Engineered Leishmania cells

Abstract

The present application relates to methods for recombinantly engineering Leishmania cells involving homologous recombination of DNA fragments. Further provided herein are Leishmania cells recombinantly engineered using the methods provided herein. Also provided herein are methods for preparing polypeptides using the Leishmania cells described herein, and polypeptides produced by the methods provided herein.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 958,088, filed January 7, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application incorporates by reference the entire Sequence Listing in computer-readable format (CRF), which is in ASCII text format. This Sequence Listing text file has the title "14197-011-228_SEQ_LISTING", its creation date is December 21, 2020, and its size is 1,115,773 bytes.

[0003] 1. Introduction The present application relates to methods for recombinantly engineering Leishmania cells involving homologous recombination of DNA fragments. Further provided herein are Leishmania cells recombinantly engineered using the methods provided herein. Also provided herein are methods for preparing polypeptides using the Leishmania cells described herein, and polypeptides produced by the methods provided herein. [Background technology]

[0004] 2. Background technology Leishmania species have unusual genetic characteristics, including a lack of transcriptional regulation at certain levels. Genes are transcribed into polycistronic pre-mRNAs, which are then processed into mature mRNAs by trans-splicing (involving the addition of splice leaders or mini-exons and polyadenylation). Regulation of gene expression does not occur at the transcriptional level, but rather at the levels of RNA stability, translation, and protein turnover (Roberts, Sigrid C. (2011) Bioeng Bugs 2(6), pp. 320-326). These processes are influenced by non-coding DNA regions between genes (intergenic regions (IR)) (Breitling, et al. (2002) Protein Expr. Purif. 25(2), pp. 209-218). For this reason, all protein-coding sequences must be separated by intergenic regions, which may originate from Leishmania tarentolae or related species. This is also true when recombinant DNA and vector plasmids are used.

[0005] Direct assembly of multiple linear DNA fragments via homologous recombination, also known as in vivo assembly or transformation-associated recombination, has been successfully applied to the assembly of DNA constructs ranging in size from several kilobases to entire synthetic microbial genomes. This assembly has even enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. Complex in vivo assembly of multiple DNA fragments has become a routine procedure using S. cerevisiae, contributing to its widespread use as a synthetic biology and biotechnology host (Shao, et al. (2009) Nucleic Acids Research 37(2), e16).

[0006] Homologous recombination occurs efficiently in Leishmania species. This homologous recombination is used, for example, to replace a target gene with a drug resistance marker, which provides a selection mechanism. In the design of a targeting construct, upstream and downstream regions corresponding to the flanking sequences of the target gene are linked to a drug resistance cassette. Previously, the generation of targeting DNA involved time-consuming cloning steps (Roberts, Sigrid C. (2011) Bioeng Bugs 2(6), pp. 320-326). Several methods have been developed for the simultaneous assembly of multiple DNA fragments, which significantly simplifies the assembly of targeting constructs. Examples include those using a PCR fusion-based strategy (Mukherjee, et al. (2009) Mol Microbiol 74(4), pp. 914-927) and a one-step multiple fragment ligation approach (Fulwiler, et al. (2011) Molecular and Biochemical Parasitology 175(2), pp. 209-212). This general strategy was reported to be applicable to the generation of targeting constructs for other parasites and genetically manipulable organisms by simply generating species-specific selectable markers flanked by appropriate SfiI sites (Fulwiler, et al. (2011) Molecular and Biochemical Parasitology 175(2), pp. 209-212). This multiple fragment ligation approach was reported for the generation of deletion strains but not for knock-in / insertion strains.

[0007] To use Leishmania as an expression host for glycosylated therapeutic proteins (International Publication No. WO2019 / 002512A2, which is incorporated herein by reference in its entirety), several recombinant elements can be inserted into the host cell genome and co-expressed simultaneously. For efficient expression, i.e., for processing and splicing of polycistronic pre-mRNA to processed mature mRNA, regulatory DNA sequences flanking the recombinantly expressed gene of interest are required. In the case of multiple gene insertion, the number of genes and regulatory sequences to be inserted is limited, since undesired recombination events can occur if identical sequences are inserted into the same genome. Methods are provided herein to address these concerns. Summary of the Invention

[0008] 3. Overview of the Invention Provided herein are methods for recombinantly manipulating Leishmania cells, Leishmania cells, kits comprising Leishmania cells, methods for preparing polypeptides using Leishmania cells, and polypeptides produced by such methods.

[0009] In one aspect, provided herein is a method for recombinantly engineering Leishmania cells, the method comprising: (a) introducing two or more DNA fragments into Leishmania cells; and (b) incubating the Leishmania cells to allow homologous recombination of the DNA fragments; wherein a first DNA fragment of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region, and the 5' homologous region is homologous to a 3' homologous region of a second DNA fragment of the two or more DNA fragments, or the 3' homologous region of the first DNA fragment is homologous to a 5' homologous region of the second DNA fragment; Nucleotide sequences located outside the homologous region(s) in the first and second DNA fragments are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or have no homology within the respective DNA fragments.

