombinant glycosyltransferase comprises a retention sequence retaining the additional recombinant glycosyltransferase in the secretory pathway. In certain embodiments, the retention sequence is derived from the same source as the additional recombinant glycosyltransferase (i.e. the retention sequence is not added to additional recombinant glycosyltransferase, but is one fused to the additional recombinant glycosyltransferase when naturally expressed in the source). In certain embodiments, the additional recombinant glycosyltransferase is retained in the secretory pathway without adding a Leishmania retention sequence to the additional recombinant glycosyltransferase. In other embodiments, the retention sequence is added to the additional recombinant glycosyltransferase. In certain embodiments, the retention sequence is fused to the C- terminus of the additional recombinant glycosyltransferase. In certain embodiments, the retention sequence is fused to the N-terminus of the additional recombinant glycosyltransferase. In certain embodiments, the retention sequence is fused to one or more amino acids within the polypeptide of the additional recombinant glycosyltransferase. In certain embodiments, the retention sequence is derived from a Leishmania species. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from a Leishmania species. In certain embodiments, the retention sequence is derived from Leishmania tarentolae. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from Leishmania tarentolae.

[00101] In certain embodiments, the GalNAc transferase described in Section 7.1.1 and the additional recombinant glycosyltransferase are co-localized in the secretory pathway. 7.1.3 Deletion, Mutation and/or Functionally Inactivation of Endogenous Enzymes from the Glycan Biosynthesis Pathway

[00102] In certain embodiments, the Leishmania host cells provided herein are characterized in that one or more endogenous enzymes from the glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated. In certain embodiments, the Leishmania host cell does not have endogenous N-glycan elongation. In certain embodiments, the Leishmania host cells do not have endogenous N-glycan elongation as described in WO 2019/002512, which is incorporated herein by reference in its entirety. In certain embodiments, the Leishmania host cell has been genetically engineered such that the formation of an O-linked GlcNAc on a polypeptide in the host cell is reduced or eliminated. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one N-acetylglucosamine (GlcNAc)- transferase. In certain embodiments, the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is functionally inactivated, downregulated, deleted, and/or mutated.

[00103] In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc is an N-acetylglucosamine (GlcNAc)-transferase. In certain embodiments, the GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2, OGNTL, and homologous GlcNAc-transf erases thereof. Without being bound by theory, OGNT1, OGNT2, OGNTL were identified based on homology to Trypanosoma enzymes and not mammalian (e.g. human) enzymes (Heise, N., et al Glycobiology, 19(8), 918-933 (2009) and Chiribao, M.L. et al Gene, 498(2), 147-154 (2012), each of which is incorporated herein by reference in its entirety). In certain embodiments, the GlcNAc-transferase is OGNTL In other embodiments, the GlcNAc-transferase is OGNT2. In yet other embodiments, the GlcNAc-transferase is OGNTL. In certain embodiments, the GlcNAc-transferase is a GlcNAc-transferase that is homologous to OGNTL In certain embodiments, the GlcNAc- transferase is a GlcNAc-transferase that is homologous to OGNT2. In certain embodiments, the GlcNAc-transferase is a GlcNAc-transferase that is homologous to OGNTL. In certain embodiments, the GlcNAc-transferase is derived from Leishmania tarentolae. In certain embodiments, the GlcNAc-transferase is derived from other Trypanosomatida species. Nonlimiting examples of GlcNAc-transferases in Trypanosomatida are listed in Table 1, in which one representative genome per species is listed.

Table 1: Exemplary GlcNAc-transferases in Trypanosomatida.

[00104] In certain embodiments, the enzyme that catalyzes the formation of O-linked

GlcNAc is derived from species other than Trypanosomatida species. In certain embodiments, the enzyme is a human O-GlcNAc transferase (OGT, Uniprot: 015294) and homologous enzymes thereof. In certain embodiments, the O-GlcNAc transferase (OGT; uridine diphospho-N-acetylglucosamine:polypeptide P-N-acetylglucosaminyltransferase; EC 2.4.1.255) can catalyze the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in cytoplasmic and nuclear proteins resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc). In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc may be different isoforms of OGT. Exemplary isoforms of OGT include but are not limited to: (1) the nucleocytoplasmic or full- length variant (ncOGT), which may be 110 kDa; (2) a short isoform of OGT (sOGT), which may be 78 kDa; and (3) a variant of OGT that is targeted to the mitochondria (mOGT; which may be 90 kDa). In certain embodiments, OGT may appear to form multimers in the nucleus and cytoplasm, consisting of one or more 110-kDa subunits and 78-kDa subunits (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)). In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc is human EOGT (Uniprot: Q5NDL2). In certain embodiments, the enzyme catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in extracellular proteins resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc). In certain embodiments, the enzyme catalyzes Specific glycosylation of the Thr residue located between the fifth and sixth conserved cysteines of folded EGF-like domains.

[00105] In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc may transfer in alpha-linkage. In other embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc may transfer in beta-linkage.

[00106] In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one enzyme as described in this Section, for example one, two, three, four, five, six, seven, eight, nine or ten enzymes as described in this Section.

[00107] In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc-transferase derived from Trypanosomatida species, for example Leishmania tarentolae. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc-transferase, for example one, two, three, four, five, six, seven, eight, nine or ten GlcNAc-transferases, one or more of which is derived from Trypanosomatida species. In certain embodiments, the number of the at least one GlcNAc- transferase is one, two or three. In certain embodiments, the at least one GlcNAc-transferase is selected from the group consisting of 0GNT1, 0GNT2, OGNTL and homologous GlcNAc-transferases thereof. In certain embodiments, at least one GlcNAc-transferase is a GlcNAc-transferase that is homologous to 0GNT1, 0GNT2 and/or OGNTL.

[00108] In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc-transferase derived from species that is other than Trypanosomatida species, for example human. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc-transferase, for example one, two, three, four, five, six, seven, eight, nine or ten GlcNAc-transferases, one or more of which is derived from human. In certain embodiments, the number of the at least one GlcNAc-transferase is one, two or three. In certain embodiments, the at least one GlcNAc-transferase is selected from the group consisting of human O-GlcNAc transferase and human EOGT and homologous enzymes thereof. In certain embodiments, at least one GlcNAc-transferase is an enzyme that is homologous to human O-GlcNAc transferase and/or human EOGT.

[00109] In certain embodiments, the enzyme catalyzes the formation of O-linked GlcNAc prior to the genetic engineering of the Leishmania host cell. In certain embodiments, the enzyme still catalyzes the formation of O-linked GlcNAc after the genetic engineering of the Leishmania host cell. In certain embodiments, the enzyme does not catalyze the formation of O-linked GlcNAc after the genetic engineering of the Leishmania host cell.

[00110] In certain embodiments, the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is functionally inactivated. In certain embodiments, the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is downregulated. In certain embodiments, the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is overexpressed.

[00111] In certain embodiments, the Leishmania host cell provided herein comprises at least one gene deletion. In certain embodiments, the gene encoding the at least one GlcNAc- transferase is deleted. In certain embodiments, the gene encoding the at least one GlcNAc- transferase is mutated. In certain embodiments, the gene encoding the at least one GlcNAc- transferase is overexpressed. In certain embodiments, additional modifications may be introduced (e.g., using recombinant techniques) into the Leishmania host cell described herein.

[00112] In certain embodiments, the genes encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 enzymes that each catalyzes the formation of O- linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 enzymes that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding three enzymes that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes and/or genetic loci that may be functionally inactivated include but are not limited to OGNT1, OGNT2, and OGNTL. [00113] In certain embodiments, the genes encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 GlcNAc-transferase that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GlcNAc- transferase that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding three GlcNAc-transferases that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.

[00114] In certain embodiments, the genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyzes the formation of O- linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.

[00115] In certain embodiments, the genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc-transferases that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc-transferases that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.

[00116] In certain embodiments, the at least one GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2 and OGNTL, and homologous GlcNAc-transferases thereof. In certain embodiments, the Leishmania host cell is a OGNT1, OGNT2 and OGNTL triple knockout.

[00117] In certain embodiments, in the Leishmania host cell, the formation of the O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a reference Leishmania cell. In certain embodiments, in the Leishmania host cell, the formation of the O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a reference Leishmania cell. In certain embodiments, the reference Leishmania cell is wild-type. In certain embodiments, the reference Leishmania cell is genetically engineered differently as the genetically engineered Leishmania cells described herein. In certain embodiments, some of the engineering of the reference Leishmania cell may be the same of the engineering of the genetically engineered Leishmania cells described herein, for example the deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc. In certain embodiments, the reference Leishmania cell may comprise a recombinant nucleic acid encoding a heterologous glycosyltransferase, for example the Leishmania cells described in International Publication No. W02019/002512 A2, incorporated by reference in its entirety herein. In certain embodiments, the formation of the O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a wild-type Leishmania cell. In certain embodiments, the formation of the O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a wild-type Leishmania cell. In certain embodiments, the formation of the O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O- linked GlcNAc in a Leishmania cell that comprises a recombinant nucleic acid encoding a heterologous glycosyltransferase. In certain embodiments, the formation of the O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a Leishmania cell that comprises a recombinant nucleic acid encoding a heterologous glycosyl transferase.

[00118] In certain embodiments, the growth rate of the Leishmania host cell described herein is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a reference Leishmania cell. In certain embodiments, the growth rate of the Leishmania host cell is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a reference Leishmania cell. In certain embodiments, the reference Leishmania cell is wildtype. In certain embodiments, the reference Leishmania cell is genetically engineered differently as the genetically engineered Leishmania cells described herein. In certain embodiments, some of the engineering of the reference Leishmania cell may be the same of the engineering of the genetically engineered Leishmania cells described herein, for example the deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc. In certain embodiments, the growth rate of the Leishmania cell is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a wild-type Leishmania cell. In certain embodiments, the growth rate of the Leishmania cell is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a wild-type Leishmania cell.

7.1.4 Heterologous UDP-GalNAc Biosynthetic Pathway Proteins

[00119] In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding UDP-GalNAc biosynthetic pathway proteins capable of generating UDP-GalNAc. In certain embodiments, the recombinant UDP-GalNAc biosynthetic pathway proteins are heterologous to the Leishmania host cell.

[00120] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc are derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens.

[00121] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting N- Acetyl galactosamine 1 -phosphate (GalNAc- 1-P) to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc- 1-P and UTP to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens, or a functionally active variant thereof. In certain embodiments, the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP- N-acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise a variant of UDP-N- acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. Without being bound by theory, the AGX1 isoform of UDP-N-acetyl hexosamine pyrophosphorylase is about two to three times more active towards GalNAc-l-P than GlcNAc-1-P, whereas the AGX2 isoform is about eight times more active towards GlcNAc-1-P than GalNAc-l-P. In certain embodiments, the UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) is the AGX1 isoform of UAP1. In other embodiments, the UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) is the AGX2 isoform of UAP1. In certain embodiments, the UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) comprises an amino acid sequence of SEQ ID NO: 15.

[00122] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc to GalNAc-l-P. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2) of Homo sapiens, or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N- acetyl galactosamine kinase (GALK2) of Homo sapiens, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2) of Homo sapiens. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise a variant of N-acetyl galactosamine kinase (GALK2) of Homo sapiens that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the N-acetyl galactosamine kinase (GALK2) comprises an amino acid sequence of SEQ ID NO: 15.

[00123] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting UDP-GlcNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein capable of converting UDP- GlcNAc to UDP-GalNAc comprises a NAD-dependent epimerase that converts UDP- GlcNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting UDP-GlcNAc to UDP-GalNAc is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP -galactose 4-epimerase (GalE), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP -galactose 4-epimerase (GalE) of Homo sapiens (hGalE), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprise hGalE, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise hGalE. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise a variant of hGalE that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the hGalE comprises an amino acid sequence of SEQ ID NO: 17. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting UDP- GlcNAc to UDP-GalNAc are derived from a bacterial source. In certain embodiments, the bacterial source is Campylobacter jejuni. In certain embodiments, the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-GlcNAc/Glc 4-epimerase of Campylobacter jejuni (CjGne), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway proteins comprise comprise a variant of CjGne that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the CjGne comprises an amino acid sequence of SEQ ID NO: 18.

[00124] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc as described in this Section; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway as described in Section 7.1.5. In certain embodiments, the recombinant nucleic acid in (a) encodes UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc- 1-P to UDP-GalNAc and UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to GalNAc- 1-P, as described in this Section.

[00125] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc as described in this Section; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway as described in Section 7.1.5. [00126] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc as described in this Section; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc as described in this Section. In certain embodiments, the recombinant nucleic acid in (a) encodes UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc- 1-P to UDP-GalNAc and UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to GalNAc- 1-P, as described in this Section.

[00127] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc as described in this Section; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc as described in this Section; and (c) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway as described in Section 7.1.5. In certain embodiments, the recombinant nucleic acid in (a) encodes UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc- 1-P to UDP-GalNAc and UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to GalNAc- 1-P, as described in this Section.

7.1.5 Heterologous UDP-GalNAc Transporter Proteins

[00128] In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway. In certain embodiments, the UDP- GalNAc transporter protein is heterologous to the Leishmania host cell.

[00129] In certain embodiments, the heterologous UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source, a mammalian source, a brachiopod source, a chordate source, choanoflagellate source, a gyrista source, a fungi source, a mollusk source, or a placozoan source. In certain embodiments, the heterologous UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source. In certain embodiments, the nematode source is C. elegans the mammalian source is Homo sapiens,' the brachiopod source is Lingula unguis,' the chordate source is Parambassis ranga, Geotrypetes seraphini, or Scophthalmus maximus,' the choanoflagellate source is Salpingoeca rosetta, the gyrista source is Fragilariopsis cylindrus,' the fungi source is Dentipellis fragilis,' the mollusk source is Octopus bimaculoides,' and/or the placozoan source is trichoplax sp. H2.

[00130] In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source. In certain embodiments, the nematode source is C. elegans.

[00131] In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises UDP-GalNAc transporter of C. elegans (CeC03H5.2), or a functionally active variant thereof. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises CeC03H5.2, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises CeC03H5.2. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is CeC03H5.2. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to the secretory pathway comprises a variant of CeC03H5.2 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to the secretory pathway is a variant of CeC03H5.2 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the CeC03H5.2 has an amino acid sequence of SEQ ID NO: 19.

[00132] In certain embodiments, the variant of CeC03H5.2 is UDP-N-acetylglucosamine transporter isoform XI of Lingula unguis (GnF). In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnF. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnF. In certain embodiments, GnF has an amino acid sequence of SEQ ID NO: 132.

[00133] In certain embodiments, the variant of CeC03H5.2 is UDP-N-acetylglucosamine transporter-like isoform XI of Parambassis ranga (GnG). In certain embodiments, the UDP- GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnG. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnG. In certain embodiments, GnG has an amino acid sequence of SEQ ID NO: 133.

[00134] In certain embodiments, the variant of CeC03H5.2 is UDP-N-acetylglucosamine transporter of Salpingoeca rosetta (GnH). In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnH. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnH. In certain embodiments, GnH has an amino acid sequence of SEQ ID NO: 134.

[00135] In certain embodiments, the variant of CeC03H5.2 is UDP-N-acetylglucosamine transporter-like protein of Fragilariopsis cylindrus (GnI). In certain embodiments, the UDP- GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnI. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnI. In certain embodiments, GnI has an amino acid sequence of SEQ ID NO: 135.

[00136] In certain embodiments, the variant of CeC03H5.2 is previously uncharacterized protein of Dentipellis fragilis (GnJ) identified by homology search of CeC03H5.2. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnJ. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnJ. In certain embodiments, GnJ has an amino acid sequence of SEQ ID NO: 136.

[00137] In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises UDP-GalNAc transporter of Homo sapiens (UGTREL7), or a functionally active variant thereof. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises UGTREL7, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises UGTREL7. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is UGTREL7. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to the secretory pathway comprises a variant of UGTREL7 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to the secretory pathway is a variant of UGTREL7 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the UGTREL7 has an amino acid sequence of SEQ ID NO: 137.

[00138] In certain embodiments, the variant of UGTREL7 is UDP -glucuronic acid/UDP- N-acetylgalactosamine transporter of Geotrypetes seraphini (GnL). In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnL. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnL. In certain embodiments, GnL has an amino acid sequence of SEQ ID NO: 138.

[00139] In certain embodiments, the variant of UGTREL7 is putative UDP-N- acetylglucosamine/UDP-glucose/GDP-mannose transporter-like protein of Scophthalmus maximus (GnM). In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnM. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnM. In certain embodiments, GnL has an amino acid sequence of SEQ ID NO: 139.

[00140] In certain embodiments, the variant of UGTREL7 is TPT domain-containing protein of Octopus bimaculoides (GnN). In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises GnN. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnN. In certain embodiments, GnL has an amino acid sequence of SEQ ID NO: 140.

[00141] In certain embodiments, the variant of UGTREL7 is UDP-N- acetylglucosamine/UDP-glucose/GDP -mannose transporter of trichoplax sp. H2 (GnO). In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to the secretory pathway comprises GnO. In certain embodiments, the UDP- GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is GnO. In certain embodiments, GnL has an amino acid sequence of SEQ ID NO: 141.

[00142] In certain embodiments, the heterologous UDP-GalNAc transporter protein comprises a signal peptide localizing the heterologous UDP-GalNAc transporter protein in the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the heterologous UDP-GalNAc transporter protein (i.e. the signal peptide is not added to the heterologous UDP-GalNAc transporter protein, but is one fused to the heterologous UDP-GalNAc transporter protein when naturally expressed in the source). In certain embodiments, the heterologous UDP-GalNAc transporter protein is localized in the secretory pathway without adding a Leishmania signal peptide to the heterologous UDP- GalNAc transporter protein. In other embodiments, the signal peptide is added to the heterologous UDP-GalNAc transporter protein. In certain embodiments, the signal peptide is derived from Leishmania species. In certain embodiments, the signal peptide is a modified version of a signal peptide from derived from a. Leishmania species. In certain embodiments, the signal peptide is derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of a signal peptide from derived from Leishmania tarentolae. In certain embodiments, the signal peptide is an invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of the invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the heterologous UDP-GalNAc transporter protein.

[00143] In certain embodiments, the heterologous UDP-GalNAc transporter protein comprises a retention sequence retaining the heterologous UDP-GalNAc transporter protein in the secretory pathway. In certain embodiments, the retention sequence is derived from the same source as the heterologous UDP-GalNAc transporter protein (i.e. the retention sequence is not added to the heterologous UDP-GalNAc transporter protein, but is one fused to the heterologous UDP-GalNAc transporter protein when naturally expressed in the source). In certain embodiments, the heterologous UDP-GalNAc transporter protein is retained in the secretory pathway without adding a Leishmania retention sequence to the heterologous UDP- GalNAc transporter protein. In other embodiments, the retention sequence is added to the heterologous UDP-GalNAc transporter protein. In certain embodiments, the retention sequence is fused to the C-terminus of the heterologous UDP-GalNAc transporter protein. In certain embodiments, the retention sequence is fused to one or more amino acids within the polypeptide of the heterologous UDP-GalNAc transporter protein. In certain embodiments, the retention sequence is derived from a Leishmania species. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from a Leishmania species. In certain embodiments, the retention sequence is derived from Leishmania tarentolae. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from Leishmania tarentolae. [00144] Without wishing to be bound by theory, host cells encoding at least two heterologous UDP-GalNAc transporter proteins, including those with two gene copies of the same transporter protein, can be beneficial for optizimizing the extent and/or homogeneity of glycosylation of a polypeptide of interest. In certain embodiments, the recombinant nucleic acid encodes at least two heterologous UDP-GalNAc transporter proteins. In certain embodiments, the recombinant nucleic acid encodes two heterologous UDP-GalNAc transporter proteins. In certain embodiments, the recombinant nucleic acid encodes two copies of the same heterologous UDP-GalNAc transporter protein. In certain embodiments, the recombinant nucleic acid encodes two different heterologous UDP-GalNAc transporter proteins. In certain embodiments, the heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2 and UGTREL7, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2, GnF, GnG, GnH, GnI, GnJ, UGTREL7, GnL, GnM, GnN, or GnO, or functionally active variants thereof. In certain embodiments, the heterologous UDP-GalNAc transporter proteins are selected as a combination of heterologous UDP-GalNAc transporters listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the heterologous UDP-GalNAc transporter proteins comprise CeC03H5.2. In certain embodiments, the heterologous UDP-GalNAc transporter protein is CeC03H5.2. In certain embodiments, the host cell has two gene copies encoding CeC03H5.2. In certain embodiments, the heterologous UDP-GalNAc transporter proteins comprise CeC03H5.2 and GnJ. In certain embodiments, the heterologous UDP- GalNAc transporter proteins are CeC03H5.2 and GnJ.

[00145] In certain embodiments, the recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter comprises a first ORF encoding a first heterologous UDP-GalNAc transporter and a second ORF encoding a second heterologous UDP-GalNAc transporter. In certain embodiments, the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module. In certain embodiments, the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in separate genetic modules. In certain embodiments, the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP- GalNAc transporter are integrated into the [ssuPolI] locus of the host cell. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter are selected as a combination of heterologous UDP-GalNAc transporters listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2 and UGTREL7, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2, GnF, GnG, GnH, GnI, GnJ, UGTREL7, GnL, GnM, GnN, or GnO, or functionally active variants thereof. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are the same UDP-GalNAc transporter protein. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are each CeC03H5.2. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are different UDP-GalNAc transporter proteins. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are CeC03H5.2 and GnJ, respectively.

[00146] Without wishing to be bound by theory, the extent and/or homogeneity of glycosylation of a polypeptide of interest may be optimized by appropriate combination of recombinant GalNAc transferase(s) and heterologous UDP-GalNAc transporter protein(s). In certain embodiments, the combination of recombinant GalNAc transferase(s) and heterologous UDP-GalNAc transporter protein(s) is selected from a combination listed in Table 12 or variants that are 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 at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof. In certain embodiments, when the one or more GalNAc transferases comprise human p4-GalNAcT3, the heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2, GnF, GnH, GnJ, UGTREL7, GnM, GnN, and GnO. In certain embodiments, when the one or more GalNAc transferases comprise human p4-GalNAcT3, the heterologous UDP-GalNAc transporter proteins comprise CeC03H5.2. In certain embodiments, when the one or more GalNAc transferases comprise human p4-GalNAcT3, the heterologous UDP-GalNAc transporter proteins comprise GnJ. In certain embodiments, when the one or more GalNAc transferases comprise human p4-GalNAcT3, the heterologous UDP-GalNAc transporter proteins comprise GnM. In certain embodiments, when the one or more GalNAc transferases comprise human p4-GalNAcT3, the heterologous UDP-GalNAc transporter proteins comprise GnN. In certain embodiments, when the one or more GalNAc transferases comprise human p4-GalNAcT3, the heterologous UDP-GalNAc transporter proteins comprise GnO. In certain embodiments, when the one or more GalNAc transferases comprise Ptp4GalNAcT, the heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2, GnF, GnJ, and GnM. In certain embodiments, when the one or more GalNAc transferases comprise Ptp4GalNAcT, the heterologous UDP- GalNAc transporter proteins comprise CeC03H5.2. In certain embodiments, when the one or more GalNAc transferases comprise Ptp4GalNAcT, the heterologous UDP-GalNAc transporter proteins comprise GnF. In certain embodiments, when the one or more GalNAc transferases comprise CeP4GalNAcT, the heterologous UDP-GalNAc transporter proteins comprise CeC03H5.2 and/or GnF.

[00147] In certain embodiments, the recombinant nucleic acids encode at least two GalNAc transferases and at least two heterologous UDP-GalNAc transporter proteins. In certain embodiments, the recombinant nucleic acids encode two GalNAc transferases and two heterologous UDP-GalNAc transporter proteins. In certain embodiments, the at least two GalNAc transferases are different. In certain embodiments, the recombinant nucleic acids encode two copies of the same heterologous UDP-GalNAc transporter protein. In certain embodiments, the recombinant nucleic acids encode two different heterologous UDP- GalNAc transporter proteins. In certain embodiments, the at least two GalNAc transferases are selected from the group consisting of human p4-GalNAcT3, human p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto; and the at least two heterologous UDP-GalNAc transporter proteins are selected from the group consisting of CeC03H5.2, GnF, GnG, GnH, GnI, GnJ, UGTREL7, GnL, GnM, GnN, or GnO, or functionally active variants thereof. In certain embodiments, the at least two GalNAc transferases and the at least two heterologous UDP-GalNAc transporter proteins are selected from a combination listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the recombinant nucleic acids encode human p4-GalNAcT3, CeP4GalNAcT, CeC03H5.2, and GnJ. In certain embodiments, the recombinant nucleic acids encode human P4-GalNAcT3, Ptp4GalNAcT, and CeC03H5.2.

[00148] In certain embodiments, the host cell is such that (a) the recombinant nucleic acid encoding one or more GalNAc transferases comprises a first ORF encoding a first GalNAc transferase and a second ORF encoding a second GalNAc transferase; and (b) the recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter comprises a third ORF encoding a first heterologous UDP-GalNAc transporter and a fourth ORF encoding a second heterologous UDP-GalNAc transporter. In certain embodiments, the host cell is such that (a) the first open reading frame (ORF) encoding the first GalNAc transferase and and the third ORF encoding the first heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module; and/or (b) the second open reading frame (ORF) encoding the second GalNAc transferase and and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module. In certain embodiments, the host cell is such that (a) the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in separate genetic modules; and/or (b) the third ORF encoding the first heterologous UDP-GalNAc transporter and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in separate genetic modules. In certain embodiments, the first ORF encoding the first GalNAc transferase, the second ORF encoding the second GalNAc transferase, the third ORF encoding the first heterologous UDP-GalNAc transporter, and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module. In certain embodiments, the first ORF encoding the first GalNAc transferase, the second ORF encoding the second GalNAc transferase, the third ORF encoding the first heterologous UDP-GalNAc transporter, and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the [ssuPolI] locus of the host cell. In certain embodiments, the first and the second GalNAc transferases are different GalNAc transferases. In certain embodiments, the first and the second heterologous UDP- GalNAc transporter proteins are the same UDP-GalNAc transporter protein. In certain embodiments, the first and the second heterologous UDP-GalNAc transporter proteins are different UDP-GalNAc transporter proteins. In certain embodiments, the first GalNAc transferase, the second GalNAc transferase, the first heterologous UDP-GalNAc transporter protein, and the second heterologous UDP-GalNAc transporter protein are selected as a combination of GalNAc transferases and heterologous UDP-GalNAc transporter proteins listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof. In certain embodiments, the first and the second GalNAc transferases are human p4-GalNAcT3 and CeP4GalNAcT, respectively; and the first and the second heterologous UDP-GalNAc transporter proteins are CeC03H5.2, and GnJ, respectively. In certain embodiments, the first and the second GalNAc transferases are human p4-GalNAcT3 and Ptp4GalNAcT, respectively; and the first and the second heterologous UDP-GalNAc transporter proteins are each CeC03H5.2.

[00149] In certain embodiments, the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein are co-localized in the secretory pathway. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP- GalNAc transporter protein each independently comprise: (a) a signal peptide localizing the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein in the secretory pathway; and/or (b) a retention sequence retaining the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein in the secretory pathway. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP- GalNAc transporter protein each independently comprise the same signal peptide and/or retention sequence. In certain embodiments, the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein comprise different signal peptides and/or retention sequences. In certain embodiments, the signal peptide is derived from the same source as the one or more GalNAc transferases (i.e. the signal peptide is not added to the one or more GalNAc transferases, but is one fused to the one or more GalNAc transferases when naturally expressed in the source). In certain embodiments, the one or more GalNAc transferases is localized in the secretory pathway without adding a Leishmania signal peptide to the one or more GalNAc transferases. In certain embodiments, the signal peptide is derived from the same source as the heterologous UDP-GalNAc transporter protein (i.e. the signal peptide is not added to the heterologous UDP-GalNAc transporter protein, but is one fused to the heterologous UDP-GalNAc transporter protein when naturally expressed in the source). In certain embodiments, the heterologous UDP-GalNAc transporter protein is localized in the secretory pathway without adding a Leishmania signal peptide to the heterologous UDP-GalNAc transporter protein. In certain embodiments, the signal peptide of the one or more GalNAc transferases and/or the signal peptide of the heterologous UDP- GalNAc transporter protein is derived from a Leishmania species. In certain embodiments, the signal peptide of the one or more GalNAc transferases and/or the signal peptide of the heterologous UDP-GalNAc transporter protein are processed and removed. In certain embodiments, the retention sequence is derived from the same source as the one or more GalNAc transferases (i.e. the retention sequence is not added to the one or more GalNAc transferases, but is one fused to the one or more GalNAc transferases when naturally expressed in the source). In certain embodiments, the one or more GalNAc transferases is retained in the secretory pathway without adding a Leishmania retention sequence to the one or more GalNAc transferases. In certain embodiments, the retention sequence is derived from the same source as the heterologous UDP-GalNAc transporter protein (i.e. the retention sequence is not added to the heterologous UDP-GalNAc transporter protein, but is one fused to the heterologous UDP-GalNAc transporter protein when naturally expressed in the source). In certain embodiments, the heterologous UDP-GalNAc transporter protein is retained in the secretory pathway without adding a Leishmania retention sequence to the heterologous UDP- GalNAc transporter protein. In certain embodiments, the retention sequence of the one or more GalNAc transferases and/or the retention sequence of the heterologous UDP-GalNAc transporter protein are derived from a Leishmania species. In certain embodiments, the Leishmania species is Leishmania tarentolae.

7.1.6 Strains of the Leishmania Host Cell

[00150] In certain embodiments, the Leishmania host cell is a Leishmania tarentolae cell. In certain embodiments, the Leishmania host cell is a Leishmania aethiopica cell. In certain embodiments, the Leishmania host cell is part of the Leishmania aethiopica species complex. In certain embodiments, the Leishmania host cell is a Leishmania aristidesi cell. In certain embodiments, the Leishmania host cell is a Leishmania deanei cell. In certain embodiments, the Leishmania host cell is part of the Leishmania donovani species complex. In certain embodiments, the Leishmania host cell is a Leishmania donovani cell. In certain embodiments, the Leishmania host cell is a Leishmania chagasi cell. In certain embodiments, the Leishmania host cell is a Leishmania infantum cell. In certain embodiments, the Leishmania host cell is a Leishmania hertigi cell. In certain embodiments, the Leishmania host cell is part of the Leishmania major species complex. In certain embodiments, the Leishmania host cell is a Leishmania major cell. In certain embodiments, the Leishmania host cell is a Leishmania martiniquensis cell. In certain embodiments, the Leishmania host cell is part of the Leishmania mexicana species complex. In certain embodiments, the Leishmania host cell is a Leishmania mexicana cell. In certain embodiments, the Leishmania host cell is a Leishmania pifanoi cell. In certain embodiments, the Leishmania host cell is part of the Leishmania tropica species complex. In certain embodiments, the Leishmania host cell is a Leishmania tropica cell.

7.2 Methods of Genetically Engineering a Leishmania Cell

[00151] Also provided herein are methods of genetically engineering Leishmania host cell as described in Section 7.1. In certain embodiments, the method may be used to accomplish the introduction of one or more genes encoding a GalNAc transferase as described in Section 7.1.1. In certain embodiments, the method may be used to accomplish the introduction of one or more genes encoding an additional recombinant glycosyltransferase as described in Section 7.1.2. In certain embodiments, the method may be used to accomplish the functional inactivation of one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc as described in Section 7.1.3. In certain embodiments, the method may be used to accomplish the introduction of one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.1.4. In certain embodiments, the method may be used to accomplish the introduction of one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.1.5. In certain embodiments, the strain of the engineered Leishmania host cell is described in Section 7.1.6.

[00152] Any method known in the art can be used to engineer the Leishmania host cell, e.g., Leishmania tarentolae. In certain embodiments, nucleic acids are introduced into the host cells described herein using a plasmid, e.g., the heterologous nucleic acids are expressed in the host cells by a plasmid (e.g., an expression vector), and the plasmid is introduced into the modified host cells by transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation. In a specific embodiment, said plasmid is introduced into the modified host cells by stable transfection. [00153] In specific embodiments, linearized nucleic acids are introduced into the host cells described herein using transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation. In a further embodiment, heterologous nucleic acids are integrated site-specifically into the host cell genome by homologous recombination.

[00154] In certain embodiments, the method of engineering the Leishmania host cell comprises introducing one or more genes encoding a GalNAc transferase as described in Section 7.1.1. [00155] In certain embodiments, the method of engineering the Leishmania host cell comprises introducing one or more genes encoding an additional recombinant glycosyltransferase as described in Section 7.1.2.

[00156] In certain embodiments, the method of engineering the Leishmania host cell comprises functionally inactivating one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc as described in Section 7.1.3. In certain embodiments, the method comprises downregulating the gene encoding the at least one GlcNAc-transferase. In certain embodiments, the method comprises deleting the gene encoding the at least one GlcNAc-transferase. In certain embodiments, the method comprises mutating the gene encoding the at least one GlcNAc-transferase. In certain embodiments, the method comprises overexpressing the gene encoding the at least one GlcNAc-transferase. In certain embodiments, the method comprises functionally inactivating the gene encoding an enzyme that catalyzes the formation of O-linked GlcNAc using the methods described in the Assay or Example Sections (Sections 7.6 and 8. , respectively). In certain embodiments, the method comprises functionally inactivating the gene encoding an enzyme that catalyzes the formation of O-linked GlcNAc using any method known in the art, for example methods described in International Publication No. W02019/002512 A2, incorporated by reference in its entirety herein.

[00157] Non-limiting exemplary mutagenesis approaches include site directed mutagenesis using targeted gene editing techniques such as TALENs , ZFNs, CRISPR/Cas9; in combination with a repair scaffold for directed, homologous recombination mediated repair (Zhang, W et al. (2017) mSphere 2 (1); Gupta, R. and Musunuru, K. (2014) The Journal of clinical investigation 124 (10):4154— 4161), transposon mutagenesis (Damasceno, J. et al. (2015) Christopher Peacock (Ed.): Parasite Genomics Protocols, vol. 1201. New York, NY : Springer New York (Methods in Molecular Biology), pp. 235-245); replacing the endogenous copy in situ with selectional markers, potentially in combination with a mutated gene version, that are integrated by homologous recombination (Roberts, S. (2011) Bioeng Bugs 2 (6): 320-326); RNA interference (RNAi) (Lye, L. et al. (2010) PLoS Pathog 6 (10), elOOl 161), conditional knock-down using Cre/LoxP or FRT/FLP (Duncan, S. (2017) Molecular and Biochemical Parasitology 216: 30-38).

[00158] Overexpression may be accomplished by the following non-limiting exemplary approaches, such as gene copy number increase by introduction of additional copies into separate loci (Beverley, S. (1991): Gene amplification in Leishmania. In d/z/w. Rev. Microbiol. 45, pp. 417-444), high expression loci (ribosomal DNA loci) or episomal constructs (Lodes, M. et al. (1995) Mol Cell Biol 15 (12), pp. 6845-6853. DOI: 10.1128/mcb.15.12.6845; Boucher, N. (2004) Nucleic Acids Res 32 (9): 2925-2936), modification of the native UTRs flanking the coding sequence; introduction of additional promoter regions such as the endogenous Poll promoter or a T7 promoter in combination with expression of bacterial T7 polymerase to increase the expression levels (Boucher, N. et al. (2002) Molecular and Biochemical Parasitology 119 (1): 153-158; Gu, P. et al. (2015) Scientific reports 5, p. 9684), use of transposable elements or recombinase based systems such as FRT-FLP or Cre/LoxP to introduce multiple copies of an expression construct (Duncan, S. et al. (2017) Molecular and Biochemical Parasitology 216, pp. 30-38), minichromosome integration (Zomerdijk, J. et al. (1992) Nucleic acids research 20 (11): 2725-2734), and forced chromosomal translocation by CRISPR (Zhang, W. et al. (2017) mSphere 2 (1). DOI: 10.1128/mSphere.00340-16).

[00159] In certain embodiments, the method of engineering the Leishmania host cell comprises introducing one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.1.4.

[00160] In certain embodiments, the method of engineering the Leishmania host cell comprises introducing one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.1.5.

[00161] In certain embodiments, the method of engineering the Leishmania host cell comprises (i) functionally inactivating one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc as described in Section 7.1.3; (ii) introducing one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.1.4; (iii) introducing one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.1.5; and (iv) introducing one or more genes encoding a GalNAc transferase as described in Section 7.1.1.

[00162] In certain embodiments, the method of engineering the Leishmania host cell comprises (i) functionally inactivating one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc as described in Section 7.1.3; (ii) introducing one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.1.4; (iii) introducing one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.1.5; (iv) introducing one or more genes encoding a GalNAc transferase as described in Section 7.1.1; and (v) introducing one or more genes encoding an additional recombinant glycosyltransferase as described in Section 7.1.2. [00163] In certain embodiments, the method comprises conducting steps (i)-(v) in sequential order. In other embodiments, steps (i)-(v) are conducted in a different order. For example, in certain embodiments, steps (ii) and (iii) are conducted before step (i). In other embodiments, steps (ii) and (iii) are conducted after step (i). In other embodiments, step (iv) and/or (v) are conducted before step (i). In certain embodiments, step (v) is conducted before step (iv). In certain embodiments, step (v) is conducted first.

[00164] In some embodiments, one or more of steps (i)-(v) may be conducted simultaneously, for example by introducing the genes in a single module. For example, in certain embodiments, steps (ii) and (iii) are conducted simultaneously. In certain embodiments, steps (ii), (iii), and (iv) are conducted simultaneously. In certain embodiments, step (v) is conducted before step (iv). In certain embodiments, step (v) is conducted first.

[00165] In a specific embodiment, step (i) is conducted, followed by steps (ii) and (iii), which are conducted simultaneously, and then step (iv) separately. In another specific embodiment, steps (ii) and (iii) are conducted simultaneously and before step (iv), and step (iv) is conducted before step (i). In yet another specific embodiment, step (i) is conducted, and steps (ii), (iii), and (iv) are conducted simultaneously after step (i). In certain embodiments, step (v) is conducted before step (iv). In certain embodiments, step (v) is conducted first.

[00166] In certain embodiments, the method comprises conducting steps (i)-(v) in the order shown in any one of FIG. 5, FIG. 6, and FIG. 14. In certain embodiments, step (v) is conducted first.

[00167] In certain embodiments, the Leishmania host cells may be engineered using the methods described in the Assay and Examples Sections (Sections 7.6 and 8. , respectively). Non-limiting exemplary Leishmania strains produced and plasmids used as donors for their production are provided in Table 3 and Table 9.

7.3 Methods of Culturing Leishmania Host Cells

[00168] Provided herein are methods for culturing Leishmania host cells described in Section 7.1.

[00169] In one embodiment, the Leishmania host cells are cultured using any of the standard culturing techniques known in the art. For example, cells are routinely grown in rich media like Brain Heart Infusion, Trypticase Soy Broth or Yeast Extract, all containing 5 pg /ml Hemin. Additionally, incubation is done at 26°C in the dark as static or shaking cultures for 2-3 days. In some embodiments, cultures of recombinant cell lines contain the appropriate selective agents. Non-limiting exemplary selective agents are provided in Table 2.

[00170] In certain embodiments, the Leishmania host cells are cultured in a growth medium comprising GalNAc. In certain embodiments, the growth medium comprises at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, or at least 20 mM GalNAc. In certain embodiments, the growth medium comprises about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 15 mM, or about 15 mM to about 20 mM GalNAc. In certain embodiments, the growth medium comprises about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM GalNAc. In certain embodiments, the growth medium comprises about about 10 mM GalNAc.

[00171] In certain embodiments, the Leishmania host cells are cultured in a growth medium comprising GlcNAc. In certain embodiments, the growth medium comprises at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, or at least 20 mM GlcNAc. In certain embodiments, the growth medium comprises about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 15 mM, or about 15 mM to about 20 mM GlcNAc. In certain embodiments, the growth medium comprises about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM GlcNAc.

[00172] In certain embodiments, the Leishmania host cells may be cultured using the methods described in the Assay and Examples Sections (Sections 7.6 and 8. , respectively). Non-limiting exemplary Leishmania strains and plasmids used as donors for their production are provided in Table 3 and Table 9.