[0010] In certain embodiments, each of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region, wherein the 5' homologous region of each of the two or more DNA fragments is homologous to the 3' homologous region of another of the two or more DNA fragments, or wherein each 3' homologous region of the two or more DNA fragments is homologous to the 5' homologous region of another of the two or more DNA fragments, and wherein nucleotide sequences located outside the homologous regions in each DNA fragment are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or have no homology within each DNA fragment.

[0011] In certain embodiments, the two or more DNA fragments are suitable for integration into the chromosome of a Leishmania cell, optionally after the two or more DNA fragments have recombined with each other. In certain embodiments, the two or more DNA fragments are integrated into the chromosome of a Leishmania cell, optionally after the two or more DNA fragments have recombined with each other. In certain embodiments, the two or more DNA fragments are integrated tandemly into the paraflagellar rod protein (Pfr) locus. In certain embodiments, the two or more DNA fragments are integrated into the start site (Ssu-PolI) of the 18S coding region.

[0012] In certain embodiments, the two or more DNA fragments do not integrate into the chromosome of the Leishmania cell before and / or after recombination with one another.

[0013] In certain embodiments, two or more DNA fragments undergo homologous recombination to generate a circular plasmid.

[0014] In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, 15,000, or 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, 15,000, or 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 nucleotides.In certain embodiments, the length of the nucleotide sequence located outside the regions of homology in all of the two or more DNA fragments is at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, 15,000, or 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 nucleotides.

[0015] In certain embodiments, the DNA fragments undergo homologous recombination to generate a nucleotide sequence having a length of 50 to 100 nucleotides, 100 to 500 nucleotides, 500 to 1000 nucleotides, 1000 to 5000 nucleotides, 5000 to 10000 nucleotides, 10000 to 15000 nucleotides, 15000 to 20000 nucleotides, 20000 to 25000 nucleotides, or 25000 to 30000 nucleotides. nucleotides, 30,000 nucleotides to 35,000 nucleotides, 35,000 nucleotides to 40,000 nucleotides, 40,000 nucleotides to 45,000 nucleotides, 45,000 nucleotides to 50,000 nucleotides, 50,000 nucleotides to 55,000 nucleotides, 55,000 nucleotides to 60,000 nucleotides, 60,000 nucleotides to 65,000 nucleotides, 65,000 nucleotides to 70,000 nucleotides, 70,000 nucleotides to 75,000 nucleotides, or 75,000 nucleotides to 80,000 nucleotides.

[0016] In certain embodiments, the length of the 5' and / or 3' homologous region of the first DNA fragment is at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In certain embodiments, the length of the 5' and / or 3' homologous region of the second DNA fragment is at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides. In certain embodiments, the length of all 5' homologous regions and / or 3' homologous regions of the two or more DNA fragments is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides.

[0017] In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the first DNA fragment is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides. In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the second DNA fragment is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides.In certain embodiments, the length of all 5' homologous regions and / or 3' homologous regions of the two or more DNA fragments is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides.

[0018] In certain embodiments, the 5' homologous region of the first DNA fragment and the 3' homologous region of the second DNA fragment share at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. In certain embodiments, the 3' homologous region of the first DNA fragment and the 5' homologous region of the second DNA fragment share at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity.

[0019] In certain embodiments, the two or more DNA fragments are introduced by transfection. In certain embodiments, the two or more DNA fragments are introduced simultaneously.

[0020] In certain embodiments, the number of DNA fragments is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0021] In certain embodiments, the nucleotide sequences located outside the homologous regions in the two or more DNA fragments are selected from the group consisting of intergenic regions (IRs), untranslated regions (UTRs), and open reading frames (ORFs) encoding polypeptides. In certain embodiments, the IRs, UTRs, and ORFs do not share homologous sequences within themselves and / or with each other.

[0022] In certain embodiments, the nucleotide sequences located outside the homologous region in two or more DNA fragments encode the same polypeptide.In certain embodiments, Leishmania cells have the ability to express two or more copies of the same polypeptide.In certain embodiments, the method increases the expression level of polypeptide.

[0023] In certain embodiments, the DNA fragments undergo homologous recombination to produce a nucleotide sequence that contains at least 50%, 60%, 70%, 80%, 90%, or 100% of the genetic information encoded by the two or more DNA fragments.

[0024] In certain embodiments, the proportion of Leishmania cells that undergo undesired deletions and / or crossing outs over at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the proportion of Leishmania cells that undergo undesired deletions and / or crossing outs over at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0025] In certain embodiments, the Leishmania cell is Leishmania tarentolae.

[0026] In another aspect, provided herein are Leishmania cells recombinantly engineered using the methods provided herein. In certain embodiments, the Leishmania cells are recombinantly engineered using the methods iteratively. In certain embodiments, the Leishmania cells are Leishmania tarentolae.

[0027] In another aspect, provided herein is a kit comprising one or more containers and instructions for use, wherein the one or more containers contain Leishmania cells provided herein.