7.4 Uses of the Leishmania Host Cell as an Expression System [00173] In certain embodiments, a Leishmania host cell described in Section 7.1 may be used as an expression system for making a polypeptide of interest. In certain embodiments, the polypeptide of interest may be a heterologous, non-Leishmania protein, such as a therapeutic protein (e.g., an antibody). The Leishmania host cells may be engineered as described in Sections 7.2 and cultured as described in Section 7.3. Other methods of producing Leishmania host cells for use as expression systems are known and may also be used, for example, see WO 2019/002512, WO 2021/140144 and WO 2021/140143, each of which are incorporated herein by reference in their entirety. Use of Leishmania host cells to make monoclonal antibodies are also known. Exemplary methods are described in WO 2022/053673, which is incorporated herein by reference in its entirety.

[00174] In certain embodiments, the Leishmania host cells may be used as an expression system for producing a polypeptide of interest according to the methods described in the Assay and Examples Sections (Sections 7.6 and 8. , respectively).

7.4.1 Compositions Comprising Host Cells

[00175] In one aspect, provided herein are compositions comprising the Leishmania host cells described in Section 7.1. Such compositions can be used in methods for generating a polypeptide of interest as described in Section 7.5. In certain embodiments, the compositions comprising Leishmania host cells can be cultured under conditions suitable for the production of polypeptides of interest. Subsequently, the polypeptides of interest can be isolated from said compositions comprising Leishmania host cells using methods known in the art.

[00176] The compositions comprising the Leishmania host cells can comprise additional components suitable for maintenance and survival of the Leishmania host cells, and can additionally comprise additional components required or beneficial to the production of polypeptides of interest by the Leishmania host cells, e.g., inducers for inducible promoters, such as arabinose, IPTG.

7.4.2 Methods of Making Polypeptides of Interest

[00177] In one aspect, provided herein are methods for making a polypeptide of interest, for example, one described in Section 7.5. In one embodiment, provided herein is a method of producing a polypeptide of interest in vivo, using a Leishmania host cell described in Section 7.1. In a specific embodiment, provided herein is a method for producing a polypeptide of interest, said method comprising (i) culturing a. Leishmania host cell described in Section 7.1 under conditions suitable for polypeptide production and (ii) isolating said polypeptide of interest. In a specific embodiment, the Leishmania host cell comprises: (a) a recombinant nucleic acid encoding a polypeptide of interest; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the Leishmania host cell is capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan. In particular, the Leishmania host cells provided herein is capable of producing polypeptides comprising an N-glycan of the following structure:

[00178] In certain embodiments, the polypeptide of interest produced by the Leishmania host cell is a therapeutic polypeptide, ie., a polypeptide used in the treatment of a disease or disorder. For example, the target polypeptide produced by the Leishmania host cell can be an enzyme, a cytokine, or an antibody. A list of non-limiting exemplary polypeptides of interest is provided in Section 7.5.

7.5 Polypeptides of Interest

[00179] In certain embodiments, the polypeptide of interest produced by the Leishmania host cell provided in Section 7.1 is a therapeutic polypeptide, z.e., a polypeptide used in the treatment of a disease or disorder. For example, the polypeptide of interest produced by the Leishmania host cell can be an enzyme, a cytokine, or an antibody. In certain embodiments, the polypeptide of interest is selected from the group consisting of adalimumab, rituximab and erythropoietin (EPO).

[00180] Any polypeptide (or peptide/polypeptide corresponding to the polypeptide) known in the art can be used as a polypeptide of interest in accordance with the methods described herein. One of skill in the art will readily appreciate that the nucleic acid sequence of a known polypeptide, as well as a newly identified polypeptide, can easily be deduced using methods known in the art, and thus it would be well within the capacity of one of skill in the art to introduce a nucleic acid that encodes any polypeptide of interest into a host cell provided herein (e.g., via an expression vector, e.g., a plasmid, e.g., a site specific integration by homologous recombination).

[00181] In certain embodiments, the polypeptide of interest is glycosylated, e.g., with an N-glycan of the following structure:

wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the polypeptide of interest. One of skill in the art will further recognize that the polypeptides of interest may be glycosylated using the methods described herein, e.g., either in vivo using Leishmania host cell in Section 7.1 or in vitro, possess therapeutic benefit (e.g., due to improved pharmacokinetics) and thus can be used in the treatment of subjects having diseases/disorders that will benefit from treatment with the glycosylated polypeptides of interest.

[00182] In certain embodiments, the polypeptide of interest comprises the amino acid sequence of human Interferon-a (INF -a), Interferon-P (INF-P), Interferon-y (INF-y), Interleukin-2 (IL2), Chimeric diphteria toxin-IL-2 (Denileukin diftitox), Interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), Tumor necrosis factor alpha (TNF-a), Insulin, Pramlintide, Growth hormone (GH), Insulin-like growth factor (IGF1), Human parathyroid hormone, Calcitonin, Glucagon-like peptide-1 agonist (GLP-1), Glucagon, Growth hormone-releasing hormone (GHRH), Secretin, Thyroid stimulating hormone (TSH), Human bone morphogenic polypeptide 2 (hBMP2), Human bone morphogenic proetin 7 (hBMP7), Gonadotropin releasing hormone (GnRH), Keratinocyte growth factor (KGF), Platelet-derived growth factor (PDGF), Fibroblast growth factor 7 (FGF7), Fibroblast growth factor 20 (FGF20), Fibroblast growth factor 21 (FGF21), Epidermal growth factor (EGF), Vascular endothelial growth factor (VEGF), Neurotrophin-3, Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-a, Erythropoietin, Granulocyte colony-stimulating factor (G-CSF), Granulocytemacrophage colony-stimulating factor (GM-CSF), the extracellular domain of CTLA4 (e.g., an FC-fusion), or the extracellular domain of TNF receptor (e.g., an FC-fusion). In a specific embodiment, the target polypeptide used in accordance with the methods and host cells described herein is an enzyme or an inhibitor. Exemplary enzymes and inhibitors that can be used as a target polypeptide include, without limitation, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor Vila, Antithrombin III (AT -III), Polypeptide C, Tissue plasminogen activator (tPA) and tPA variants, Urokinase, Hirudin, Streptokinase, Glucocerebrosidase, Alglucosidase-a, Laronidase (a-L-iduronidase), Idursulphase (Iduronate- 2-sulphatase), Galsulphase, Agalsidase-P (human a-galactosidase A), Botulinum toxin, Collagenase, Human DNAse-I, Hyaluronidase, Papain, L- Asparaginase, Uricase (Urate oxidase), glutamate carboxypeptidase (glucarpidase), al Protease inhibitor (al antitrypsin), Lactase, Pancreatic enzymes (lipase, amylase, protease), and Adenosine deaminase.

[00183] In a specific embodiment, the polypeptide of interest used in accordance with the methods and Leishmania host cells provided herein is a cytokine. Exemplary cytokines that can be used as a polypeptide of interest include, without limitation, Interferon-a (INF-a), Interferon-P (INF-P), Interferon -y (INF-y), Interleukin-2 (IL2), Chimeric diphteria toxin-IL-2 (Denileukin diftitox), Interleukin- 1 (IL1), IL IB, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), and Tumor necrosis factor alpha (TNF-a).

[00184] In a specific embodiment, the polypeptide of interest used in accordance with the methods and Leishmania host cells provided herein is a hormone or growth factor. Exemplary hormones and growth factors that can be used as a polypeptides of interest include, without limitation, Insulin, Pramlintide, Growth hormone (GH), Insulin-like growth factor (IGF1), Human parathyroid hormone, Calcitonin, Glucagon-like peptide- 1 agonist (GLP-1), Glucagon, Growth horm one-releasing hormone (GHRH), Secretin, Thyroid stimulating hormone (TSH), Human bone morphogenic polypeptide 2 (hBMP2), Human bone morphogenic proetin 7 (hBMP7), Gonadotropin releasing hormone (GnRH), Keratinocyte growth factor (KGF), Platelet-derived growth factor (PDGF), Fibroblast growth factor 7 (FGF7), Fibroblast growth factor 20 (FGF20), Fibroblast growth factor 21 (FGF21), Epidermal growth factor (EGF), Vascular endothelial growth factor (VEGF), Neurotrophin-3, Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-a, Erythropoietin, Granulocyte colony-stimulating factor (G-CSF), and Granulocyte-macrophage colony-stimulating factor (GM-CSF).

[00185] In a specific embodiment, the polypeptide of interest used in accordance with the methods and Leishmania host cells provided herein is a receptor. Exemplary receptors that can be used as a polypeptide of interest include, without limitation, the extracellular domain of human CTLA4 (e.g., fused to an Fc) and the soluble TNF receptor (e.g., fused to an Fc).

[00186] In other embodiments, the polypeptide of interest is a therapeutic polypeptide. In other embodiments, the polypeptide of interest is an approved biologic drug. In another embodiment, the therapeutic polypeptide comprises the amino acid sequence of Abatacept (e.g., Orencia® (Bristol-Myers Squibb)), Aducanumab-avwa (e.g., Aduhelm® (Biogen Corporation)), Aflibercept (e.g., Eylea® (Regeneron Corp.)), Agalsidase beta (e.g., Fabrazyme® (Genzyme Corp.)), Albiglutide (e.g., Eperzan® (GlaxoSmithKline Corp.)), Aldesleukin (e.g., Proleukin® (Clinigen, Inc.)), Alefacept (e.g., Amevive® (Astellas Pharma, Inc.)), Alglucerase (e.g., Ceredase® (Genzyme Corp.)), Alglucosidase alfa (e.g., Lumizyme® (Genzyme Corp.)), Aliskiren (e.g., Tekturna® (Noden Pharma)), Alpha- 1-polypeptidease inhibitor (e.g., Aralast® (Takeda Pharmaceuticals, Inc.)), Alteplase (e.g., Activase® (Genentech)), Anakinra (e.g., Kineret® (Sobi, Inc.)), Anistreplase (e.g., Eminase® (SmithKlineBeecham)), Anthrax immune globulin human (e.g., Anthrasil® (Cangene Corp.)), Antihemophilic Factor (e.g., Advate® (Baxter Healthcare Corp.)), Antihemophelic Factor Fc- VWF-XTEN fusion protein (e.g., Altuviiio® (Bioverativ Therapeutics, Inc.)), Anti-inhibitor coagulant complex (e.g., Feiba Nf® (Takeda Pharmaceuticals, Inc.)), Antithrombin Alfa, Antithrombin III human, Antithymocyte globulin (e.g., Antithymocyte globulin), Antithymocyte Globulin (Equine) (e.g., ATGAM® (Pfizer)), Anti -thymocyte Globulin (Rabbit) (e.g., ATG-Fresenius), Aprotinin (e.g., Trasylol® (Bayer AG)), Asfotase Alfa (e.g., Strensiq® (AstraZeneca)),, Asparaginase (e.g., Elspar® (Merck & Co., Inc.)), Asparaginase erwinia chrysanthemi (e.g., Erwinaze® (EUSA Pharma, Inc.)), Becaplermin (e.g., Regranex® (Smith & Nephew, Inc.)), Belatacept (e.g., Nulojix® (Bristol-Myers Squibb)), Beractant, Bivalirudin (e.g., Angiomax® (The Medicines Co.)), Botulinum Toxin Type A (e.g., Botox® (Allergan, Inc.)), Botulinum Toxin Type B (e.g., Myobloc® (Supernus Pharmaceuticals)), Brentuximab vedotin (e.g., Adcetris® (Seagen Inc.)), Buserelin (e.g., Suprecur® (Sanofi-Aventis)), Cl Esterase Inhibitor (Human) (e.g., Cinryze® (Takeda Corporation)), Cl Esterase Inhibitor (Recombinant) (e.g., Ruconest® (Salix Pharmaceuticals, Inc.)), Cerliponase alfa (e.g.,Brineura® Biomarin Pharmaceutical, Inc.)), Certolizumab pegol (e.g., Cimzia® (UCB Pharma Ltd.)), Choriogonadotropin alfa (e.g., Choriogonadotropin alfa), Chorionic Gonadotropin (Human) (e.g., Ovidrel® (EMD Serono)), Chorionic Gonadotropin (Recombinant) (e.g., Ovitrelle® (Merck Serono)), Coagulation factor ix (e.g., Alprolix® (Bioverativ Therapeutics, Inc.)), Coagulation factor Vila (e.g., NovoSeven® (Novo Nordisk A/S)), Coagulation factor X human (e.g., Coagadex® (Bio Products Laboratory, Ltd.)), Coagulation Factor XIII A-Subunit (Recombinant), Collagenase (e.g., Cordase® (Headway Pharma PVT Ltd.)), Conestat alfa, Corticotropin (e.g., H.P. Acthar® (Mallinckrodt Pharmaceuticals)), Cosyntropin (e.g., Cortrosyn® (Amphastar Pharmaceuticals, Inc.)), Darbepoetin alfa (e.g., Aranesp® (Amgen Inc.)), Defibrotide (e.g., Noravid® (Gentium S.p.A.)), Denileukin diftitox (e.g., Ontak® (Eisai Medical Research)), Desirudin, Digoxin Immune Fab (Ovine) (e.g., Digibind® (GlaxoSmithKline LLC)), Domase alfa (e.g., Pulmozyme® (Genentech Inc.)), Drotrecogin alfa (e.g., Xigris® (Eli Lilly & Co.)), Dulaglutide (e.g., Trulicity® (Eli Lilly and Co.)), Efgartigimod alfa (e.g., Vyvgart® Hytrulo (Argenx, US, Inc.)), Ecallantide (e.g., Kalbitor® (Dyax Corp.)), Elapegademase (e.g., Revcovi® (Leadiant Biosciences, Inc.)), Efmoroctocog alfa (e.g., Elocta® (Swedish Orphan Biovitrum AB)), Elosulfase alfa (e.g., Vimizim® (Biomarin Pharmaceutical, Inc.)), Enfuvirtide (e.g., Fuzeon® (Genentech)), Eptinezumab (e.g., Vyepti® (Lundbeck Seattle Biopharmaceuticals, Inc.)), Epoetin alfa (e.g., Binocrit® (Sandoz GmbH)), Epoetin zeta (e.g., Retacrit® (Pfizer)), Eptifibatide (e.g., Integrilin® (COR Therapeutics, Inc.)), Etanercept (e.g., Enbrel® (Amgen Inc.)), Exenatide (e.g., Byetta® (AstraZeneca)), Factor IX Complex (Human) (e.g., AlphaNine® (Grifols Biologicals LLC)), Fibrinolysin aka plasmin (e.g., Elase® (Parke-Davis)), Filgrastim (e.g., N.A.), Filgrastim-sndz, Follitropin alfa (e.g., Gonal- F® (EMD Serono)), Follitropin beta (e.g., Follistim AQ® (Organon & Co.)), Galsulfase (e.g., Naglazyme® (Biomarin Pharmaceutical Inc.)), Gastric intrinsic factor, Gemtuzumab ozogamicin (e.g., Mylotarg® (Pfizer)), Glatiramer acetate (e.g., Copaxone® (Teva Neuroscience)), Glucagon recombinant (e.g., GlucaGen® (Novo Nordisk, Inc.)), Glucarpidase (e.g., Voraxaze® (BTG Pharmaceuticals)), Gramicidin D (e.g., Neosporin® (Johnson & Johnson Consumer, Inc.)), Hepatitis B immune globulin, Human calcitonin, Human Clostridium tetani toxoid immune globulin, Human rabies virus immune globulin (e.g., Hyperab Rabies Immune Globulin Human), Human Rho(D) immune globulin (e.g., Hyp Rho D Inj 16.5%), Human Serum Albumin (e.g., Albuminar® (CSL Behring LLC)), Human Varicella-Zoster Immune Globulin (e.g., Varizig® (Cangene Corporation)), Hyaluronidase (e.g., Hylenex® (Henry Schein, Inc.)), Hyaluronidase (Human Recombinant), Ibritumomab tiuxetan (e.g., Zevalin® (Acrotech Biopharma Inc.)), Idursulfase (e.g., Elaprase® (Takeda Pharmaceuticals, Inc.)), Imiglucerase (e.g., Cerezyme® (Genzyme Corporation)), Immune Globulin Human, Insulin aspart (e.g., NovoLog® (Novo Nordisk A/S)), Insulin Beef, Insulin Degludec (e.g., Tresiba® (Novo Nordisk A/S)), Insulin detemir (e.g., Levemir® (Novo Nordisk A/S)), Insulin Glargine (e.g., Lantus® (Sanofi-Aventis US LLC)), Insulin glulisine (e.g., Apidra® (Sanofi-Aventis US LLC)), Insulin Lispro (e.g., Humalog® (Eli Lilly Corp.)), Insulin Pork (e.g., Iletin II® (Eli Lilly Corporation)), Insulin Regular (e.g., Humulin R® (Eli Lilly Corp.), Insulin, porcine (e.g., Vetsulin® (Merck & Co.)), Insulin, isophane (e.g., Novolin N® (Novo Nordisk A/S)), Interferon Alfa-2a, Recombinant (e.g., Roferon A® (Hoffman- LaRoche, Inc.)), Interferon alfa-2b (e.g., Intron A® (Merck & Co., Inc.)), Interferon alfacon-1 (e.g., Infergen® (Three Rivers Pharmaceuticals, LLC)), Interferon alfa-nl (e.g., Wellferon® (GlaxoSmithKline)), Interferon alfa-n3 (e.g., Alferon® (AIM Immunotech Inc.)), Interferon beta-la (e.g., Avonex® (Biogen-Idec Corporation)), Interferon beta-lb (e.g., Betaseron® (Bayer Healthcare Pharmaceuticals)), Interferon gamma- lb (e.g., Actimmune® (Horizon Pharma USA, Inc.)), Intravenous Immunoglobulin (e.g., Xembify® (Griffols Therapeutics LLC)), Laronidase (e.g., Aldurazyme® (Genzyme Corporation)), Lenograstim (e.g., Granocyte® (Chugai Pharmaceuticals, Inc.)), Lepirudin (e.g., Refludan® (Behring GmbH)), Leuprolide (e.g., Eligard® (Sanofi -Aventis US, LLC)), Liraglutide (e.g., Saxenda® (Novo- Nordisk, Inc.)), Lucinactant (e.g., Surfaxin®), Lutropin alfa (e.g., Luveris® (EMD Serono)), Mecasermin (e.g., N.A.), Menotropins (e.g., Menopur® (F erring Pharmaceuticals)), Methoxy polyethylene glycol-epoetin beta (e.g., Mircera® (Vifor Pharma)), Metreleptin (e.g., Myalept® (AstraZeneca)), Natural alpha interferon OR multiferon (e.g., Intron/Roferon-A® (Merck & Co./Hoffman-LaRoche, Inc.)), Nesiritide (e.g., Natrecor® (Scios, Inc.)), Ocriplasmin (e.g., Jetrea® (ThromboGenics, Inc.)), Oprelvekin (e.g., Neumega® (Wyeth Pharmaceuticals, Inc.)), OspA lipopolypeptide (e.g., Lymerix® (GlaxoSmithKline)), Oxytocin (e.g., Pitocin® (Pfizer)), Palifermin (e.g., Kepivance® (Amgen, Inc.)), Pancrelipase (e.g., Creon® (Abb Vie Inc.)), Pegademase bovine (e.g., Adagen® (Enzon Pharmaceuticals, Inc.)), Pegaspargase (e.g., Oncaspar® (Sigma-Tau Pharmaceuticals, Inc.)), Pegfilgrastim (e.g., Neulasta® (Amgen, Inc.)), Peginterferon alfa-2a (e.g., Pegasys® (Genentech USA, Inc.)), Peginterferon alfa-2b (e.g., PEG-Intron® (Merck & Co.)), Peginterferon beta-la (e.g., Plegridy® (Biogen Corporation)), Pegloticase (e.g., Krystexxa® (Horizon Therapeutics)), Pegvisomant (e.g., Somavert® (Pfizer)), Poractant alfa (e.g., Curosurf® (Chiesi USA, Inc/Cornerstone Therapeutics, Inc.)), Pramlintide (e.g., Symlin® (AstraZeneca Pharmaceuticals)), Preotact (e.g., Preotact® (Nycomed A/S)), Protamine sulfate (e.g., Protamine Sulfate Injection, USP), Polypeptide S human (e.g., Polypeptide S human), Prothrombin (e.g., Feiba Nf® (Takeda Pharmaceuticals, Inc.)), Prothrombin complex (e.g., Cofact® (Sanquin Plasma Products B.V.)), Prothrombin complex concentrate (e.g., Kcentra® (CSL Behring LLC)), Rasburicase (e.g., Elitek® (Sanofi-Aventis US, LLC)), Reteplase (e.g., Retavase® (Chiesi USA, Inc.)), Rilonacept (e.g., Arcalyst® (Kiniksa Pharmaceuticals, Ltd.)), Romiplostim (e.g., Nplate® (Amgen, Inc.)), Sacrosidase (e.g., Sucraid® (QOL Medical, LLC)), Salmon Calcitonin (e.g., Calcimar® (Sandoz GmbH)), Sargramostim (e.g., Leucomax® (Novartis)), Satumomab Pendetide (e.g., OncoScint® (Cytogen Corporation)), Sebelipase alfa (e.g., Kanuma® (Alexion Pharmaceuticals, Inc.)), Secretin (e.g., SecreFlo® (Repligen Corp.)), Sermorelin (e.g., Sermorelin acetate), Serum albumin (e.g., Albunex® (Mallinckrodt Medical, Inc.)), Serum albumin iodonated (e.g., Megatope® (Iso-Tex Diagnostics, Inc.)), Simoctocog Alfa (e.g., Nuwiq® (Octapharma USA, Inc.)), Sipuleucel-T (e.g., Provenge® (Dendreon Corporation)), Somatotropin Recombinant (e.g., NutropinAQ® (Genentech)), Somatropin recombinant (e.g., BioTropin® (Bio-Technology General )), Streptokinase (e.g., Streptase® (CSL Behring LLC)), Susoctocog alfa (e.g., Obizur® (Baxalta US, Inc.)), Taliglucerase alfa (e.g., Elelyso® (Pfizer, Inc.)), Teduglutide (e.g., Gattex® (NPS Pharmaceuticals, Inc.)), Tenecteplase (e.g., TNKase® (Genentech, Inc.)), Teriparatide (e.g., Forteo® (Lilly US, LLC)), Tesamorelin (e.g., Egrifta® (Theratechnologies, Inc.)), Thrombomodulin Alfa (e.g., Recomodulin® (Asahi Kasei Pharma)), Thymalfasin (e.g., Zadaxin® (SciClone Pharmaceuticals, IntT.)), Thyroglobulin, Thyrotropin Alfa (e.g., Thyrogen® (Genzyme Corporation)), Tuberculin Purified Polypeptide Derivative (e.g., Aplisol® (Par Pharmaceuticals)), Turoctocog alfa (e.g., Zonovate® (Novo Nordisk)), Urofollitropin (e.g., Bravelle® (Ferring Pharmaceuticals, Inc.)), Urokinase (e.g., Kinlytic® (ImaRx Therapeutics, Inc.)), Vasopressin (e.g., Pitressin® (JHP Pharmaceuticals, LLC)), Velaglucerase alfa (e.g., Vpriv® (Takeda Pharmaceuticals)), Abciximab (e.g., ReoPro® (Janssen Biotech, Inc.)), Adalimumab (e.g., Humira® (Abb Vie, Inc.)), Alemtuzumab (e.g., Campath® (Takeda Oncology, Inc.)), Alirocumab (e.g., Praluent® (Regeneron/Sanofi)), Arcitumomab (e.g., CEA-Scan® (Immunomedics, Inc.)), Atezolizumab (e.g., Tecentriq® (Genentech)), Basiliximab (e.g., Simulect® (Novartis Pharmaceuticals Corporation)), Belimumab (e.g., Benlysta® (GlaxoSmithKline Inc.)), Benralizumab (e.g., Fasenra® (AstraZeneca)), Bevacizumab (e.g., Avastin® (Genentech, Inc.)), Bezlotoxumab (e.g., Zinplava® (Merck & Co., Inc.)), Blinatumomab (e.g., Blincyto® (Amgen, Inc.)), Brodalumab (e.g., Siliq® (Valeant Pharmaceuticals)), Brolucizumab (e.g., Beovu® (Novartis Pharmaceuticals Corporation)), Burosumab (e.g., Crysvita® (Ultragenyx, Inc.)), Calaspargase pegol (e.g., Asparlas® (Servier Pharmaceuticals, LLC)), Canakinumab (e.g., Haris® (Novartis Pharaceuticals Corporation)), Caplacizumab (e.g., Cablivi® (Ablynx, N.V.)), Capromab (e.g., ProstaScint® (EUSA Pharma (USA), Inc.)), Cemiplimab (e.g., Libtayo® (Regeneron Pharmaceuticals, Inc.)), Cetuximab (e.g., Erbitux® (Lilly USA, LLC)), Crizanlizumab (e.g., Adakveo® (Novartis Pharmaceuticals Corporation)), Daclizumab (e.g., Zenapax® (Hoffmann-LaRoche, Inc.)), Daratumumab (e.g., Darzalex® (Janssen Biotech, Inc.)), Denosumab (e.g., Prolia® (Amgen, Inc.)), Xgeva® (Amgen, Inc.)), Dinutuximab (e.g., Unituxin® (United Therapeutics Corp.)), Dostarlimab (e.g., Jemperli® (GlaxoSmithKline, LLC)), Durvalumab (e.g., Imfinzi® (AstraZeneca)), Dupilimab (e.g., Dupixent® (Regeneron Pharmaceuticals, Inc.)), Eculizumab (e.g., Soliris® (Alexion Pharmaceuticals, Inc.)), Efalizumab (e.g., Raptiva® (Genentech, Inc.)), Elotuzumab (e.g., Empliciti® (Bristol-Myers Squibb)), Elranatamab (e.g., Elrexfio® (Pfizer, Inc.)), Emapalumab (e.g., Gamifant® (Sobi, Inc.)), Emicizumab (e.g., Hemlibra® (Genentech, Inc.)), Erenumab (e.g., Aimovig® (Amgen, Inc.)), Evinacumab (e.g., Evkeeza® (Regeneron Pharmaceuticals, Inc.)), Evolocumab (e.g., Repatha® (Amgen, Inc.)), fam-trastuzumab deruxtecan-nxki (e.g., Enhertu® (Daiichi Sankyo, Inc.)), Fremanezumab (e.g., Ajovy® (Teva Pharmaceuticals, Inc.)), Galcanezumab (e.g., Emgality® (Eli Lilly and Company)), Golimumab (e.g., Simponi® (Janssen Biotech, Inc.)), Guselkumab (e.g., Tremfya® (Janssen Biotech, Inc.)), Ibalizumab (e.g., Trogarzo® (Theratechnologies, Inc.)), Ibritumomab (e.g., Zevalin® (Acrotech Biopharma Inc.)), Idarucizumab (e.g., Praxbind® (Boehringer Ingelheim Pharmaceuticals, Inc.)), Infliximab (e.g., Remicade® (Janssen Biotech, Inc.)), Ipilimumab (e.g., Yervoy® (Bristol-Myers Squibb)), Isatuximab (e.g., Sarclisa® (Sanofi -Aventis, US, LLC)), Ixekizumab (e.g., Taltz® (Eli Lilly & Co.)), Lanadelumab (e.g., Takhzyro® (Takeda Pharmaceuticals, USA, Inc.)), Magrolimab (Gilead Sciences, Inc.), Margetuximab (e.g., Margenza® (Macrogenics, Inc.)), Mepolizumab (e.g., Nucala® (GlaxoSmithKline)), Muromonab (e.g., Orthoclone 0KT3® (Centocor Ortho Biotech Products, LP)), Natalizumab (e.g., Tysabri® (Biogen Idee Corporation)), Necitumumab (e.g., Portrazza® (Eli Lilly and Company)), Nivolumab (e.g., Opdivo® (Bristol-Myers Squibb)), Obiltoxaximab (e.g., Anthim® (Elysys Therapeutics, Inc.)), Obinutuzumab (e.g., Gazyva® (Genentech, Inc.)), Ofatumumab (e.g., Arzerra® (GlaxoSmithKline)), Omalizumab (e.g., Xolair® (Genentech, Inc.)), Palivizumab (e.g., Synagis® (Medimmune, LLC)), Panitumumab (e.g. Vectibix® (Amgen, Inc.)), Pembrolizumab (e.g., Keytruda® (Merck & Co.)), Pertuzumab (e.g., Perjeta® (Genentech, Inc.)), Polatuzumab (e.g., Polivy® (Genentech, Inc.)), Pozelimab® (e.g., Veopoz (Regeneron Pharmaceuticals, Inc.)), Ramucirumab (e.g., Cyramza® (Eli Lilly and Company)), Ranibizumab (e.g., Lucentis® (Genentech, Inc.)), Ravulizumab-cwvz (e.g., Ultomoris® (AstraZeneca)), Raxibacumab (e.g., Raxibacumab (GlaxoSmithKline)), Risankizumab (e.g., Risanizumab-rzaa, Skyrizi® (Abb Vie Inc.)), Rituximab (e.g., Rituxan® (Genentech, Inc.)), Rozanolixizumab (e.g., Rystiggo® (UCB, Inc.)), Sarilumab (e.g., Kevzara® (Sanofi-Aventis, US, LLC)), Satrilizumab® (e.g., Enspryng (Genentech, Inc.)), Secukinumab (e.g., Cosentyx® (Novartis Pharmaceuticals Corporation)), Siltuximab (e.g., Sylvant® (Janssen Biotech, Inc.)), Tildrakizumab (e.g., Ilumya® (Merck & Co.), Talquetamab (e.g., Talvey® (Janssen Biotech, Inc.)), Teclistamab (e.g., Tecvayli® (Janssen Biotech, Inc.)), Tocilizumab (e.g., Actemra® (Genentech, Inc.)), Tositumomab (e.g., Bexxar® (GlaxoSmithKline)), Trastuzumab (e.g., Herceptin® (Genentech, Inc.)), Ustekinumab (e.g., Stelara® (Janssen Biotech, Inc.)), or Vedolizumab (e.g., Entyvio® (Takeda Pharmaceuticals, USA, Inc.)).