[0028] In another aspect, provided herein are methods for preparing a polypeptide, the methods comprising: (a) culturing a Leishmania cell provided herein under conditions suitable for polypeptide production; and (b) isolating the polypeptide. In certain embodiments, the method further comprises introducing a nucleotide sequence encoding the polypeptide.

[0029] In yet another aspect, provided herein are polypeptides produced by the methods for preparing polypeptides provided herein.

[0030] 3.1 Definition As used herein, the term "end" refers to the region located at the 5' or 3' end of a DNA fragment, unless otherwise specified.

[0031] As used herein, the term "about," when used in connection with a number, unless otherwise specified, refers to the variability of any number within ±1%, ±5%, or ±10% of the stated number.

[0032] The term "subject," as used herein, refers to animals (e.g., birds, reptiles, and mammals), unless otherwise specified. In another embodiment, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In certain embodiments, the subject is a non-human animal. In some embodiments, the subject is a livestock or pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken). In certain embodiments, the subject is a human. The terms "subject" and "patient" may be used interchangeably herein.

[0033] As used herein, the term "effective amount" in the context of administering a therapeutic (e.g., a composition described herein) to a subject, unless otherwise specified, refers to an amount of the therapeutic that has a prophylactic and / or therapeutic effect(s). In certain embodiments, an "effective amount" refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of a disease / disorder or symptoms associated therewith, (ii) reducing the duration of a disease / disorder or symptoms associated therewith, (iii) preventing the progression of a disease / disorder or symptoms associated therewith, (iv) inducing regression of a disease / disorder or symptoms associated therewith, (v) preventing the onset or occurrence of a disease / disorder or symptoms associated therewith, (vi) preventing the recurrence of a disease / disorder or symptoms associated therewith, (vii) reducing organ failure associated with a disease / disorder, (viii) reducing the number of hospitalizations of a subject with a disease / disorder, (ix) reducing the length of hospitalization of a subject with a disease / disorder, (x) increasing the survival time of a subject with a disease / disorder, (xi) eliminating the disease / disorder in a subject, and / or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another treatment. 3.2 Common Terms and Abbreviations [Table 1] TIFF0007720307000002.tif45165 [Brief explanation of the drawings]

[0034] 4. Brief description of the drawings [Figure 1A]Leishmania tarentolae can assemble chromosomal integration constructs from multiple DNA fragments by homologous recombination. A schematic diagram of a full-length monoclonal antibody (mAb) expression construct is shown. The rituximab integration construct (top) contains homologous recombination sites (Lhr[ssu] and Rhr[ssu]) for integration into the ssu locus, four intergenic regions (IR1 = aprtIR; IR2 = aTubIR from L. enrietti; IR3 = CamIR from L. tarentolae; IR4 = dhfr-ts) that ensure correct mRNA transcription and splicing, and open reading frames (ORFs) for the rituximab light chain (ORF1), the rituximab heavy chain (ORF2), and the selectable marker NTC (ORF3). The complete construct is present on the plasmid pLMTB5026 or as separate fragments on the donor plasmids pLMTB5024 and pLMTB5025. The overlap regions for homologous recombination into the genome (>500 bp; bottom of the figure) and between the fragments (250 bp) are shown as gray bars. [Figure 1B] Leishmania tarentolae can assemble chromosomally integrated constructs from multiple DNA fragments by homologous recombination. Western blot analysis of cell culture supernatants from strains obtained by cotransfection of two DNA fragments that recombine in vivo to form an expression construct for a monoclonal antibody (rituximab). Positive control = rituximab (anti-CD20, Lubio: A1049-100). Expression of full-length monoclonal antibodies can be directly detected in cell culture supernatants using antibodies specific for the light or heavy chain.

[0035] [Figure 2]Multiple homologous recombination events of heterologous coding sequences result in functionally engineered Leishmania host cells. Schematic representation of the integration construct (top). This integration construct contains homologous recombination sites for integration into the AQP locus (Lhr[AQP] and Rhr[AQP]), regulatory elements (PolA; promoter) and intergenic regions (aTubIR from L. enrietti; Pfr IR = IR for paraflagellar rod protein from L. tarentolae; Val IR (IR from valosin from L. tarentolae); Cam IR from L. tarentolae) that ensure correct mRNA transcription and splicing. The construct contains coding sequences for heterologous glycosyltransferases (sfGntI, rnMGAT2, drMGAT1, hsB4GalT1 (see, e.g., International Publication No. WO 2019 / 002512 A2, which is incorporated herein by reference in its entirety) and a hygromycin selectable marker (Sm[hyg]). The complete construct is divided into 10 fragments, which were excised from 10 donor plasmids. The overlap regions (500 bp) for homologous recombination into the genome are located in black boxes at the ends, and the homologous recombination regions (200 bp) between the fragments are shown as gray bars. The graph at the bottom shows the functional readout of glycosylation efficiency (expressed as the relative % of N-glycans), which were released from cellular proteins and measured by routine N-glycan analysis (e.g., RF-MS or PC labeling).

[0036] [Figure 3A]Multiple homologous events of heterologous coding sequences interspersed with Leishmania tarentolae regulatory elements and intergenic regions (IRs). (A) Functional readouts of glycosylation efficiency are shown as the relative percentage of N-glycans released from cellular proteins and measured by routine RF-MS. These readouts demonstrate the presence of activity at all enzymatic steps in St15368. The absence of activity beyond the second glycosylation enzymatic step by MGAT2 in St15448 suggests phenotypic differences based on desired and undesired integration events. (B) Schematic diagram of the integrants (top). The integration contains homologous recombination sites (dark gray boxes (Lhr and Rhr)) for reverse disruption of aquaporins (AQPs), the regulatory element PolA and intergenic regions (striped boxes) that ensure correct mRNA transcription and splicing (aTubIR from L. enrietti; Pfr IR = IR for the paraflagellar rod protein from L. tarentolae; Val IR (IR from valosin from L. tarentolae); 60S ribosomal protein L23 from L. tarentolae), and the 3'UTR located downstream of the selectable marker gene (SmA). rnMGAT2 (GtD), hsB4GalT1 (GtE), sfGntI (GtA), and drMGAT1 (GtB) are coding sequences for heterologous glycosyltransferases, and SmA is the coding sequence for the hygromycin selectable marker. The inserted genetic element region is indicated by shading on a gray dotted background. An example of correct integration is shown for St15368 (top). The region marked with a black background and white dots indicates an undesired crossover of the Pfr IR with the identical Pfr IR sequence in chromosome 29 in St15448, resulting in the elimination of the recombinant genetic elements (rnMGAT2(GtD) and hsB4GalT1(GtE)) (bottom) and (C) the generation of a hybrid chromosome (identified by PacBio sequencing (PacBio raw subreads m54073_181001_130829 / 9307006 / 0_32110)). [Figure 3B] Multiple homologous events of heterologous coding sequences interspersed with Leishmania tarentolae regulatory elements and intergenic regions (IRs). (A) Functional readouts of glycosylation efficiency are shown as the relative percentage of N-glycans released from cellular proteins and measured by routine RF-MS. These readouts demonstrate the presence of activity at all enzymatic steps in St15368. The absence of activity beyond the second glycosylation enzymatic step by MGAT2 in St15448 suggests phenotypic differences based on desired and undesired integration events. (B) Schematic diagram of the integrants (top). The integration contains homologous recombination sites (dark gray boxes (Lhr and Rhr)) for reverse disruption of aquaporins (AQPs), the regulatory element PolA and intergenic regions (striped boxes) that ensure correct mRNA transcription and splicing (aTubIR from L. enrietti; Pfr IR = IR for the paraflagellar rod protein from L. tarentolae; Val IR (IR from valosin from L. tarentolae); 60S ribosomal protein L23 from L. tarentolae), and the 3'UTR located downstream of the selectable marker gene (SmA). rnMGAT2 (GtD), hsB4GalT1 (GtE), sfGntI (GtA), and drMGAT1 (GtB) are coding sequences for heterologous glycosyltransferases, and SmA is the coding sequence for the hygromycin selectable marker. The inserted genetic element region is indicated by shading on a gray dotted background. An example of correct integration is shown for St15368 (top). The region marked with a black background and white dots indicates an undesired crossover of the Pfr IR with the identical Pfr IR sequence in chromosome 29 in St15448, resulting in the elimination of the recombinant genetic elements (rnMGAT2(GtD) and hsB4GalT1(GtE)) (bottom) and (C) the generation of a hybrid chromosome (identified by PacBio sequencing (PacBio raw subreads m54073_181001_130829 / 9307006 / 0_32110)). [Figure 3C]Multiple homologous events of heterologous coding sequences interspersed with Leishmania tarentolae regulatory elements and intergenic regions (IRs). (A) Functional readouts of glycosylation efficiency are shown as the relative percentage of N-glycans released from cellular proteins and measured by routine RF-MS. These readouts demonstrate the presence of activity at all enzymatic steps in St15368. The absence of activity beyond the second glycosylation enzymatic step by MGAT2 in St15448 suggests phenotypic differences based on desired and undesired integration events. (B) Schematic diagram of the integrants (top). The integration contains homologous recombination sites (dark gray boxes (Lhr and Rhr)) for reverse disruption of aquaporins (AQPs), the regulatory element PolA and intergenic regions (striped boxes) that ensure correct mRNA transcription and splicing (aTubIR from L. enrietti; Pfr IR = IR for the paraflagellar rod protein from L. tarentolae; Val IR (IR from valosin from L. tarentolae); 60S ribosomal protein L23 from L. tarentolae), and the 3'UTR located downstream of the selectable marker gene (SmA). rnMGAT2 (GtD), hsB4GalT1 (GtE), sfGntI (GtA), and drMGAT1 (GtB) are coding sequences for heterologous glycosyltransferases, and SmA is the coding sequence for the hygromycin selectable marker. The inserted genetic element region is indicated by shading on a gray dotted background. An example of correct integration is shown for St15368 (top). The region marked with a black background and white dots indicates an undesired crossover of the Pfr IR with the identical Pfr IR sequence in chromosome 29 in St15448, resulting in the elimination of the recombinant genetic elements (rnMGAT2(GtD) and hsB4GalT1(GtE)) (bottom) and (C) the generation of a hybrid chromosome (identified by PacBio sequencing (PacBio raw subreads m54073_181001_130829 / 9307006 / 0_32110)).