[00187] In other embodiments, the polypeptide of interest is an antibody. In further embodiments, the antibody has the amino acid sequence of adalimumab (Humira® (Abb Vie Inc.)); Remicade® (Janssen Biotech, Inc.) (Infliximab); ReoPro® (Janssen Biotech, Inc.) (Abciximab); Rituxan® (Genentech, Inc.) (Rituximab); Simulect® (Novartis Pharmaceuticals Corporation) (Basiliximab); Synagis® (Medimmune, LLC) (Palivizumab); Herceptin® (Genentech, Inc.) (Trastuzumab); Mylotarg® (Pfizer) (Gemtuzumab ozogamicin); Campath® (Takeda Oncology Corporation) (Alemtuzumab); Zevalin® (Acrotech Biopharma Inc.) (Ibritumomab tiuxetan); Xolair® (Genentech, Inc.) (Omalizumab); Bexxar® (GlaxoSmithKline) (Tositumomab-I-131); Erbitux® (Lilly USA, Inc.) (Cetuximab); Avastin® (Genentech, Inc.) (Bevacizumab); Tysabri® (Biogen Idee Corporation) (Natalizumab); Actemra® (Genentech) (Tocilizumab); Vectibix® (Amgen, Inc.) (Panitumumab); Lucentis® (Genentech, Inc.) (Ranibizumab); Soliris® (Alexion Pharmaceuticals Inc.) (Eculizumab);

Cimzia® (UCB Pharma Ltd.) (Certolizumab pegol); Simponi® (Janssen Biotech, Inc.) (Golimumab); Haris® (Novartis Pharmaceuticals Corporation) (Canakinumab); Stelara® (Janssen Biotech, Inc.) (Ustekinumab); Arzerra® (GlaxoSmithKline) (Ofatumumab); Prolia® and Xgeva® (Amgen, Inc.) (Denosumab); Numax® (Medimmune, LLC) (Motavizumab); ABThrax® (GlaxoSmithKline) (Raxibacumab); Benlysta® (GlaxoSmithKline) (Belimumab); Yervoy® (Bristol-Myers Squibb) (Ipilimumab); Adcetris® (Seagen, Inc.) (Brentuximab Vedotin); Perjeta® (Genentech, Inc.) (Pertuzumab); Kadcyla® (Genentech, Inc.) (Ado- trastuzumab emtansine); or Gazyva® (Genentech, Inc.) (Obinutuzumab).

[00188] In other embodiments, the antibody is a full length antibody, an Fab, an F(ab')2, an Scfv, or a sdAb. In certain embodiments, the polypeptide of interest is a camelid heavy-chain antibody domain, such as a VHH, sdAbs, or nanobody. In other embodiments, the polypeptide of interest comprises the amino acid sequence of an enzyme or an inhibitor thereof. In another embodiment, the polypeptide of interest comprises the amino acid sequence of Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor Vila, Antithrombin III (AT-III), Polypeptide C, Tissue plasminogen activator (tPA) and tPA variants, Urokinase, Hirudin, Streptokinase, Glucocerebrosidase, Alglucosidase-a, Laronidase (a-L-iduronidase), Idursulphase (Iduronate-2-sulphatase), Galsulphase, Agalsidase-P (human a-galactosidase A), Botulinum toxin, Collagenase, Human DNAse-I, Hyaluronidase, Papain, L- Asparaginase, Uricase (Urate oxidase), glutamate carboxypeptidase (glucarpidase), al Protease inhibitor (al antitrypsin), Lactase, Pancreatic enzymes (lipase, amylase, protease), and Adenosine deaminase.

[00189] In a specific embodiment, the polypeptide of interest used in accordance with the methods and Leishmania host cells provided herein is a receptor. Exemplary receptors that can be used as a polypeptide of interest include, without limitation, the extracellular domain of human CTLA4 (e.g., fused to an Fc) and the soluble TNF receptor (e.g., fused to an Fc).

[00190] In another embodiment, the polypeptide of interest is secreted into the culture media. In certain embodiments, the polypeptide of interest is purified from the culture media. In another embodiment, the polypeptide of interest is purified from the culture media via affinity purification or ion exchange chromatography. In another embodiment, the polypeptide of interest contains an FC domain and is affinity purified from the culture media via polypeptide-A. In another embodiment, the polypeptide of interest contains an affinity tag and is affinity purified.

[00191] In certain embodiments, the polypeptide of interest used in accordance with the methods and Leishmania host cells provided herein can be a full length polypeptide, a truncation, a polypeptide domain, a region, a motif or a peptide thereof.

[00192] In certain embodiments, the polypeptide of interest is an Fc-fusion polypeptide.

[00193] In certain embodiments, the polypeptide of interest is a biologic comprising an Fc domain of an IgG.

[00194] In certain embodiments, the polypeptide of interest produced by a method described herein is a bifunctional degrader described in ‘GLYCAN-MEDIATED PROTEIN DEGRADATION’, filed September 27, 2023 as an international application with the European Receiving Office, which claims priority to U.S. Provisional Application Nos. 63/410,955 and 63/410,936, and which is incorporated herein in its entirety. [00195] In certain embodiments, the polypeptide of interest is localized in the secretory pathway. Without being bound by theory, localization within the secretory pathway includes, but is not limited to, localization to one or more of the following sub-cellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface anchored proteins, and membrane proteins. In certain embodiments, localization in the secretory pathway comprises localization to one or more of said sub-cellular compartments.

[00196] In certain embodiments, the polypeptide of interest comprises a signal peptide localizing the polypeptide of interest in the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the polypeptide of interest (i.e. the signal peptide is not added to the polypeptide of interest, but is one fused to the polypeptide of interest when naturally expressed in the source). In certain embodiments, the polypeptide of interest is localized in the secretory pathway without adding a Leishmania signal peptide to the polypeptide of interest. In other embodiments, the signal peptide is added to the polypeptide of interest. In certain embodiments, the signal peptide is derived from a Leishmania species. In certain embodiments, the signal peptide is a modified version of a signal peptide from derived from a Leishmania species. In certain embodiments, the signal peptide is derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of a signal peptide from derived from Leishmania tarentolae. In certain embodiments, the signal peptide is an invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide is a modified version of the invertase signal peptide derived from Leishmania tarentolae. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the polypeptide of interest.

[00197] In certain embodiments, the polypeptide of interest comprises a retention sequence retaining the polypeptide of interest in the secretory pathway. In certain embodiments, the retention sequence is derived from the same source as the polypeptide of interest (i.e. the retention sequence is not added to the polypeptide of interest, but is one fused to the polypeptide of interest when naturally expressed in the source). In certain embodiments, the polypeptide of interest is retained in the secretory pathway without adding a Leishmania retention sequence to the polypeptide of interest. In other embodiments, the retention sequence is added to the polypeptide of interest. In certain embodiments, the retention sequence is fused to the C-terminus of the polypeptide of interest. In certain embodiments, the retention sequence is fused to the N-terminus of the polypeptide of interest. In certain embodiments, the retention sequence is fused to one or more amino acids within the polypeptide of the polypeptide of interest. In certain embodiments, the retention sequence is derived from a Leishmania species. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from a Leishmania species. In certain embodiments, the retention sequence is derived from Leishmania tarentolae. In certain embodiments, the retention sequence is a modified version of a retention sequence derived from Leishmania tarentolae.

[00198] In another embodiment, the polypeptide of interest has been engineered to comprise one or more tag(s). In other embodiments, the tag is processed and removed from the polypeptide of interest.

[00199] In certain embodiments, the GalNAc transferase described in Section 7.1.1 and the polypeptide of interest are co-localized in the secretory pathway.

7.5.1 Composition and/or Formulation Comprising the Polypeptide

[00200] In another aspect, provided herein are compositions (e.g., pharmaceutical compositions) comprising one or more of the polypeptides of interest described herein. The compositions described herein are useful in the treatment and/or prevention of diseases/disorders in subjects (e.g., human subjects) (see Section 7.5.2).

[00201] In certain embodiments, in addition to comprising a polypeptide of interest described herein, the compositions (e.g., pharmaceutical compositions) described herein comprise a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term “carrier,” as used herein in the context of a pharmaceutically acceptable carrier, refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. [00202] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration. In a specific embodiment, the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration.

[00203] In certain embodiments, the compositions described herein additionally comprise one or more buffers, e.g., phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the compositions described herein do not comprise buffers.

[00204] In certain embodiments, the compositions described herein additionally comprise one or more salts, e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts). In other embodiments, the compositions described herein do not comprise salts.

[00205] The compositions described herein can be included in a kit, container, pack, or dispenser together with instructions for administration.

[00206] The compositions described herein can be stored before use, e.g., the compositions can be stored frozen (e.g., at about -20°C or at about -70°C); stored in refrigerated conditions (e.g., at about 4°C); or stored at room temperature.

7.5.2 Prophylactic and Therapeutic Uses

[00207] In one aspect, provided herein are methods of preventing or treating a disease or disorder in a subject comprising administering to the subject a polypeptide of interest described herein or a composition thereof. Further provided herein are methods of preventing a disease or disorder in a subject comprising administering to the subject a polypeptide of interest described herein or a composition thereof.

[00208] In one aspect, provided herein are methods of treating a disease or disorder in a subject comprising administering to the subject a polypeptide of interest described herein or a composition thereof. In another aspect, provided herein are methods of preventing a disease or disorder in a subject comprising administering to the subject a polypeptide of interest described herein or a composition thereof. In a specific embodiment, provided herein is a method for treating or preventing a disease or disorder in a subject comprising administering to the subject a polypeptide of interest produced according to the methods described herein, wherein the polypeptide of interest is glycosylated with an N-glycan of the following structure:

wherein the black square represents an N-acetylgalactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the polypeptide of interest.

[00209] In certain embodiments, the disease or disorder may be caused by the presence of a defective version of a polypeptide of interest in a subject, the absence of a polypeptide of interest in a subject, diminished expression of a polypeptide of interest in a subject can be treated or prevented using the polypeptides of interest produced using the methods described herein. In certain embodiments, the diseases or disorder may be mediated by a receptor that is bound by a polypeptide of interest produced using the methods described herein, or mediated by a ligand that is bound by a polypeptide of interest produced using the methods described herein (e.g., where the polypeptide of interest is a receptor for the ligand).