[0037] [Figure 4] Figure 1 shows a schematic diagram of the intended integrant. This integrant contains 500-bp homologous recombination sites (dark gray boxes) for the reverse disruption of Ptr1 in chromosome 23 (Lhr and Rhr), the regulatory element PolI ("PolA") and intergenic region (IR (striped box)) that ensure correct mRNA transcription and splicing, and the 3' UTR downstream of the selectable marker gene (SmA). hsB4GalT1 (ORF1), hsMGAT1 (ORF2), and rnMGAT2 (ORF3) are coding sequences for heterologous glycosyltransferases, and SM is the coding sequence for the hygromycin selectable marker. The complete expression cassette is divided into eight fragments, which were excised from their donor plasmids. The overlap regions for homologous recombination into the genome (500 bp located at the termini) and between fragments (200 bp) are shown as gray boxes. The dark gray shaded area with white dots within black brackets indicates the identical 93-bp stretch derived from the 3xHA tag located at the C-terminus of the hsMGAT1 ORF in donor fragment GtC_5IrLmM(8081) and the identical sequence in IrLmO_5GtD(8085) derived from the 3xHA tag also present at the C-terminus of the rnMGAT2 ORF. Homologous recombination of these fragments results in the undesired deletion and loss of the IR2 and rnMGAT2 genetic information. The phenotype is expressed by the glycosylation-modifying activity of GT; the absence of G0 and G2 glycans indicates the absence of MGAT2 activity (shown as the relative % N-glycan graph (top left)).

[0038] [Figure 5]Schematic diagram of different chromosomal integration strategies. Light gray arrows indicate chromosomal coding sequences, and dark gray arrows indicate heterologous coding sequences, which are inserted via homologous ends located at their ends (shown in gray shading). The regulatory element Pol I is the promoter region for Pol I transcription and is used for transcription initiation in the reverse integration construct.

[0039] [Figure 6A] Non-identical heterologous sequences ensure correct chromosomal integration and internal fragment recombination, and the selected heterologous control sequences are functional in Leishmania tarentolae custom glycan host cells. GT glycosylation-modifying activity was assessed as the relative % of N-glycans derived from total surface glycoproteins and compared between different strains with different IRs but identical GT and SM coding sequences. [Figure 6B] Non-identical heterologous sequences ensure correct chromosomal integration and internal fragment recombination, and the selected heterologous control sequences are functional in Leishmania tarentolae custom glycan host cells. Schematic representation of Nanopore sequencing analysis results for different integrants and resulting strains (St17212 = LmIR, St17311 = LdIR, St17176 = LiIR, St17180 = LmxIR). Each integrant contains homologous recombination sites (LhrD[GP63] and RhrD[GP63]) for integration into the GP63 locus on chromosome 10. Intergenic regions (IRs) from different Leishmania species (L. major (Lm), L. donovani (Ld), L. infantum (Li), and L. mexicana (Lmx)) that ensure correct mRNA transcription and splicing are shown as striped boxes, along with a text description for each IR. The coding sequences of heterologous glycosyltransferases (GTs) (sfGntI, rnMGAT2, drMGAT1, hsB4GalT1) are shown as white arrows, and the coding sequence of the hygromycin selection marker is shown in gray; these coding sequences are identical in all four strains.

[0040] [Figure 7] Transfection of several genetic modules resulted in a stepwise increase in N-glycan conversion efficiency, as shown by the relative percentage of N-glycans obtained from surface glycoproteins in three successively derived strains: St17238 (first), St17294 (second), and St17826 (third). Increased glycosyltransferase copy numbers were achieved by using codon-diversifying enzymes and homologs from different species (hs, Homo sapiens; rn, Rattus norvegicus; dr, Danio rerio; gj, Gekko japonicus; ag, Anopheles gambiae).

[0041] [Figure 8A]Generation of the cell line St17527, capable of N-glycan sialylation, by integrating 13 fragments into the parent cell line (St17311) containing glycosyltransferases via multiple homologous recombination. Schematic diagram of the genome modification of St17527. The top shows the genomic integrated expression cassette present at the gp63 locus (chromosome 10) from the parent cell line St17311. The new integrant contains homologous recombination sites (Lhr[aTub] and Rhr[aTub]) for the alpha-tubulin locus on chromosome 13, intergenic regions (Ir) for mRNA transcription and splicing (Ir from L. infantum (IrLi) and Ir from L. major (IrLm)) (shown as striped boxes). Coding sequences for sialic acid (Neu5Ac) biosynthesis, Golgi import, and transfer to N-glycans (e.g., NeuC3×Myc: UDP-N-acetylglucosamine 2-epimerase, CgNal: N-acetylneuraminic acid lyase supporting N-acetylneuraminic acid synthesis, NeuB3×HA:CMP-sialic acid synthase, 3×HAST6: beta-galactoside alpha-2,6-sialyltransferase 1, NeuA3×HA:CMP-sialic acid synthase, and CST3×mycCMP-Neu5Ac transporter) are shown as white arrows, and the coding sequence for the selectable marker (pac) is shown in gray. [Figure 8B] Generation of the N-glycan sialylation-competent cell line St17527 by multiple homologous recombination of 13 fragments into the glycosyltransferase-containing parent cell line (St17311). HPLC tracing of DMB-labeled total sialic acid (Neu5Ac + CMP-Neu5Ac) and CMP-Neu5Ac extracted from cell pellets of St17527 demonstrates the presence of Neu5Ac and the activated sugar CMP-Neu5Ac, thus demonstrating functional sialic acid precursor biosynthesis. [Figure 8C]Generation of cell line St17527 with N-glycan sialylation capabilities by multiple homologous recombination of 13 fragments into the parent cell line (St17311) containing glycosyltransferases. GT glycosylation modification activity is expressed as a relative % of N-glycans derived from total surface glycoproteins. Total galactosylation and total sialylation rates are also shown, demonstrating customized L. tarentolae host cell function.

[0042] [Figure 9A] Chromosomal integration of the same glycosylation modification construct into different chromosomal loci to increase the glycosylation modification activity level of the expressed glycosyltransferase. Schematic diagram of a chromosomal integration strategy targeting the Pfr locus on chromosome 29 and a chromosomal integration strategy targeting the rDNA expression locus on chromosome 27. Light gray arrows indicate chromosomal coding sequences, and dark gray arrows indicate heterologous coding sequences, which are inserted via homologous ends located at their ends (indicated by gray shading). Because the chromosomal region shown is a multicopy locus, integration can occur at several different locations, as indicated by differently shaded gray bars. Regulatory elements ensuring correct pre-mRNA processing are indicated by boxes adjacent to the 5' end of the first integrated heterologous coding sequence (in the case of integration into the rDNA loci (Ssu and Ssu-PolI)) and the 3' end of the last heterologous coding sequence in the integration construct; these boxes are marked with different stripes. [Figure 9B] Chromosomal integration of the same glycosylation-engineering construct into different chromosomal loci to increase the glycosylation-engineering activity level of the expressed glycosyltransferase. Comparison of glycosylation-engineering activity of GTs encoded by the same G0 integration construct targeted to different chromosomal integration loci ("Pfr," "Ssu," or "Ssu-PolI"). The relative percentage of N-glycans derived from total surface glycoproteins for each strain is shown. [Figure 9C] Chromosomal integration of the same glycosylation-modifying construct into different chromosomal loci to increase the glycosylation-modifying activity level of the expressed glycosyltransferase. The glycosylation-modifying activity of GT encoded by the same G0 integration construct targeting different variants of the rDNA chromosomal integration locus ("Ssu" vs. "Ssu-PolI") is compared. The relative percentage of N-glycans released from the N-glycosylation site of the Fc of a co-expressed monoclonal antibody (adalimumab) is shown.

[0043] [Figure 10A]Multiple homologous recombination events of heterologous coding sequences result in functionally engineered Leishmania host cells. Schematic representation of the integration construct (top), which contains homologous recombination sites (LhrP and RhrP) for integration into the "Pfr" locus, intergenic regions (15 different IRs: IR from L. major (Lm), L. donovani (Ld), L. infantum (Li), and L. tarentolae (Lt)) that ensure correct mRNA transcription and splicing, and UtrA = dhfr-ts, shown as striped boxes. ), as well as coding sequences for heterologous glycosyltransferases (different orthologs of MGAT1, MGAT2, and hsB4GalT1 are shown as white boxes; see, e.g., International Publication No. WO2019 / 002512A2, which is incorporated herein by reference in its entirety), coding sequences for enzymes of the sialic acid biosynthesis and transport pathway (shown as dark gray boxes), and coding sequences for a selectable marker for puromycin resistance (SmD, shown as a light gray box). The complete construct is divided into 25 fragments, which were excised from 25 donor plasmids. The overlapping regions (500 bp) for homologous recombination into the genome are located within black boxes at the ends, and the homologous recombination regions (≥200 bp) between the fragments are shown as gray bars. The bottom graph shows the functional readout of glycosylation efficiency (shown as relative % of N-glycans), which were released from cellular proteins and measured by routine N-glycan analysis (PC labeling). [Figure 10B]Multiple homologous recombination events of heterologous coding sequences result in functionally engineered Leishmania host cells. Transfection of several genetic modules increases the efficiency of N-glycan conversion. This is shown as the N-glycans (shown as relative %) obtained from total surface glycoproteins of three successively derived strains: St18700 (first), St19084 (second), and St19384 (third). Increasing the copy number of glycosyltransferases was achieved by using codon-diversifying enzymes and homologs from different species. [Figure 10C] Multiple homologous recombination events of heterologous coding sequences result in functionally engineered Leishmania host cells. Alternate strains with different genetic compositions are capable of nearly uniformly converting N-glycans to G2S2. This figure shows the N-glycans (shown as relative %) obtained from total surface glycoproteins of three alternate strains (St20157, St20208, and St20224).