[00210] In certain embodiments, the methods of preventing or treating a disease or disorder in a subject comprise administering to the subject an effective amount of a polypeptide of interest described herein or a composition thereof. In certain embodiments, the effective amount is the amount of a therapy which has a prophylactic and/or therapeutic effect(s). In certain embodiments, an “effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a disease/disorder or symptom associated therewith; (ii) reduce the duration of a disease/disorder or symptom associated therewith; (iii) prevent the progression of a disease/disorder or symptom associated therewith; (iv) cause regression of a disease/disorder or symptom associated therewith; (v) prevent the development or onset of a disease/disorder, or symptom associated therewith; (vi) prevent the recurrence of a disease/disorder or symptom associated therewith; (vii) reduce organ failure associated with a disease/disorder; (viii) reduce hospitalization of a subject having a disease/disorder; (ix) reduce hospitalization length of a subject having a disease/disorder; (x) increase the survival of a subject with a disease/disorder; (xi) eliminate a disease/disorder in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

7.6 Assay

7.6.1 Strains, Growth and Genetic Methods

[00211] Provided herein are methods for culturing Leishmania host cells described in Section 7.1.

[00212] Host cells are cultured using any of the standard culturing techniques known in the art. For example, cells are routinely grown in rich media like Brain Heart Infusion, Trypticase Soy Broth or Yeast Extract, all containing 5 pg/ml Hemin. In some embodiments, incubation is done at 26°C in the dark as static or shaking cultures for 2-3 days. In some embodiments, cultures of recombinant cell lines contain the appropriate selective agents. In some embodiments, cultures contain Biopterin at a final concentration of 10 pM to support growth.

[00213] A non-limiting list of selective agents is provided in Table 2.

Table 2: Selective agents used during transfection (50% concentration for preselection and 100% concentration for main selection) and standard culturing of L. tarentolae. Double amounts of the selective agents could be used if higher selection pressure was intended.

Attorney Docket No. 14688-006-228

Table 3: Summary of strains presented in the examples. Some of the strains were produced by several rounds of transfection building on top of each other.

Attorney Docket No. 14688-006-228

(i) Plasmids

[00214] Plasmids were derived from a pUC57 vector backbone for E. coli propagation and contained an ampicillin or kanamycin section marker. The expression cassettes are flanked by restriction sites suitable for excision. The composition of the cassettes depends on the intended use and is described in the respective methods and examples. The genes of interest are included as ORFs that were codon usage optimized for L. tarentolae. Optimized sequences were manually curated for avoidance of restriction sites and deletion of repeats or homopolymer stretches. The plasmids were generated and sequenced by a gene synthesis provider. Plasmids and descriptions are found in the sequence listings.

[00215] For codon usage optimization, protein sequences were back-translated to nucleotide sequences using a custom Python3 script that stochastically selects codons based on the /.. tarentolae codon usage frequency while excluding rare codons (frequency <10%). The codon usage has been calculated using cusp (Rice, et al. (2000) Trends in genetics : TIG 16 (6), pp. 276-277) on all annotated L. tarentolae nucleotide coding sequences.

(ii) Transfection Method

(A) Preparation of DNA

[00216] Restriction digest (12 pg DNA in total volume of 240 pL) was performed using standard restriction enzymes (ThermoFisher, preferably FastDigest) according to the manufacturer’s instructions. The restriction digest was performed until completion or o/n at 30°C and DNA was purified by EtOH precipitation (2 volume 100% ice cold EtOH was added to 1 volume digested DNA, incubated 30min on ice, centrifuged for 30 min 17'500 x g at 4°C. Pellet was washed with 70% EtOH and subsequently dried for maximum 15 min and resuspended in ddH20. For optimized removal of circularized plasmid, 1 or 2 restriction enzymes with recognition sites in the vector backbones were chosen and digest was done for Ih at 37°C and purified by EtOH as described above. The digest was analyzed by agarose gel electrophoresis in 0.7-2% agarose gels (TAE buffer). Optionally, gel extraction was performed with the NucleoSpin® Gel and PCR Clean-up kit (Macherey&Nagel) according to manufacturer’s instructions to remove undigested plasmid from the preparation.

(B) Transfection with Nucleofactor

[00217] One day before transfection, a densely grown culture of the parental strain was diluted 1 : 10 into fresh media (Brain Heart Infusion plus Hemin, “BHIH”; or Yeast Extract plus Hemin, “YEH”) containing all antibiotics for which selection markers were previously integrated and cultured overnight at 26°C. [00218] The linear DNA fragments for integration are mixed for transfection in the needed combinations for multiple DNA fragment- homologous recombination at 1 pg per fragment. The volume of the mix was reduced to approximately 2 pl per transfection in a vacuum concentrator at 30°C. For episomal transfection of plasmids, 0.1-1 pg of plasmid DNA were directly used for transfection.

[00219] Transfection was performed using the 4D-Nucleofector™_Core_X with the P3 Primary Cell 4D-NucleofectorTM X Kit (Lonza). For this, DNA as prepared above was mixed with 16.4 pl P3 Primary Cell solution and 3.6 pl Supplement Solution. The equivalent culture volume of 10⁷ cells (OD should be around 0.3-1.0/ml, cell shape round to drop-like) was pelleted by centrifugation at 1800 g for 5 min and the supernatant was removed. The cell pellet was resuspended in the DNA mix and transfected using a 16-well electroporation strip with pulse FI-158 (in some examples alternative pulses FP167, CM150, EO115, DN100, FP 158, FBI 58 were used). As negative control, an additional culture was transfected with ddH2O only.

[00220] 80 pl of fresh media (BHIH or YEH plus parental selection markers) was added to each well and 2x45 pl of the mix were transferred to individual wells of a 96 well culture plate that were prefilled with 200 pl of fresh medium. After incubation for 24 h at 26°C in the dark (recovery), the new selection marker was added at 50% concentration (preselection; see Table 2). After further incubation for 1-2 days, the selection marker was topped up to 100% (main selection, see Table 2) and several dilutions between 1 :2 and 1 : 10 were performed in 96 well format (final volume 250 pl). Cultures were further incubated at 26°C in the dark for up to 7 days. If no growth was observed, the culture medium was replaced (centrifugation at 1800 g, 10 min, RT) and cultures were again incubated for up to 7 days. This step was repeated if necessary. Growing cultures were expanded in to higher culture volumes by dilutions in the range of 1 :5 and 1 :20 before analysis.

[00221] For clonal selection, cells were streaked on BHIH or YEH plates (containing 1.4% agar and the appropriate 100% selective agent) as soon as the liquid culture turned turbid. Plates were sealed with parafilm and incubated 7-10 days upside down in dark at 26°C. Single colonies (1-2 mm size) were transferred into 24-well plates containing 1 ml BHIH or YEH, sealed with parafilm and incubated in dark at 26°C for around 7-10 days. 1 ml culture was then transferred from 24-well plate into 10 ml BHI or YEH in a flask and further grown statically as usual. (iii) Method of Engineering Leishmania tarentolae Cells Devoid of O-glycosylation

[00222] Without wishing to be bound by theory, one of the most effective methods to control O-linked GlcNAc modification in L. tarentolae has been found to be an RNP transfection for CRISPR/Cas9 mediated replacement of all three OGNT genes with a single selection marker. See WO 2021/140143. However, other methods have been reported and may be employed, See WO 2021/140143 which is incorporated herein by reference in its entirety. In one embodiment, a ribonucleoprotein complex formed of the endonuclease SpCas9 and bipartite guideRNAs (gRNA) are transfected into L. tarentolae to introduce double-strand breaks in the 5’ and 3’ regions of the open reading frames encoding OGNT1, OGNT2 and OGNTL. The gRNAs are formed by a scaffold RNA (tracrRNA) and one of the six sequence specific targeting RNAs (crRNA), used in this method. Along with the RNP complexes a selection marker expression construct consisting of two linear DNA fragments is transfected into the cells. During double-strand break repair in L. tarentolae the linear DNA pieces are integrated at the former OGNT expression sites by homologous recombination with each other and the 5’ and 3’ untranslated regions of the OGNT gene. In the setup described here, the selection marker expression construct does not introduce additional flanking untranslated regions and thus results in transcription of the marker by endogenous PolII.

(A) Preparation of Ribonucleoprotein (RNP) Complexes for Transfection

[00223] gRNA for CRISPR/Cas9 mediated genome editing was assembled from equimolar amounts of tracrRNA and crRNA (Microsynth) as above by denaturation for 5 min at 95°C and subsequent slow cool down at 0. l°C/s in a thermo cycler. This was done separately for every crRNA used before the different gRNAs were subsequently mixed in equimolar amounts. Next, 122 pmol recombinantly expressed Cas9 protein (i.e. Alt-R® S.p. HiFi Cas9 Nuclease V3 (IDT, #1081061) were added to 360 pmol of the gRNA mix and incubated for 15 min at RT to allow RNP formation. The final volume used for a transfection by Nucleofector (as described in this Section) should not exceed 6 pl. Lastly, the RNP mix was added to the repair DNA containing transfection solution described below along with 1 pl of Alt-R® Cas9 Electroporation Enhancer (IDT, #1081072).

(B) DNA Preparation for Transfection [00224] The linear DNA fragments for integration are mixed for transfection in the needed combinations at 1 pg per fragment and the gRNA was prepared as described above and mixed with the integration fragments. The volume of the mix was reduced to maximum 2 pl per transfection in a vacuum concentrator at 30°C.

(C) Transfection with Nucleofector

[00225] One day before transfection, a densely grown culture of the parental strain was diluted 1 : 10 into fresh media (BHIH) containing all antibiotics for which selection markers were previously integrated and cultured over night at 26°C.

[00226] Transfection was performed using the 4D-Nucleofector™_Core_X with the P3 Primary Cell 4D-NucleofectorTM X Kit (Lonza). For this, DNA (or DNA/RNA or DNA/RNP) as prepared above was mixed with 16.4 pl P3 Primary Cell solution and 3.6 pl Supplement Solution. The equivalent culture volume of 10⁷ cells (OD should be around 0.3- 1.0/ml, cell shape round to drop-like) was pelleted by centrifugation at 1800 g for 5 min and the supernatant was removed. The cell pellet was resuspended in 20 pl of the DNA (or DNA/RNA or DNA/RNP) mix and transfected using a 16-well electroporation strip with pulse FI-158 (in some examples alternative pulses FP167, CM150, EO115, DN100, FP158, FBI 58 were used). As negative control, an additional culture was transfected with ddH2O only.

[00227] 80 pl of fresh media (containing selection markers for the parental cell line) was added to each well and 2x45 pl of the mix were transferred to individual wells of a 96 well culture plate that were prefilled with 200 pl of fresh medium. After incubation for 24 h at 26°C in the dark (recovery), the new selection marker was added at 50% concentration (preselection; see Table 2). After further incubation for 1-2 days, the selection marker was topped up to 100% (main selection) and several dilutions between 1 :2 and 1 : 10 were performed in 96 well format (final volume 250 pl). Cultures were further incubated at 26°C in the dark for up to 7 days. If no growth was observed, the culture medium was replaced (centrifugation at 1800 g, 10 min, RT) and cultures were again incubated for up to 7 days. This step was repeated if necessary. Growing cultures were expanded into higher culture volumes by dilutions in the range of 1 :5 and 1 :20 before analysis.

(D) crRNA Design

[00228] crRNAs were designed based on the target regions (usually coding sequences of OGNT genes) for use with SpCas9 (PAM=NGG) by EuPaGDT (http://grna.ctegd.uga.edu/) and were counterchecked for on-/off-target effects by blast against the whole genome of L. tarentolae. crRNAs were then selected such that they are ideally targeting the extremities of the coding sequence to be replaced.

Table 4: crRNAs designed for knockout of OGNT genes. Information about stabilizing modifications of tracrRNA and crRNA ordered at Microsynth. * = PTO bond (Phosphorothioate bond); 5 = 2'-O-Methyl-RNA-A; 6 = 2'-O-Methyl-RNA-C, 7 = 2'-O- Methyl-RNA-G; 8 = 2'-O-Methyl-RNA-U; HPL = IEX-HPLC purified.

(iv) PCR and Sequence Analysis of Deletion Strains

(A) Preparation of gDNA - Genomic DNA Isolation by Tissue Kit

[00229] 2 ml of densely grown L. tarentolae culture were pelleted at 1800 g and the supernatant was discarded. The pellet was used for preparation of genomic DNA by the NucleoSpin® Tissue Kit (Macherey-Nagel). For this, the pellet is resuspended in 200 pl of Buffer T1 and further treated according to the manufacturer’s instructions until elution. For efficient elution, 50 pl of prewarmed (50°C) Buffer BE are added to the column and incubated at RT for 3 min. The eluate is collected by centrifugation for 1 min at 11000g. Repetition of this step as well as reloading of the eluate can be used to increase the yield.

(B) Preparation of Crude Cell Extracts for PCR Analysis

[00230] 50 pl of culture were washed in 1ml PBS and pelleted at 1800 g for 5 min. The supernatant was removed and the pellet was resuspended in 50 pl of PBS and boiled at 95°C for 5 min with intermitted vortexing. 1 pl was used instead of template DNA in the PCR reaction.

(C) PCR Analysis of OGNT KOs

[00231] PCR confirmation of OGNT knock-outs was performed by either amplification of the complete locus (OGNT1, OGNT2, OGNTL or OGNT1+L, where OGNT1+L comprises OGNT1 and OGNTL in tandem on the chromosome) or by amplification of the shorter fragments covering the integration sites.

[00232] Usually the successful replacement of the wildtype OGNT sequence (OGNT1 = 3.4 kbp, OGNT2=1.9 kbp and OGNTL=3.4 kbp) by the selection marker coding sequences (0.4- 1.0 kbp) with or without the additional intergenic regions (together approx. 0.9 kbp) could be easily confirmed by the size of the amplicon resulting from the PCR targeting the whole native locus, since correct replacement of the wt gene would lead to a much shorter amplicon. For these PCRs LATaq DNA polymerase (TaKaRa) was used in combination with a buffer for amplification of GC rich sequences that allowed the amplification of the long wildtype regions (see Table 5). In some cases, amplification of shorter regions with primer binding within the OGNT coding sequence was preferred to test for presence of remaining wt genes (see Table 6). For these, the DreamTaq DNA polymerase (Thermo Fisher Scientific) was used. Alternatively, the correct integration of the selection marker gene into the respective OGNT locus could be tested by combinations of a primer binding in the genome with one primer binding to the selection marker CDS or the intergenic regions of the integrated construct and the other one targeting the genome.

Table 5: PCRs for analysis of OGNT deletions. PCR primers used for confirmation OGNT knock-outs by absence of the respective OGNT wt gene and the expected amplicon sizes are summarized. * KO amplicon length for whole locus PCRs depends on the combination of selection marker and intergenic regions used.

Table 6: Listing of primer sequences used in the described examples.

(v) Expression analysis

(A) Sample Preparation from Leishmania tarentolae

[00233] Cells were grown for 2-3 days at 26°C, static (e.g.in 3 ml in a 6-well plates). Whole cell extract (WCE) cell free culture supernatants were analyzed by Western blot. For supernatant analysis, grown culture was centrifuged at 1800 g at RT for 5 min and cell free supernatant was transferred to a new tube and mixed with Laemmli dye under reducing or non-reducing conditions. Cell pellets for WCE were washed with lx PBS, centrifuged again at 1800 g at RT for 5 min and frozen at -80°C for minimally 30 min. After thawing it again at RT pellet was then resolved in Leammli (reducing) buffer, boiled again at 95 °C for 10 min and vortexed intensively.

(B) Expression Analysis by Western Blot

[00234] Samples were run on 4-12% Bis-Tris SDS PAGE, using a MOPS running buffer with 200 V for 60 min. Gels were blotted using an Iblot device for 7 min on PVDF membranes. Membranes were blocked for at least 30 min at RT in 10% milk. Primary antibodies (i.e. goat anti-Human IgG-HRP (A6029, Sigma) 1 :2000 diluted, mouse antiHuman Kappa Light Chain (K4377, Sigma) 1 :5000 diluted were used diluted in 1% milk, lx PBST for o/n incubation at 4°C. Afterwards, the blot was washed with lx PBST three times for 5 min before detection with horse reddish peroxidase (HRP) coupled secondary antibodies (anti-mouse polyvalent-HRP (A0412, Sigma) 1 :2000 diluted or anti-rabbit-HRP conjugate (Jackson ImmunoResearch #111-035-008) 1 :2000 diluted) in 1% milk, lx PBST for 3h rotating at 30°C, followed by three washes for 5 min in IxPBST and one component 3,3’,5,5’-tetramethylbenzidine (TMB) substrate staining for colorimetric detection (TMBM- 1000-01, Surmodics).

7.6.2 Small Scale Expression and Purification of Monoclonal Antibodies

[00235] Host cells were routinely grown in 50 ml culture in BHIH or YEH for 48 h at 26°C shaking at 140 rpm. Cultures were harvested and centrifuged for 10 min at 1800*g at RT. Media SN was filtered through 0.22 pm filter (Steriflip, SCGP00525) and EDTA(0.5 M pH8) was added to each load in a 1 : 100 dilution. Media SNs of each strain were subjected to 4h incubation with 100 pl of proteinA resin (ProteinA-Sepharose 4B Fast Flow, Sigma Aldrich, P9424) per Falcon tube in batch while rotating at RT. After treatment with ProteinA resin, the samples were centrifuged at 500*g for 5 min, the FT was discarded and the resin was transferred to spin columns. Washes were performed with 3 x 5 CV using Buffer A (pH 7.2, 20 mM Na2HPO4, 150 mM NaCl, pH was adjusted with HC1 to 7.20) using 500 pl for 100 pl resin; with centrifugation at 1000*g, RT, 1 min between each step. Elution was performed with several CV of Buffer B (0.1 M acetic acid, 100 mM NaCl, pH was adjusted with 1 M NaOH to 3.20) using 100 pl for 100 pl resin, with centrifugation at 1000*g, RT, 1 min between each step (e.g. 3xlCV and 1x0.5 CV). Elution fractions were pooled and immediately neutralized by adding 100 mM Tris-HCl (1 M pH8). Afterwards, the pooled elutions were buffer exchanged to PBS pH 6 using 2 ml 7K ZebaSpin desalting columns and optionally concentrated using Amicon 0.5 ml 30 K concentrators.

[00236] Elution fractions were pooled and immediately neutralized by adding 100 mM Tris-HCl (1 M pH8). Afterwards, the pooled elutions were buffer exchanged to PBS pH 6 using 2 ml 7K ZebaSpin desalting columns and optionally concentrated using Amicon 0.5 ml 30 K concentrators.

[00237] Samples were then subjected to analyses such as HILIC-UPLC-MS as described below.

7.6.3 Analysis

(i) SDS PAGE and Capillary Gel Electrophoresis

[00238] SDS PAGE was performed under reduced or non-reducing conditions using 10 pg for Coomassie, 2.5 pg for WB, separated on 4-12% Gel with MOPS buffer for 55 minutes. Determination of Protein purity was done by Coomassie Stained SDS-PAGE with 10 pg protein sample and compared to a BSA standard curve. Impurities were quantified by ImageQuant. Capillary Gel Electrophoresis (CGE) was performed using an Agilent Protein 230 Kit (5067-1518), according to protocol.

(ii) Analytical SEC

[00239] MAbPac SEC-1 (4x300 mm) is a size exclusion chromatography (SEC) column specifically designed for separation and characterization of monoclonal antibodies (mAbs) and was used according to manufacturer’s recommendation (Temperature: 30 °C; Eluent: PBS 50mM NaPO4, 300 mM NaCl pH 6.8; Elution: isocratic, 30 minutes; Flow: 0.2 mL/minute; Detection: 215 nm; Injection V: 5 pL corresponding to 5 pg protein).

(iii) O-HexNAc identification

[00240] Intact monoclonal antibodies were analyzed by mass spectrometry employing the state-of-the-art instrumentation (Orbitrap FTMS), data processing and data analysis (bioinformatics) tools by SpectroSwiss. In addition to the intact measurement the antibody was reduced with TCEP or enzymatically cleaved (IdeS) to generate Fd, LC and Fc/2 subunits, which were analyzed using the same instrumentation.

(iv) Sample preparation for IdeS derived subunit analysis

[00241] To generate mAb subunits of about 25 kDa each, IdeS (FabRICATOR, Genovis, Lund, Sweden) digestion of mAbs was performed in formulation buffers. One unit of IdeS was added to each pg of mAbs and left to react for 30 minutes at 37° C. Then, mAbs were denatured and reduced by incubation with 6 M GdnCl and 30 mM TCEP at room temperature for 30 minutes. Finally, the reaction was quenched by acidifying the solution to 1 % TFA. For the analysis samples were diluted with 0.1% FA in water to a final concentration of 1 pg/pl.

(v) Mass spectrometry for intact and subunit analysis, Bioinformatics and Data processing.

[00242] A standard Orbitrap-based intact protein mass measurement set-up (a triplicate LC-MS experiment) was employed using:

[00243] - Analytical HPLC (Dionex) with 30 min run duration,

[00244] - Analytical column (Waters), C4 Aquity, BEH, 90 pl/min @60C,

[00245] - Q Exactive HF FTMS (Thermo),

[00246] - FTMS Booster (Spectroswiss), advanced data acquisition system [00247] - Full MS resolution: 30’000 @ m/z 200 for intact mAb and 60’000 @ m/z 200 for subunit analysis

[00248] - Charge target number (ion number): AGC of 3e6

[00249] Intact Mass (Protein Metrics) was employed for mass spectra deconvolution of intact mAbs. MASH Suite software tool (open access, Wisconsin University, Ying Ge Group) was used for deconvolution of the mass spectra of the subunit analysis. The employed resolution enabled obtaining isotopically-resolved data. Peak-by-Peak software tool (Spectroswiss) was employed for time domain signals and mass spectra processing. The employed methods enabled obtaining high spectral dynamic range data.

(vi) Analysis of N-glycans released from purified proteins and cells surfaces by HILIC-UPLC-MS

[00250] Enzymatic release of N-glycans from cell surfaces was performed using PNGase F (New England Biolabs). Cells (grown for 48 or 72 h at 26°C shaking at 140 rpm) were harvested and washed with PBS by centrifugation for 10 min at 1800*g at RT.50 mg of cell pellet were re-suspended in Glyco Buffer 2 and incubated with 1 pl PNGase F for 1 h at 37 °C and 650 rpm. Cells were again pelleted by centrifugation and 75 pl of the supernatant was dried down in a SpeedVac concentrator. Glycans were resuspended in 10 pl of water.

Following release, glycans were directly labeled with procainamide as described previously (Behrens, et al. (2018) Glycobiology 28 (11), pp. 825-831). Briefly, released glycans were mixed with 1 pl acetic acid, 8 pl of a procainamide stock solution (550 mg/ml in DMSO) and 12 pl of a sodium cyanoborohydride stock solution (200 mg/ml in H2O). Samples were incubated for 60 min at 65 °C and cleaned up using LC-PROC-96 clean up plates (Ludger Ltd) according to the manufacturer’s instructions.

[00251] Enzymatic release of N-glycans from purified proteins was performed using Rapid PNGase F (New England Biolabs) as recommended by the supplier. 8 pl of sample (15 pg of protein) were mixed with 2 pl Rapid Buffer and 1 pl of Rapid PNGase F. The mixture was incubated at 50 °C for 10 min followed by 1 min at 90 °C.

[00252] For site specific N-glycan analysis of monoclonal antibody subunits, IgGl mAb was either cleaved with IdeZ to F(ab’)2 and Fc/2, or heavy and light chains were reduced before separation on SDS PAGE. Bands were excised and enzymatic release of N-glycans from the monoclonal antibody was performed using PNGase F. Following release, glycans were directly labeled with procainamide (PC).

[00253] Procainamide-labeled N-glycans were analyzed by hydrophilic interaction chromatography-ultra performance liquid chromatography-mass spectrometry (HILIC- UPLC-MS) using am Acquity UPLC System (Waters) with fluorescence detection coupled to a Synapt G2-Si mass spectrometer (Waters). Glycans were separated using an Acquity BEH Amide column (130 A, 1.7 pm, 2.1 mM x 150 mM; Waters) with 50 mM ammonium formate, pH 4.4 as solvent A and acetonitrile as solvent B. The separation was performed using a linear gradient of 72-55 % solvent B at 0.5 ml/min for 40 min. Fluorescence was detected at an excitation wavelength of 310 nm and a detection wavelength of 370 nm. The Synapt G2-Si mass spectrometer fitted with a Zspray electrospray source was used for mass detection in positive resolution mode using the following parameters: Scan range: m/z 300- 3500; scan time: 1 sec; capillary: 2.2 kV; source temperature: 120 °C and sampling cone: 75 V. MassLynx 4.2 (Waters) was used for data acquisition. Data processing and analysis was performed using Unifi 1.9.4.053 (Waters). Glucose units were assigned using a fifth-order polynomial distribution curve based on the retention times of a procainamide-labeled dextran ladder (Ludger Ltd). Glycan structures were assigned based on their m/z values and their retention times and matched against a previously constructed N-glycan library. For individual samples the UPLC was coupled to a Synapt HDMS mass spectrometer using comparable settings.

7.6.4 Analysis of UDP-GalNAc by High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD)

[00254] MeOH/Chloroform extraction procedure for L. tarentolae cell pellets was performed on 2 OD of each sample, which were harvested by centrifugation and washed 2x with IxPBS (2200 g, 10 min, RT) and frozen. For extraction, pellets were thawed, resuspended in 480 pl MeOH, supplemented with 20 pl water and sonicated in a water bath at RT for 15 min. The samples were spun in a table-top centrifuge at 18000 g and 4°C for 10 min. The SN was transferred into a glass vial, supplemented with 268 pl chloroform and vortexed. Next, 500 pl H2O (MS grade) was added and the sample was vortexed again. The MeOH/chloroform/H2O (1/0.54/1) mixture was spun at 2200 g and RT for 20 min to remove proteins, lipids and DNA in the CHC13 phase. Approximately half (525 pl) of the upper Me0H/H20 phase was collected and transferred into Eppendorf tubes, corresponding to extracted material from 0.5 OD pellet. The samples were dried in a speed-vac and stored at - 20°C until analysis. Thawed samples were resuspended in H2O and amounts corresponding to 0.5 OD analyzed by High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) using CarboPac PAI, 4x250 mm (Thermo), based on Tomiya et al. 2001 : Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Celles and Mammalian Cells. Analytical Biochemistry 293, p. 129-137, with an adjusted gradient. A standard of UDP-GalNAc/- GlcNAc mix (each 25 ug/mL) in H2O was prepared in addition to the cell extract samples -> 1000 ug/mL UDP-GlcNAc and -GlcNAc solutions were pre-diluted 1 : 10 to 100 ug/mL solutions. 50 uL of each 100 ug/mL solution was added to 100 uL H2O for a solution containing 25 ug/mL from each component (corresponds to 38.4 uM from each component). Spiked UDP-GalNAc was detectable in cell extract samples as set for another control. Variability for three independent replicates of 0.5 OD injection is at 17.2% CV (n=3). Average recovery lies at 69%. For the 1.2 OD injection, spike recovery lies at 62% (n=l).

8. EXAMPLES

8.1 EXAMPLE 1

[00255] Glycoengineered Leishmania larenlolae. the CGP (CustomGlycan Platform) host cells have performed efficiently to generate specific human N-glycans such as A2, A2G2 or A2G2S2, and for expression of different recombinant therapeutic proteins such as monoclonal antibodies (mAbs) and Erythropoietin, as described in detail in WO 2019/002512, WO 2021/140144, WO 2021/140143 and WO 2022/053673. Described herein is the engineering of host cells to produce native and recombinant proteins having a biantennary GalNAc terminated N-glycan, namely A2GalNAc2 (for nomenclature, see Section 5.3). The Leishmania host cells provided herein produce surprisingly efficient ASGPR ligands that can be exploited for generating innovative therapeutics designed, for example, but not limited to, targeted protein degradation, such as clearing soluble targets quickly from circulation.

[00256] As a first step, the enzymatic steps required to generate A2GalNAc2 containing N-glycans in Leishmania host cells (as referred to herein as CGP, or CGP host cells) were identified. First, there are enzymes required for generating UDP-GalNAc and second, the enzymes that mediate the transfer of GalNAc from UDP-GalNAc to GlcNAc acceptors with a beta-l,4-linkage, to form monoantennary and biantennary GalNAc-beta-l,4-GlcNAc (A2GalNAc2) structures in N-linked glycans.

[00257] A genomic search was performed to identify enzymes involved in the generation of nucleotide activated sugar donors in L. tarentolae '.

[00258] For UDP-Glc and UDP-Gal the enzymes and reactions are as follows: a-D-Glc-1- P + UTP is converted by UDP -glucose pyrophosphorylase (UGP) to UDP-Glc, which can be further epimerized by GalE (UDP -galactose 4-epimerase) to UDP-Gal. In addition to this de novo pathway, Leishmania parasites and plants possess also a UDP-sugar pyrophosphorylase (USP), which has broad substrate specificity and is involved in monosaccharide salvage. This means that a-D-Gal-l-P + UTP can be formed by USP to UDP-Gal. The UDP-sugar pyrophosphorylase can therefore activate both Gal-l-P and Glc-l-P n Lei hmania major. Deletion of the UGP encoding gene in L. major strongly reduced but did not abolish the biosynthesis of UDP-Gal and its immediate precursor UDP-Glc, since USP enabled the limited biosynthesis of both nucleotide sugars. (Roper 2002, Urbaniak 2006, Capul 2007, Damerow 2004). For illustration, see FIG. 1.

Table 7: Bioinformatic Evaluation on USP, UGP and GalE Orthologues

[00259] UDP-Gal is readily available in Leishmania tarentolae, as determined by (1) the presence of USP, UGP and GalE orthologues (see Table 7) and (2) the efficient generation of galactosylated N-glycans from engineered L. tarentolae, for which the recombinant galactosyltransferases need UDP-Gal as activated sugar donor for their activity (see A2G2 and A2G2S2 in WO 2019/002512, WO 2021/140144, WO 2021/140143, and WO 2022/053673). While UDP-Gal is natively produced by L. tarentolae presumably by a predominant de novo process involving epimerization of the abundant nucleotide sugar UDP- glucose by the UDP -galactose 4-epimerase (GalE), there was no evidence of genes generating UDP-GalNAc found by the bioinformatic search.

8.1.1 UDP-GalNAc Biosynthesis Pathway: the Salvage Pathway

[00260] UDP-GalNAc can be generated by the salvage pathway in mammalian cells. UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) converts UTP and GlcNAc-1-P into UDP-GlcNAc, and UTP and GalNAc-l-P into UDP-GalNAc. Without wishing to be bound by theory, the isoform AGX1 is about 2 to 3 times more active towards GalNAc-l-P, while the isoform AGX2 is about 8 times more active towards GlcNAc-1-P. GalNAc-l-P is formed by a specific kinase which is GALK2 (N-acetyl galactosamine kinase) that is distinct from galactose- 1 -kinase.

[00261] These GalNAc Salvage pathway enzymes are not present in L. tarentolae and were selected to be recombinantly expressed in CGP host cells.

8.1.2 UDP-GalNAc Biosynthesis Pathway: Epimerization Pathway

[00262] On the other hand, UDP-GalNAc can also be formed by epimerization of UDP- GlcNAc using the same NAD-dependent epimerase that converts UDP-Glc to UDP-Gal. The human GalE catalyzes two distinct but analogous reactions: the reversible epimerization of UDP-Glc to UDP-Gal and the reversible epimerization of UDP-GlcNAc to UDP-GalNAc. The reaction with UDP-Gal plays a critical role in the Leloir pathway of galactose catabolism in which galactose is converted to the glycolytic intermediate Glc-6-P. It contributes to the catabolism of dietary Gal and enables the endogenous biosynthesis of both UDP-Gal and UDP-GalNAc when exogenous sources are limited. The human GalE was selected to be recombinantly expressed in CGP host cells.

[00263] Another epimerase was selected from a bacterial source. The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni contain Gal and/or GalNAc residues. The presence of GalNAc residues in these carbohydrates suggested that GalE, the initially annotated UDP-glucose 4-epimerase might be a UDP-GlcNAc 4-epimerase. GalE was indeed shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne (Bematchez et al., (2005) J Biol Chem.; 280(6):4792-802. doi: 10.1074/jbc.M407767200).

8.1.3 UDP-GalNAc Biosynthesis Pathway: Transporter Protein

[00264] A transporter of UDP-GlcNAc and UDP-GalNAc is encoded by the Caenorhabditis elegans gene C03H5.2. Surprisingly, translocation of these substrates occurs in an independent and simultaneous manner that is neither a competitive nor a symport transport (Caffaro et al., (2006). Proc Natl Acad Sci U S A;103(44): 16176-81). This transporter was selected to be recombinantly expressed along with the salvage and epimerization pathway enzymes. 8.1.4 GalNAc Transferases

[00265] N-acetyl-beta-glucosaminyl-glycoprotein 4-P-N-acetyl galactosaminyl-transferase activity was described by Sato et al., (2003): Journal of Biological Chemistry 278 (48), pp. 47534-47544. This human p4GalNAc-T3 (“P4-GalNAcT3”) effectively synthesizes N,N'-diacetylgalactosediamine, GalNAc P 1-4 GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. These results suggest that p4-GalNAcT3 could transfer GalNAc residues, producing N,N'-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans. p4-GalNAcT3, (Sato et al., (2003): Journal of Biological Chemistry 278 (48), pp. 47534-47544), and P4- GalNAcT4 (Gotoh et al., (2004): FEBS Letters 562 (1-3), pp. 134-140) were therefore identified for GalNAc transfer and described have a broad tissue expression coverage including fetal kidney and brain in human.

[00266] It has also been reported that adding a carboxyl-terminal 19-amino-acid a-helix stretch with several basic amino acids is sufficient to mediate GalNAc transfer to N-linked oligosaccharides. Other GalNAc motifs involve three structural loops with aromatic side chains, as well as additional unidentified motifs. Some glycohormones contain N,N'- diacetylgalactosediamine structures in their N-glycan. Fiete et al., (2012): J. Biol. Chem. 287 (34), pp. 29194-29203. Bonar et al., (2014): J. Biol. Chem. 289 (43), pp. 29677-29690.

[00267] Human p4-GalNAcT3 and p4-GalNAcT4, were selected to be recombinantly expressed in CGP host cells.

[00268] The A2GalNAc2 motif occurs in mammalian pituitary glycoprotein hormones, where the terminal GalNAc residues are 4-O-sulfated functions as a recognition marker for clearance by the endothelial cell CD206 (ManR) receptor. However, nonpituitary mammalian glycoproteins also contain A2GalNAc2 determinants, indicating that expression of A2GalNAc2 determinants in vertebrate glycoconjugates is more widespread than once thought. Noteworthy, A2GalNAc2 and modifications of A2GalNAc2 sequences are common antigenic determinants in many parasitic nematodes and trematodes. Caenorhabditis elegans CeP4GalNAcT is a member of the P4GalT family, a 383-amino acid type 2 membrane glycoprotein. Its soluble, epitope-tagged recombinant form of CeP4GalNAcT expressed in CHO-Lec8 cells was functional using UDP-GalNAc, but not UDP-Gal, as a donor toward different acceptor substrates containing terminal P-linked GlcNAc in both A- and ( -glycan type structures (Kawar et al., (2002). J Biol Chem.;277(38): 34924-32 Kawar et al., (2005). J Biol Chem. (13): 12810-9). This enzyme was also selected to be recombinantly expressed in CGP host cells.

[00269] The UDP-GalNAc biosynthesis pathway enzymes and GalNAc transferases recombinantly expressed in CGP are listed in Table 8. “Tr” means truncated, as the candidate GalNAc-transferase was N-terminally truncated, to yield a soluble form, and a Leishmania tarentolae derived signal peptide was added.

Table 8: The UDP-GalNAc Biosynthesis Pathway Enzymes and GalNAc Transferases

Recombinantly Expressed in CGP

[00270] To summarize, these candidates were primarily selected from sufficiently well described human source but extended to other species such as bacterial organisms or nematodes, that do generate GalNAc containing glycoconjugates.

8.2 EXAMPLE 2 - Strains for UDP-GalNAc Biosynthesis Pathway

[00271] While there was no evidence of genes generating UDP-GalNAc found by the bioinformatic search, the absence of UDP-GalNAc in HPAEC-PAD performed on MeOH/Chloroform extraction of L. tarentolae cell pellets was confirmed. (See FIG. 3A) [00272] To then evaluate GalNAc biosynthesis pathways for CGP glycoengineering of A2GalNAc2 N-glycans, different genetic combinations were tested, e.g. Transporter +Salvage + Epimerase (“T+S+E” or “TSE”); or Transporter + Salvage (“T+S” or “TS”) ; or Transporter^- Epimerization (“T+E” or “TE”) with genetic constructs of codon usage optimized (cuo) coding sequences spaced by different intergenic regions derived from Leishmania major (J, AC, AJ, T, AF vs Y, Z, AJ, T, AF) and different site-specific integration loci (pfr and aTub). These different genetic constructs were integrated into StLMTB19915, a strain expressing an mAb with 2 exposed glycosites (Adalimumab K84N, D86N), >98% A2 N-glycans and devoid of O-glycosylation. The resulting strains (StCGP2801, 2803, 2805, 2807, 2847/2848) were not yet containing any GalNAc transferase, but their suitability to generate UDP-GalNAc as nucleotide activated sugar donor was tested by HPAEC-PAD on Chloroform/MetOH extracted cell pellets. The cells containing the salvage pathway enzymes (“T+S”) in StCGP2805 and one cell line StCGP2848, containing T+S+E, were grown in media supplemented with lOmM GalNAc.

[00273] Extracts from strains StCGP2805 and StCGP2848 clearly show detectable levels of UDP-GalNAc whereas in other extract samples, UDP-GalNAc is not or only detectable at minor amounts. Highest level of UDP-GalNAc was determined for the extract from StCGP2848. Interestingly, StCGP2847, another clone tested containing the same “T+S+E” construct (a second clone from the same transfection), but was not supplemented with GalNAc in the medium, did not show UDP-GalNAc. Also for StCGP2801 and StCGP2803 (both “T+S+E”) were not fed with GalNAc and also did not show any significant amounts of UDP-GalNAc, See FIG. 3B.

[00274] These results suggest that the salvage pathway is active in CGP when media is supplemented with GalNAc. This also means that no or only minor amount of GalNAc is present in the used Yeast extract media (YEH). Notably, Epimerization only (“T+E”) also did not show measurable levels of accumulated UDP-GalNAc, or if at all at very minor levels.

8.3 EXAMPLE 3

8.3.1 Testing Human GalNAc Transferases

[00275] The cell lines discussed in Example 2 containing different UDP-GalNAc biosynthesis pathways were further transfected with different human GalNAc transferase constructs (P4-GalNAcT3 and/or p4-GalNAcT4), and N-glycan profiles on the purified mAb were evaluated. First, the mAb was cleaved by IdeZ to generate F(ab’)2 and Fc subunits, which were separated on SDS PAGE. Bands were cut from the gel and subjected to PNGaseF N-glycan release and labeling with PC (see method vi). Surprisingly, >80% A2GalNAc2 N- glycans on exposed (Fab) glycosites in one of the strains, StCGP2879 was found (see FIGS. 4A and 4B). In many of the strains, 50 to 80% A2GalNAc2 on Fab sites was detected. While on the Fab glycosites up to 80% A2GalNAc2 was observed, the conversion up to A2GalNAc2 seems to be much higher on the Fab glycosite compared to the native surface glycoproteins. The results of this study are illustrated in FIG. 5 and discussed below. The cell lines are described in Table 3.

[00276] Furthermore, on the Fc glycosite only minor amount of A2GalNAc2 (up to 10%) was observed. However, this was expected to be low since it was already seen before that the galactosylation is not very efficient on the sterically constraint N297 Fc glycosite.

8.3.2 Strains Containing Transporter, Salvage and Epimerization Pathways and (34- GalNAcT3

[00277] The targeted locus of the UDP-GalNAc pathway did not seem to make a difference comparing StCGP2853 [Pfr] with StCGP2855 [atub]. The intergenic region variants IrLmJ and IrLmAC seemed to be beneficial over IrLmY and IrLmZ.

8.3.3 Strains Containing Transporter and Salvage Pathways and |J4-GalNAcT3

[00278] The intergenic region variants IrLmY and IrLmZ seemed to be beneficial over IrLmJ and IrLmAC.

8.3.4 Strains Containing Transporter and Epimerization Pathways and |J4-GalNAcT3

[00279] The non-fed strain (StCGP2860) showed a good A2GalNAc2 amount (33% on Fab sites) despite no major UDP-GalNAc level having been observed in its parental strain (StCGP2807) by HPAEC-PAD.