[0044] [Figure 11A] Assembly of a hybrid prokaryotic gene cluster in Leishmania tarentolae with an Escherichia coli cosmid. Schematic showing the designed fragments and predicted recombination through the 200 bp homologous region, shaded or striped in gray. [Figure 11B]Assembly of a hybrid prokaryotic gene cluster in an Escherichia coli cosmid for Leishmania tarentolae. Western blot analysis of E. coli DH5a transformed with plasmids isolated from several polyclones for expression of S. pneumoniae serotype 1 polysaccharide. Lane 1: PageRuler™ prestained protein ladder (10-180 kDa (ThermoFischer scientific)), Lane 2: polyclone 1.2, Lane 3: polyclone 1.3, Lane 4: polyclone 1.4, Lane 5: polyclone 1.5, Lane 6: polyclone 1.6, Lane 7: polyclone 1.7, Lane 8: polyclone 1.8, Lane 9: polyclone 2.3. [Figure 11C] Assembly of a hybrid prokaryotic gene cluster in Leishmania tarentolae with an Escherichia coli cosmid. Restriction enzyme digestion of plasmids isolated from different polyclones. A: Restriction enzyme digestion of polyclones obtained from transfections #1 and #2. Upper panel: BstBI restriction enzyme digestion (expected sizes of correct constructs: 22,210 bp, 9,042 bp, 3,787 bp; expected size of empty vector: 20,801 bp). Lower panel: BsiWI restriction enzyme digestion (expected sizes of correct constructs: 32,536 bp, 2,503 bp; expected size of empty vector without digestion: 20,801 bp). Lane 1: GeneRuler™ 1 kb DNA ladder (Thermo-Fischer scientific), Lane 2: polyclone 1.1, Lane 3: polyclone 1.7, Lane 4: polyclone 2.1, Lane 5: polyclone 2.2, Lane 6: pGVXN775. [Figure 11D]Assembly of a hybrid prokaryotic gene cluster in Leishmania tarentolae with an Escherichia coli cosmid. Restriction enzyme digestion of multiple clones obtained from transfection #3. SacI restriction enzyme digest (expected sizes of correct constructs: 19628 bp, 3723 bp; expected size of empty vector: 20801 bp). Lane 1: multiple clone 3.1, lane 2: multiple clone 3.2, lane 3: pLMTB6412, lane 4: GeneRuler™ 1 kb DNA ladder (Thermo-Fischer scientific). DETAILED DESCRIPTION OF THE INVENTION

[0045] 5. Detailed Description of the Invention Provided herein are methods for recombinantly engineering Leishmania cells, Leishmania cells engineered using the methods provided herein, methods for preparing target polypeptides using the Leishmania cells provided herein, and target polypeptides produced by the methods. Methods for recombinantly engineering Leishmania cells are described in Section 5.1. Properties of the resulting Leishmania cells are described in Section 5.2. Use of such Leishmania cells as expression systems for target polypeptides (e.g., therapeutic proteins) is described in Section 5.3. Properties of target polypeptides expressed in the Leishmania host cells provided herein are described in Section 5.4.

[0046] Provided herein are: i) rapid, multi-fragment homologous recombination to create large artificial chromosome inserts of at least about 20 kb in Leishmania tarentolae host cells; ii) specific strategies to avoid undesired deletion of inserted genetic elements during recombination; and iii) the use of multiple homologous recombination to assemble circular DNA that is applicable for use with any heterologous shuttle vector. Also provided herein are methods for increasing polypeptide expression by inserting multiple copies of an expression gene into the same host cell.

[0047] When the inserted DNA sequences are homologous / identical, the use of multiple homologous recombination events for host cell engineering presents problems: even short stretches of less than 100 bp can result in unwanted crossovers due to the very high natural recombination efficiency in Leishmania tarentolae.

[0048] Without being bound by theory, methods provided herein reduce or eliminate such undesired crossovers. Specifically, described herein are: i) multiple regulatory DNA sequences (obtained from related species, but not from Leishmania tarentolae) that are sufficiently different from each other and from any chromosomal sequence to avoid undesired crossovers while allowing for the processing and splicing of polycistronic pre-mRNA to form processed mature mRNA for protein expression; and ii) strategies for diversifying the coding sequences of genes intended to be inserted in multiple copies in such a way that they themselves do not recombine and are still efficiently expressed.

[0049] The multi-fragment ligation strategy described herein for creating engineered host cells is significantly faster than traditional approaches and, furthermore, allows for the simultaneous integration of multiple ORFs with only one associated selectable marker, thereby expanding the previously limited capabilities of genetic engineering. Furthermore, by selecting different insertion elements (intergenic regulatory sequences or codon-diversifying genes of interest), it is possible to express glycosylation-modified therapeutics and increase yields by increasing gene copy number. This application describes fully functional customized host cells, which are created by the described genetic methods.