[00280] Taken together, these results suggest that the epimerization pathway is active in CGP, albeit with lower efficiency than the salvage pathway (if media contains lOmM GalNAc supplement in salvage pathway strains) showing 45% A2GalNAc2 on exposed Fab sites (16% on cell surface) on StCGP2857. When both pathways are used in combination (like in parental strain StCGP2801 and 2803) the A2GalNAc2 levels found on Adalimumab Fab sites was higher with 67% and 80%, in strains StCGP2853 and StCGP2879, respectively.

8.3.5 Strains with UDP-GalNAc Biosynthesis and |J4-GalNAcT4

[00281] None of strains of the different UDP-GalNAc biosynthesis pathways containing only p4-GalNAcT4 had significant conversion to A2GalNAc2 (0.3-2.5%; not shown), indicating that P4-GalNAcT4 is not very active since the same parental strains were showing conversion up to A2GalNAc2 upon transfection of P-GalNAcT3. The reason could be either because p4-GalNAcT4 is not well expressed in this genetic context or because this acceptor glycosites with A2 were not optimal for p4-GalNAcT4.

8.3.6 Strains Containing Transporter, Salvage and Epimerization Pathways, and P4- GalNAcT3 and p4-GalNAcT4

[00282] Targeting the [atub] locus seems to be slightly better than the [Pfir] locus comparing StCGP2877 with StCGP2879, based on cell surface A2GalNAc2 levels with 20% and 25%, respectively.

8.3.7 Strains containing transporter and salvage pathway, p4-GalNAcT3 and P4- GalNAcT4

[00283] The intergenic region variants IrLmJ and IrLmAC seem to be beneficial over IrLmY and IrLmZ.

8.4 EXAMPLE 4

8.4.1 Strain StCGP3169

[00284] To obtain a suitable parental cell line harboring the capacity to modify N-glycans to A2GalNAc2, StCGP3169 was generated using the following modifications, also described in FIG. 6

[00285] For the first cell line named StLMTB 18407 expressing different glycosyltransferases for conversion of the endogenous Man3 to A2 N-glycans by using functionally redundant enzymes for the first glycoengineering step (hsMGAT, drMGATl, hsMGAT2 and mMGAT2), wild type L. tarentolae (Stl0569) cells were transfected with an expression construct, called “G0-long_hyg[Pfr]”, formed by homologous recombination of 13 DNA fragments. These DNA fragments were designed with intergenic regions interspersing the coding sequences in the assembled synthetic polycistron. Homologous recombination in-between the fragments and between fragments and the Pfr locus on the genome was enabled by 200 bp to 500 bp homologous regions, respectively. StLMTB 18407 was analyzed with 1.7% Man3, 0.8% Al and 97.5% A2 N-glycans on its surface glycoproteins.

[00286] To produce a strain showing minimal amounts of Man3 and maximal amount of A2 on any polypeptide of interest, another genetic module, called “GO M3.2_pac[ssu-Poll]”, encoding several MGAT1 and MGAT2 copies (drMGATlb, SfGntl and rnMGAT2) spaced by intergenic regions, was integrated into the [ssuPolI] locus of StLMTB 18407 by multiple homologous recombination of 10 fragments. The newly generated cell line StLMTB 19462 showed indeed complete, 100%, A2 on cell surface glycoproteins.

[00287] The next engineering step was integrating the UDP-GalNAc biosynthesis module, called “GalNAc synth (T+S+E)_ blefPfr]” into the Pfr locus, using 7 fragments. This resulting strain, StCGP3023, was further transfected to integrate the P4-GalNAcT3 enzyme by using genetic module “GalNAcT3_ bsd [ssuPolI]” by recombining 5 fragments and integrating into the ribosomal rDNA locus [ssu-PolI], benefitting from a high-level expression of the integrated construct by the RNA Poll polymerase. The strain called StCGP3053 showed 37% A2GalNAc2 on its cell surface glycoproteins.

[00288] Next, a OGNT triple deletion mutant, devoid of O-glycosylation was generated by the method of CRISPR/Cas9 based knock-outs for L. tarentolae (as described in the Assays above). The initial cell surface N-glycans of newly generated StCGP3169 had 29% A2GalNAc2.

[00289] Then we transfected StCGP3053 and StCGP3169 with Adalimumab (K84N/D86N) expression cassette to test for A2GalNAc2 levels on the exposed Fab glycosites. Both strains led to 82% A2GalNAc2 on the Fab sites (See FIGS. 7A and 7B). In order to expand the polypeptides of interest containing similar positioned glycosites, expression cassettes for mAb 5C9, a human IgG4 PAA mAb containing D86N Fab framework (FR) glycosite, and for 1 lk2, also a human IgG4 PAA mAb, containing D86N. Interestingly, while 5C9(D86N) showed 90% A2GalNAc2, 1 lk2 acceptor site D86N showed only 48% A2GalNAc2, along with 43% A2GalNAcl glycans (See FIGS. 8A and 8B). Pertuzumab, another human IgGl (like Adalimumab) was engineered with D86N and this Fab contained 60% A2GalNAc2 (data not shown). This could indicate that, despite similar expression levels of the antibodies secreted to the supernatant, the A2GalNAc2 efficiency varied due to different acceptor sequences (glycosite and/or surrounding framework regions), possibly due to the described peptide specificity of p4-GalNAcT3. However, another hypothesis could be the protein folding and structure surrounding the glycosite, posing some steric constraints for the transferase activity. On the other hand, it can’t be ruled out that slight expression levels of the UDP-GalNAc biosynthesis genes and of the transferases could also lead to this variability.

8.4.2 Strain StCGP3558

[00290] To generate another parental strain capable of generating A2GalNAc2 N-glycans, StCGP3558 was constructed as follows and described in FIG. 6. [00291] StLMTB 19462 was engineered with a triple OGNT deletion, to yield

StLMTB20097. StLMTB20097 was created by RNP mediated replacement of all three OGNTs with the selection marker for geneticin resistance. Comparison of the N-glycan profiles released from L. tarentolae surface proteins of the parental cell line Stl9462 and the triple OGNT knock-out cell line St20097 demonstrated that the efficiency of N-glycan conversion to A2 was not affected by OGNT knock-outs introduced by the RNP transfection method (see, e.g., FIG. 12C in WO 2021/140143).

[00292] Then, a combined genetic module (named “GalNAc synth (T+S+E)_GalNAcT3_ bsd(C) [ssuPolI]) containing both, the UDP-GalNAc biosynthesis pathway and the P4- GalNAcT3 enzyme was introduced by multiple homologous recombination of 10 fragments into the ssu-PolI locus. The resulting strain StCGP3558 was then also tested for its capacity to generate A2GalNAc2 N-glycans on different polypeptides of interest:

[00293] Adalimumab (K84N/D86N) had 73% A2GalNAc2, and 1 lk2 D86N 40% A2GalNAc2 on the Fab glycosites. This is comparable to the other strain performance. Interestingly, both strains, StCGP3169 and StCGP3558, had -70% A2GalNAc2 levels on Fab site of Adalimumab even without supplementing GalNAc to the medium.

8.5 EXAMPLE 5 - Evaluation of Different GalNAc Transferases

[00294] First, different parental strain were generated, similar to StCGP3558, except that the genetic information of the human P-GalNAcT3 was exchanged to alternative GalNAc transferases (see Table 8). N-glycan profiles of cell surfaces glycoproteins showed around 50% A2GalNAc2 content when C. elegans GalNAcT (“CeGalNAcT”) was encoded, which suggests a better activity than the human transferase, p4-GalNAcT3 (40% A2GalNAc2). When both enzymes were combined in one parental strain, also 50% A2GalNAc2 were reached, however with additionally 10% of incomplete N-glycan, A2GalNAcl. Interestingly, when those cell lines were transfected with two different mAb constructs, either Adalimumab (K84N/D86N) or 1 lk2 (S84N/D86N), the conversion efficiency of CeGalNAcT as well as the other GalNAc transferases (St/Hh, derived from Salmo trutta and Hucho hucho) on the engineered Fab glycosites were lower than by the human p4-GalNAcT3 (See FIGS. 9A, 9B, and 9C). However, the GalNAc transferase derived from Parasteatoda tepidariorum (PtGalNAcT) reached also 67% A2GalNAc2 on Fab of Adali(K84N/D86N), shown in FIG. 9B. This varying efficiencies can either be explained by differences in their localization/retention in the secretory pathway, or differences in expression levels influenced for example by the coding sequences themselves or mRNA stability, or by the specific

I l l activity on those peptide acceptor sequences such as primary sequences or steric constraints by the protein domains.

8.6 EXAMPLE 6 - Evaluation of uncharacterized UDP-GalNAc Transporter homologs

[00295] In eukaryotes, nucleotide sugars are synthesized in the cytoplasm or nucleus, whereas most glycosylation occurs inside the ER or Golgi compartments. Therefore, newly synthesized nucleotide sugars must be transported into the ER and Golgi lumen. Negative charge prevents these donors from simply diffusing into these compartments. To overcome this hurdle, eukaryotic cells have a set of energy-independent nucleotide sugar antiporters that deliver nucleotide sugars into the lumen of these organelles, with the simultaneous exiting of nucleoside monophosphates. The Km of the transporters ranges from 1 to 10 pm. Using in vitro systems, the transporters have been shown to increase the concentration of the nucleotide sugars within the Golgi lumen by 10- to 50-fold. This is usually sufficient to reach or exceed the calculated Km of glycosyltransferases that use these donors.

[00296] Theoretically, glycosylation may be controlled in part by regulating the availability of nucleotide sugars within the Golgi, presumably by regulating the transporters. The subcompartmental location (cis, medial, trans) of the transporters in the Golgi is not known nor are the physical relationships of the transporters to the various glycosyltransferases they service. Many putative transporters were previously identified by homology in the genomes of mammals, Drosophila melanogaster, C. elegans, plants, and yeast, but the level of amino acid identity does not provide any information to the substrate specificity. The UDP-GlcNAc transporters from mammalian cells and yeast are 22% identical, whereas mammalian CMP-Sia, UDP-Gal, and UDP-GlcNAc transporters have 40%-50% identity (Essentials of Glycobiology, 4th edition PMID: 35536922 DOI: 10.1101/9781621824213)

[00297] While there is limited biochemical information on UDP-GalNAc transporters available, it however poses another opportunity to maximize the levels of A2GalNAc2 N- glycan biosynthesis in engineered Leishmania host cells by increasing the availability of UDP-GalNAc in the Golgi by testing previously uncharacterized UDP-GalNAc transporter homologs. A wide sequence homology search using CeC03H5.2 as query was performed. However, the sequence homology search using CeC03H5.2 alone was believed unlikely to differentiate between UDP-GlcNAc and UDP-GalNAc transporters, especially in view of the fact that the C. elegans transporter CeC03H5.2 is also multifunctional (Caffaro, C. E., Hirschberg, C. B. & Beminsone, P. M. Independent and simultaneous translocation of two substrates by a nucleotide sugar transporter. Proc National Acad Sci 103, 16176-16181 (2006)). Accordingly, we extended the homology search to a second transporter called UGTREL7 (Uniprot Q9NTN3, also known as SLC35D1). UGTREL7 is described as a UDP- GalNAc and UDP -glucuronic acid transporter according to Muraoka (Muraoka, M., Kawakita, M. & Ishida, N. Molecular characterization of human UDP-glucuronic acid/UDP- N-acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity. FEBS Lett. 495, 87-93 (2001). The sequence homology search was performed using either CeC03H5.2 or UGTREL7 as representative sequence and their reciprocal best hits were clustered for 80% sequence identity. From this, a subset of sequences for CeC03H5.2 homologs (Table 10) and for UGTREL7 (Table 11) was chosen to test experimentally in glycoengineered Leishmania cells.

[00298] StCGP4334 containing a GalNAc synthesis module with Salvage and epimerization enzymes (“S+E”) and P4-GalNAcT3 of Homo sapiens (HsGalNAcT3), integrated into the ssu-PolI genomic locus, was transfected with the different transporter homologs to CeC03H5.2 (FIG. 10A) or with the transporter homologs to UGTREL7 (FIG. 10B) These cell lines were evaluated for their capacity to generate A2GalNAc2 N-glycans on the recombinant Adalimumab (K84N/D86N) expressed and secreted by these cell lines. The parental strain StCGP4334 without any designated UDP-GalNAc transporter already showed 39% A2GalNAc2 N-glycans on the glycosites K84N and D86N of Adalimumab Fab, which indicates that there is some availability of UDP-GalNAc within the secretory pathway, either by being transported by a different (native) Leishmania tarentolae nucleotide- sugar transporter, or due to some other mechanism. However, when a designated UDP-GalNAc transporter was introduced in StCGP4334, such as CeC03H5.2, an increase to 53% A2GalNAc2 was observed. When the homologs to CeC03H5.2 were compared, GnF, GnH and GnJ performed better (57%, 56% and 62%, respectively), while GnG and GnI behaved similar to CeC03H5.2 or slightly less (50% and 49%, respectively). With 62% A2GalNAc2 achieved by GnJ, and thereby leading to almost 10% more A2GalNAc2 glycans compared to CeC03H5.2, an improvement by replacing the UDP-GalNAc transporter was demonstrated. GnJ (A0A4Y9Z8V0 Uncharacterized protein from Dentipellis fragilis) is therefore a suitable UDP-GalNAc transporter for incorporating to Leishmania host cell lines producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2. [00299] Furthermore, UGTREL7 containing cell line showed 54% A2GalNAc2 N- glycans, which is a similar improvement as seen for CeC03H5.2 when compared to 39% of the parental cell line without any designated transporter. When assessing UGTREL7 homologs, the GnM, GnN and GnO generated even higher A2GalNAc2 % with up to 67% for GnN. Only GnL was achieving less (FIG.ll).

[00300] Next, a different strain composition was tested by introducing the UDP-GalNAc transporter or homolog into StCGP4106, with a GalNAc biosynthesis expression module that contains the P4GalNAcT of Parasteatoda tepidariorum (PtGalNAcT) transferase instead of the human GalNAcT3. The combined genetic module containing PtGalNAcT, the UDP- GalNAc biosynthesis pathway and the GalNAc transporter CeC03H5.2 or a UDP-GalNAc transporter homolog, either GnF, GnJ or GnM, ^GalNAc wtA(Transporter +5'+E PtGalNAcT ble ”) was introduced by multiple homologous recombination of 10 fragments into the ssu-PolI locus (FIG. 12). In StCGP5351 expressing the UPD-GalNAc transporter GnF and the P. tepidariorum GalNAc transferase (“PtGalNAcT”), the Adalimumab (K84N/D86N) had 77% A2GalNAc2 as compared to 53% with the CeC03H5.2 UDP- GalNAc transporter in StCGP4978. While GnF, GnJ and GnM expression increased the A2GalNAc2 glycans on Adalimumab (K84N/D86N), GnF was led to the highest increase, suggesting that for the PtGalNAcT expressing strain, transporter GnF might be preferred over CeC03H5.2 in this genetic context (FIG. 13).

[00301] Taken together, this study indicates that (1) incorporating a heterologous UDP- GalNAc transporter protein is beneficial for obtaining high proportions of A2GalNAc2 glycans in glycoengineered Leishmania cells; (2) appropriate transporters can be selected based on sequence homology to known UDP-GalNAc transporters from other organisms; and (3) specific combinations of heterologous GalNAc transferases and heterologous UDP- GalNAc transporter proteins can be advantageous to optimize UDP-GalNAc availability and/or obtain high proportions of A2GalNAc2 glycans in glycoengineered Leishmania cells.

8.7 EXAMPLE 7 Combining different GalNAc Transferases and UDP-GalNAc Transporters

[00302] The results from Example 6 prompted to combine different UDP-GalNAc transporter homologs with different GalNAc transferases to create potent entry cell lines for introducing different polypeptides and therapeutic modalities that contain one or more N- glycosylation consensus sites (including but not limited to Ab scaffolds like mAbs, Fabs, scFvs, VHH, nanobodies, etc.) comprising A2GalNAc2 N-glycans. 8.7.1 Strain StCGP5359

[00303] To engineer a parental strain combining different GalNAc transferases and UPD- GalNAc transporters to generate A2GalNAc2 N-glycans, StCGP5359 was constructed as follows and described in FIG. 14. StCGP3558 contains the GalNAc biosynthesis module with the UDP-GalNAc transporter CeC03H5.2 and the human GalNAcT3 transferase (“HsGalNAcT3”) as described in 6.4.2 and FIG. 6. A second combined module containing both, the UDP-GalNAc biosynthesis pathway and the GalNAc transferase enzyme was introduced by multiple homologous recombination of 10 fragments into the ssu-PolI locus. For StCGP5359 the UPD-GalNAc transporter GnJ (A0A4Y9Z8V0 Uncharacterized protein from Dentipellis fragilis) and the C. elegans GalNAc transferase (“CeGalNAcT”) were used as a second combined genetic module. To evaluate the potential of the parental strain StCGP5359, it was transfected with an expression cassette for a camelid VHH scaffold (“hVHH2-l 1-FHGT4”) to test for A2GalNAc2 levels on a C-terminal glycotag, which is a peptide stretch harboring a N-glycosylation consensus site N-X-S/T, optimized for high site occupancy and tag integrity. High amounts of 88% A2GalNAc2 were detected on purified the VHH protein and proved the high potency of the selected biosynthetic machinery engineered into Leishmania cells for A2GalNAc2 N-glycans. Importantly, when the strain was passaged over time without any selection pressure, the high levels of A2GalNAc2 on the VHH protein were maintained for 37 and 66 generations, respectively, indicating that the multiple genetic insertions are stable and that the phenotype like viability, growth, protein expression yields and engineered N-glycosylation remained unchanged (FIG. 15). StCGP5359 is therefore a suitable host cell line producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2 which mediates protein degradation.

8.7.2 Strain StCGP5942

[00304] StCGP5942 was constructed as follows and is described in FIG. 14. A combined module containing both the UDP-GalNAc biosynthesis pathway with the UPD-GalNAc transporter CeC03H5.2 and a combination of human and P. tepidariorum GalNAc transferases (“HsGalNAcT3” and “PtGalNAcT”, respectively) was introduced in StCGP552 by multiple homologous recombination of 10 fragments into the ssu-PolI locus. Here despite not using any additional UDP-GalNAc transporter homolog, a significant amount of A2GalNAc2 was achieved by the combination of HsGalNAcT3 and PtGalNAcT only, which led to 90% A2GalNAc2 on the VHH protein in StCGP6631. Moreover, StCGP6631 was passaged over time without any selection pressure, and the high levels of A2GalNAc2 on the VHH protein were stable for 35 and 63 generations (FIG. 16). This strongly indicates that the genetic insertions of StCGP6631 are stable and the phenotype like viability, growth, protein expression yield and engineered N-glycosylation is maintained, thereby proving the applicability of parental entry strain StCGP5942 as host cell line for expressing therapeutic protein scaffolds harboring one or more glycosylation sites. These Leishmania host cells are capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2, which mediates protein degradation.

[00305] Table 9 Genetic constructs used for generating StCGP2879, StCGP3169, StCGP3558, StCGP2801, StCGP2803, StCGP2805, StCGP2807, StCGP2847, StCGP5003, StCGP5006, StCGP5007, StCGP5010, StCGP5012, StCGP5013, StCGP5016, StCGP5017, StCGP5049, StCGP5019, StCGP5021, StCGP4978, StCGP5351, StCGP5352, StCGP5694, StCGP5359, StCGP6044, StCGP5942, and StCGP6631. Flowcharts of strain construction are shown in FIGS. 5, 6, 10A, 10B, 12, and 14.

[00306] Table 10: Selected UDP-GalNAc transporter homologs. Query sequences CeC03H5.2 in bold.

[00307] Table 11: Selected UDP-GalNAc transporter homologs. Query sequence UGTREL7 in bold.

[00308] Table 12: A multitude of parental (entry) strains harboring different combinations of GalNAc transferases and different UDP-GalNAc transporters were tested for their capability of expressing A2GalNAc2 N-glycans by transfecting test scaffolds Adalimumab (K84N/D86N) and/or hVHH2-ll-FHGT4. Strains were rated for % A2GalNAc2 by "+++” for >80%, “++” for 60-80%, “+” for 30-60%, and “+/-“ for 1-30% A2GalNAc2. Abbreviations are as follows: cuo = codon usage optimized, FL = full length, tr = N-terminally truncated variant; “GalNAc synth” contains CeC03H5.2, salvage, and epimerization biosynthesis pathway. If another transporter was used instead of CeC03H5.2, it is captured in the description e.g. “GalNAc synth(cuo, GnJ)” harbors GnJ instead of CeC03H5.2.

9. EQUIVALENTS

[00309] The viruses, nucleic acids, methods, host cells, and compositions disclosed herein are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the viruses, nucleic acids, methods, host cells, and compositions in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[00310] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

10. SEQUENCE LISTING

Claims

WHAT IS CLAIMED:

1. A Leishmania host cell comprising: a. a recombinant nucleic acid encoding a polypeptide of interest; and b. a recombinant nucleic acid encoding one or more recombinant N- acetylgalactosamine (GalNAc) transferases, or functionally active variants thereof.

2. The host cell of claim 1, wherein the GalNAc transferase is heterologous to the host cell.

3. The host cell of claim 1 or 2, wherein the GalNAc transferase is P4- GalNAcT3, or a functionally active variant thereof.

4. The host cell of claim 3, wherein the P4-GalNAcT3 is human p4-GalNAcT3, or a functionally active variant thereof.

5. The host cell of claim 1 or 2, wherein the GalNAc transferases are P4- GalNAcT3 and P4-GalNAcT4, or functionally active variants thereof.

6. The host cell of claim 5, wherein the p4-GalNAcT3 and p4-GalNAcT4 are human p4-GalNAcT3 and P4-GalNAcT4, or functionally active variants thereof.

7. The host cell of claim 1 or 2, wherein the host cell further comprises: a. a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc; and b. a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

8. The host cell of claim 1 or 2, wherein the host cell further comprises: a. a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP- GalNAc; and b. a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

9. The host cell of claim 1 or 2, wherein the host cell further comprises: a. a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc; and b. a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP- GalNAc; and c. a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

10. A Leishmania host cell comprising: a. a recombinant nucleic acid encoding a polypeptide of interest; b. a recombinant nucleic acid encoding one or more recombinant N- acetylgalactosamine (GalNAc) transferases, or functionally active variants thereof; and c. a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

11. The host cell of claim 10 further comprises one or more recombinant nucleic acids encoding heterologous UDP-GalNAc biosynthetic pathway proteins capable of generating UDP-GalNAc.

12. The host cell of claim 10 or 11 comprising: a. a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc; and/or b. a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP- GalNAc.

13. The host cell of any one of claims 10-12, wherein the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein are co-localized in the secretory pathway.

14. The host cell of claim 13, wherein the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein each independently comprise: a. a signal peptide localizing the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein in the secretory pathway; and/or b. a retention sequence retaining the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein in the secretory pathway.

15. The host cell of claim 14, wherein the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein each independently comprise the same signal peptide and/or retention sequence.

16. The host cell of claim 14, wherein the one or more GalNAc transferases and the heterologous UDP-GalNAc transporter protein comprise different signal peptides and/or retention sequences.

17. The host cell of any one of claims 14-16, wherein the signal peptide of the one or more GalNAc transferases and/or the signal peptide of the heterologous UDP-GalNAc transporter protein are derived from a. Leishmania species.

18. The host cell of any one of claims 14-17, wherein the retention sequence of the one or more GalNAc transferases and/or the retention sequence of the heterologous UDP- GalNAc transporter protein are derived from a Leishmania species.

19. The host cell of claim 17 or 18, wherein the Leishmania species is Leishmania tarentolae.

20. The host cell of any one of claims 14-19, wherein the signal peptide of the one or more GalNAc transferases and/or the signal peptide of the heterologous UDP-GalNAc transporter protein are processed and removed.

21. The host cell of any one of claims 7-20, wherein recombinant UDP-GalNAc biosynthetic pathway proteins and/or recombinant UDP-GalNAc transporter protein are heterologous to the host cell.

22. The host cell of any one of claims 9-21, wherein one or more of the recombinant nucleic acids are integrated into the [ssuPolI] locus of the host cell.

23. The host cell of any one of claims 1, 2 or 7-22, wherein the one or more GalNAc transferases are derived from a mammalian source.

24. The host cell of claim 23, wherein the mammalian source is Homo sapiens.

25. The host cell of any one of claims 1-24, wherein the one or more GalNAc transferases, or a functionally active variants thereof, are capable of catalyzing the addition of a GalNAc to a N-acetyl glucosamine-terminated glycan.

26. The host cell of any one of claims 1, 2 o 7-25, wherein the one or more GalNAc transferases are selected from the group consisting of p4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or functionally active variants thereof.

27. The host cell of any one of claims 1, 2 o 7-26, wherein the one or more GalNAc transferases is selected from the group consisting of p4-GalNAcT3, p4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto.

28. The host cell of claim 26, wherein the one or more GalNAc transferases comprise P4-GalNAcT3, or an N-terminally truncated variant thereof.

29. The host cell of claim 28, wherein the one or more GalNAc transferases comprise P4-GalNAcT3.

30. The host cell of claim 28, wherein the one or more GalNAc transferases comprise an N-terminally truncated variant of P4-GalNAcT3.

31. The host cell of claim 30, wherein the N-terminally truncated variant comprises an amino acid sequence of SEQ ID NO: 2.

32. The host cell of claim 27, wherein the one or more GalNAc transferases comprise a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to p4-GalNAcT3.

33. The host cell of any one of claims 26-32, wherein the one or more GalNAc transferases comprise P4-GalNAcT4, or an N-terminally truncated variant thereof.

34. The host cell of claim 33, wherein the one or more GalNAc transferases comprise P4-GalNAcT4.

35. The host cell of claim 33, wherein the one or more GalNAc transferases comprise an N-terminally truncated variant of P4-GalNAcT4.

36. The host cell of claim 35, wherein the N-terminally truncated variant is comprises an amino acid sequence of SEQ ID NO: 4.

37. The host cell of claim 27, wherein the one or more GalNAc transferases comprise a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to p4-GalNAcT4.

38. The host of claim 27, wherein the one or more GalNAc transferases comprise CeP4GalNAcT, or a variant that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto.

39. The host of claim 27, wherein the one or more GalNAc transferases comprise CeP4GalNAcT.

40. The host of claim 27, wherein the one or more GalNAc transferases comprise Ptp4GalNAcT, or a variant that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto.

41. The host of claim 27, wherein the one or more GalNAc transferases comprise Ptp4GalNAcT.

42. The host cell of any one of claims 1-41, wherein the recombinant nucleic acid encoding one or more GalNAc transferases comprises a first open reading frame (ORF) encoding a first GalNAc transferase and a second ORF encoding a second GalNAc transferase.

43. The host cell of claim 42, wherein the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in the same genetic module.

44. The host cell of claim 42, wherein the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in separate genetic modules.

45. The host cell of any one of claims 42-44, wherein the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the [ssuPolI] locus of the host cell.

46. The host cell of any one of claims 42-45, wherein the first and the second GalNAc transferases are different GalNAc transferases.

47. The host cell of any one of claims 42-46, wherein the first and the second GalNAc transferases are selected as a combination of GalNAc transferases listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof.

48. The host cell of any one of claims 42-47, wherein the first and the second GalNAc transferases are human p4-GalNAcT3 and human p4-GalNAcT4, respectively; or human p4-GalNAcT3 and CeP4GalNAcT, respectively; or human p4-GalNAcT3 and Ptp4GalNAcT, respectively; or functionally active variants thereof.

49. The host cell of any one of claims 1-48, wherein the host cell comprises a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases.

50. The host cell of claim 49, wherein the additional recombinant glycosyltransferase is heterologous to the host cell.

51. The host cell of claim 49 or 50, wherein the additional recombinant glycosyltransferase comprises one or more N-acetyl glucosamine transferases.

52. The host cell of claim 51, wherein the N-acetyl glucosamine transferase is selected from the group consisting of MGAT1 and MGAT2, or functionally active variants thereof.

53. The host cell of claim 51, herein the additional recombinant glycosyltransferase comprises MGAT1 and MGAT2.

54. The host cell of any one of claims 1-53, wherein the host cell is capable of producing polypepetides comprising a biantennary, GalNAc-terminated N-glycan.

55. The host cell of any one of claims 1-54, wherein the host cell is capable of producing polypeptides comprising an N-glycan of the following structure:

56. The host cell of claim 54 or 55, wherein the host cell attaches the N-glycan to an N-glycosylation site of the polypeptide.

57. The host cell of claim 55, wherein the amino acid residue is Asn.

58. The host cell of claim 56, wherein the N-glycosylation site comprises a consensus sequence of N-X-S/T or N-X-C, wherein X is any amino acid except proline.

59. The host cell of any one of claims 1-58, wherein one or more endogenous enzymes from the glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated.

60. The host cell of claim 59, wherein the host cell does not have endogenous N- glycan elongation.

61. The host cell of any one of claims 1-60, wherein the host cell has been genetically engineered such that the formation of an O-linked GlcNAc on a polypeptide in the host cell is reduced or eliminated.

62. The host cell of claim 61, wherein the formation of O-linked GlcNAc in the Leishmania cell prior to genetic engineering is catalyzed by at least one N-acetylglucosamine (GlcNAc)-transferase.

63. The host cell of claim 62, wherein the gene encoding the at least one GlcNAc- transferase is functionally inactivated, downregulated, deleted, or mutated.

64. The host cell of any one of claims 61-63, wherein the formation of the O- linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of the O-linked GlcNAc in a reference Leishmania cell.

65. The host cell of any one of claims 62-64, the at least one GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2 and OGNTL, and homologous GlcNAc-transferases thereof.

66. The host cell of any one of claims 61-65, wherein the host cell is a OGNT1, OGNT2 and OGNTL triple knockout.

67. The host cell of any one of claims 1-6 or 23-66, wherein the host cell further comprises a recombinant nucleic acid encoding heterologous UDP-GalNAc biosynthetic pathway proteins capable of generating UDP-GalNAc.

68. The host cell of claim 67, wherein the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc to UDP-GalNAc.

69. The host cell of any one of claims 7-68, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc are derived from a mammalian source.

70. The host cell of claim 69, wherein the mammalian source is Homo sapiens.

71. The host cell of any one of claims 7-70, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1), or a functionally active variant thereof.

72. The host cell of any one of claims 7-71, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1), or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.

73. The host cell of any one of claims 7-72, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1).

74. The host cell of any one of claims 7-73, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2), or a functionally active variant thereof.

75. The host cell of any one of claims 7-74, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2), or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.

76. The host cell of any one of claims 7-75, wherein the heterologous UDP-

GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2).

77. The host cell of any one of claims 67-76, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins are capable of converting UDP-GlcNAc to UDP- GalNAc.

78. The host cell of any one of claims 8-77, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins capable of converting UDP-GlcNAc to UDP-GalNAc are derived from a mammalian source.

79. The host cell of claim 78, wherein the mammalian source is Homo sapiens.

80. The host cell of any one of claims 8-79, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise hGalE, or a functionally active variant thereof.

81. The host cell of any one of claims 8-80, wherein the heterologous UDP- GalNAc biosynthetic pathway protein comprise hGalE, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.

82. The host cell of any one of claims 8-81, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins comprise hGalE.

83. The host cell of any one of claims 8-77, wherein the heterologous UDP- GalNAc biosynthetic pathway proteins capable of converting UDP-GlcNAc to UDP-GalNAc are derived from a bacterial source.

84. The host cell of claim 83, wherein the bacterial source is Campylobacter jejuni.

85. The host cell of any one of claims 8-77, 83, or 84, wherein the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne, or a functionally active variant thereof.

86. The host cell of any one of claims 8-77 or 83-85, wherein the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.

87. The host cell of any one of claims 8-77 or 83-86, wherein the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne.

88. The host cell of any one of claims 1-5 or 23-87, wherein the host cell further comprises a recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.

89. The host cell of any one of claims 7-88, wherein the heterologous UDP-

GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source, a mammalian source, a brachiopod source, a chordate source, choanoflagellate source, a gyrista source, a fungi source, a mollusk source, or a placozoan source.

90. The host cell of claim 89, wherein the nematode source is C. elegans,' the mammalian source is Homo sapiens,' the brachiopod source is Lingula unguis,' the chordate source is Parambassis ranga, Geotrypetes seraphini, or Scophthalmus maximus,' the choanoflagellate source is Salpingoeca rosetta, the gyrista source is Fragilariopsis cylindrus,' the fungi source is Dentipellis fragilis,' the mollusk source is Octopus bimaculoides; and/or the placozoan source is trichoplax sp. H2.

91. The host cell of any one of claims 7-90, wherein the heterologous UDP- GalNAc transporter protein is CeC03H5.2, or a functionally active variant thereof.

92. The host cell of any one of claims 7-91, wherein the heterologous UDP- GalNAc transporter protein is CeC03H5.2, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.

93. The host cell of any one of claims 7-92, wherein the heterologous UDP- GalNAc transporter protein is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to CeC03H5.2.

94. The host cell of any one of claims 7-92, wherein the heterologous UDP- GalNAc transporter protein is CeC03H5.2.

95. The host cell of any one of claims 7-93, wherein the heterologous UDP- GalNAc transporter protein is GnF, GnG, GnH, GnI, or GnJ.

96. The host cell of any one of claims 7-90, wherein the heterologous UDP- GalNAc transporter protein is UGTREL7, or a functionally active variant thereof.

97. The host cell of any one of claims 7-90 or 96, wherein the heterologous UDP- GalNAc transporter protein is UGTREL7, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.

98. The host cell of any one of claims 7-90, 96, or 97, wherein the heterologous UDP-GalNAc transporter protein is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to UGTREL7.

99. The host cell of any one of claims 7-90, 96, or 97, wherein the heterologous UDP-GalNAc transporter protein is UGTREL7.

100. The host cell of any one of claims 7-90, or 96-98, wherein the heterologous UDP-GalNAc transporter protein is GnL, GnM, GnN, or GnO.

101. The host cell of any one of claims 7-100, wherein the recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter comprises a first ORF encoding a first heterologous UDP-GalNAc transporter and a second ORF encoding a second heterologous UDP-GalNAc transporter.

102. The host cell of claim 101, wherein the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module.

103. The host cell of claim 101, wherein the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in separate genetic modules.

104. The host cell of any one of claims 101-103, wherein the first ORF encoding the first heterologous UDP-GalNAc transporter and the second ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the [ssuPolI] locus of the host cell.

105. The host cell of any one of claims 101-104, wherein the first and the second heterologous UDP-GalNAc transporter are selected as a combination of heterologous UDP- GalNAc transporters listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof.

106. The host cell of any one of claims 101-105, wherein the first and the second heterologous UDP-GalNAc transporter proteins are the same UDP-GalNAc transporter protein.

107. The host cell of claim 106, wherein the first and the second heterologous UDP-GalNAc transporter proteins are each CeC03H5.2.

108. The host cell of any one of claims 101-105, wherein the first and the second heterologous UDP-GalNAc transporter proteins are different UDP-GalNAc transporter proteins.

109. The host cell of claim 108, wherein the first and the second heterologous UDP-GalNAc transporter proteins are CeC03H5.2 and GnJ, respectively.

110. The host cell of any one of claims 7-109, wherein: a. the recombinant nucleic acid encoding one or more GalNAc transferases comprises a first ORF encoding a first GalNAc transferase and a second ORF encoding a second GalNAc transferase; and b. the recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter comprises a third ORF encoding a first heterologous UDP-GalNAc transporter and a fourth ORF encoding a second heterologous UDP-GalNAc transporter.

111. The host cell of claim 110, wherein: a. the first open reading frame (ORF) encoding the first GalNAc transferase and and the third ORF encoding the first heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module; and/or b. the second open reading frame (ORF) encoding the second GalNAc transferase and and the fourth ORF encoding the second heterologous UDP- GalNAc transporter are integrated into the host cell in the same genetic module.

112. The host cell of claim 110 or 111, wherein: a. the first ORF encoding the first GalNAc transferase and the second ORF encoding the second GalNAc transferase are integrated into the host cell in separate genetic modules; and/or b. the third ORF encoding the first heterologous UDP-GalNAc transporter and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in separate genetic modules.

113. The host cell of claim 110 or 111, wherein the first ORF encoding the first GalNAc transferase, the second ORF encoding the second GalNAc transferase, the third ORF encoding the first heterologous UDP-GalNAc transporter, and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the host cell in the same genetic module.

114. The host cell of any one of claims 110-113, wherein the first ORF encoding the first GalNAc transferase, the second ORF encoding the second GalNAc transferase, the third ORF encoding the first heterologous UDP-GalNAc transporter, and the fourth ORF encoding the second heterologous UDP-GalNAc transporter are integrated into the [ssuPolI] locus of the host cell.

115. The host cell of any one of claims 110-114, wherein the first and the second

GalNAc transferases are different GalNAc transferases.

116. The host cell of any one of claims 110-115, wherein the first and the second heterologous UDP-GalNAc transporter proteins are the same UDP-GalNAc transporter protein.

117. The host cell of any one of claims 110-115, wherein the first and the second heterologous UDP-GalNAc transporter proteins are different UDP-GalNAc transporter proteins.

118. The host cell of any one of claims 110-117, wherein the first GalNAc transferase, the second GalNAc transferase, the first heterologous UDP-GalNAc transporter protein, and the second heterologous UDP-GalNAc transporter protein are selected as a combination of GalNAc transferases and heterologous UDP-GalNAc transporter proteins listed in Table 12, or variants that are 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%, at least 98%, or at least 99% homologous thereof.

119. The host cell of any one of claims 110-118, wherein the first and the second GalNAc transferases are human p4-GalNAcT3 and CeP4GalNAcT, respectively; and the first and the second heterologous UDP-GalNAc transporter proteins are CeC03H5.2, and GnJ, respectively.

120. The host cell of any one of claims 110-118, wherein the first and the second GalNAc transferases are human p4-GalNAcT3 and Ptp4GalNAcT, respectively; and the first and the second heterologous UDP-GalNAc transporter proteins are each CeC03H5.2.

121. The host cell of any one of claims 1-120, wherein the one or more GalNAc transferases and the polypeptide of interest are co-localized in the secretory pathway.

122. The host cell of any one of claims 49-121, wherein the one or more GalNAc transferases and the additional recombinant glycosyltransferase are co-localized in the secretory pathway.

123. The host cell of any one of claims 1-122, wherein the one or more GalNAc transferases each independently comprise a signal peptide localizing the one or more GalNAc transferases in the secretory pathway.

124. The host cell of any one of claims 1-123, wherein the one or more GalNAc transferases each independently comprise a retention sequence retaining the one or more GalNAc transferases in the secretory pathway.

125. The host cell of any one of claims 49-123, wherein the additional recombinant glycosyltransferase comprises a signal peptide localizing the additional recombinant glycosyltransferase in the secretory pathway.

126. The host cell of any one of claims 49-125, wherein the additional recombinant glycosyltransferase comprises a retention sequence retaining the additional recombinant glycosyltransferase in the secretory pathway.

127. The host cell of any one of claims 123-126, wherein the signal peptide is added to an N-terminally truncated variant of the GalNAc transferase and/or the additional recombinant glycosyltransferase.

128. The host cell of any one of claims 124-127, wherein the retention sequence is added to an N-terminally truncated variant of the GalNAc transferase and/or the additional recombinant glycosyltransferase.

129. The host cell of any one of claims 1-128, wherein the polypeptide of interest comprises a signal peptide localizing the polypeptide of interest to the secretory pathway and/or a retention sequence retaining the polypeptide of interest in the secretory pathway.

130. The host cell of any one of claims 123-129, wherein the signal peptide and/or retention sequence is derived from a Leishmania species.

131. The host cell of claim 130, wherein the signal peptide and/or retention sequence is derived from Leishmania tarentolae.

132. The host cell of claim 130, wherein the signal peptide is an invertase signal peptide from derived from Leishmania tarentolae.

133. The host cell of any one of claim 123-131, wherein the signal peptide is processed and removed.

134. The host cell of any one of claims 1-133, wherein the host cell is a Leishmania tarentolae host cell.

135. The host cell of any one of claims 1-134, wherein culturing the Leishmania host cell produces a composition of the polypeptide of interest, wherein said composition of the polypeptide of interest comprises an N-glycan of the following structure:

136. The host cell of any one of claims 1-135, wherein the host cell is stable to passaging and/or continuous fermentation for 100 or more generations.

137. The host cell of any one of claims 1-136, wherein the host cell is any one of the strains listed in Table 3 or Table 9.