[0050] Furthermore, because the natural homologous recombination system of L. tarentolae is highly efficient, it can be used to assemble multiple heterologous DNA fragments into circular DNA in the absence of sites homologous to the L. tarentolae chromosome. L. tarentolae can propagate episomal (plasmid) DNA without an origin of replication. This method involves cotransfecting a donor plasmid and a series of DNA fragments (which share homology between their ends and between their ends and the recipient vector). A selectable marker for L. tarentolae is added, which is also split into two fragments to select positive transfectants of L. tarentolae host cells. Nucleic acid can be extracted from PCR-positive L. tarentolae cells, and the extracted material is transformed / transfected into a target microorganism that propagates with the desired selectable marker present in the recipient vector. This technique allows the use of any unmodified recipient circular DNA and does not require the availability of restriction enzyme recognition sites.

[0051] In summary, the efficient occurrence of multiple homologous recombination events in Leishmania allows the introduction of 2–20, and potentially even more than 20, DNA fragments (sharing homology between their ends) to create host cells with site-specifically integrated genetic information, including regulatory elements and flanking coding sequences. The regulatory elements were identified from related species and remain functional when introduced into Leishmania tarentolae. The final genetic information contains 20 kb / span stretches site-specifically inserted into the chromosome of Leishmania host cells, although insert sizes can be increased. This application describes a novel tool for efficiently generating functionally engineered Leishmania host cells using "plug-and-play" modules. Furthermore, these efficient site-specific homologous recombination events allow the assembly of multiple DNA fragments into episomal constructs suitable as shuttle vectors for different, unrelated host organisms, potentially providing a useful cloning tool for complex shuttle vectors.

[0052] 5.1 Methods for Recombinant Engineering of Leishmania Cells In one aspect, there is provided herein a method for recombinantly engineering a Leishmania cell, the method comprising: (a) introducing two or more DNA fragments into a Leishmania cell; and (b) incubating the Leishmania cell to allow homologous recombination of the DNA fragments, wherein a first DNA fragment of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region, and the 5' homologous region is homologous to a 3' homologous region of a second DNA fragment of the two or more DNA fragments, or the 3' homologous region of the first DNA fragment is homologous to the 5' homologous region of the second DNA fragment, and wherein nucleotide sequences located outside the homologous region(s) in the first DNA fragment and in the second DNA fragment are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or have no homology within the respective DNA fragments.

[0053] 5.1.1 DNA fragments In certain embodiments, the DNA fragments described herein comprise 5' or 3' homologous regions that are homologous to the 5' or 3' homologous regions of another DNA fragment. In certain embodiments, the DNA fragments described herein comprise 5' homologous regions that are homologous to the 3' homologous regions of another DNA fragment. In certain embodiments, the DNA fragments described herein comprise 3' homologous regions that are homologous to the 5' homologous regions of another DNA fragment. In certain embodiments, the DNA fragments described herein comprise a 5' homologous region that is homologous to the 3' homologous region of another DNA fragment and a 3' homologous region that is homologous to the 5' homologous region of a third DNA fragment. In certain embodiments, the nucleotide sequences located outside the homologous regions in the DNA fragments described herein are not homologous to each other. In certain embodiments, the nucleotide sequences located outside the homologous regions in the DNA fragments described herein are not homologous to sequences in the genome of a Leishmania cell. In certain embodiments, the nucleotide sequences located outside the homologous regions in the DNA fragments described herein do not share homology within the respective DNA fragments.

[0054] In certain embodiments, a first DNA fragment of the two or more DNA fragments comprises a 5' region of homology and / or a 3' region of homology. In certain embodiments, the 5' region of homology of the first DNA fragment is homologous to the 3' region of homology of a second DNA fragment of the two or more DNA fragments. In certain embodiments, the 3' region of homology of the first DNA fragment is homologous to the 5' region of homology of a second DNA fragment of the two or more DNA fragments. In certain embodiments, nucleotide sequences located outside the region(s) of homology in the first DNA fragment and the second DNA fragment are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or have no homology within the respective DNA fragments.

[0055] In certain embodiments, each of the two or more DNA fragments comprises a 5' region of homology and / or a 3' region of homology. In certain embodiments, the 5' region of homology of each of the two or more DNA fragments is homologous to the 3' region of homology of another of the two or more DNA fragments. In certain embodiments, the 3' region of homology of each of the two or more DNA fragments is homologous to the 5' region of homology of another of the two or more DNA fragments. In certain embodiments, nucleotide sequences located outside the regions of homology in each DNA fragment are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or share no homology within each DNA fragment.

[0056] In certain embodiments, the DNA fragments described herein comprise a 5' homologous region or a 3' homologous region that is homologous to a region in a chromosome of a Leishmania cell. In certain embodiments, such homologous regions allow the DNA fragment to be integrated into a chromosome of a Leishmania cell. In certain embodiments, the DNA fragments comprise a 5' homologous region that is homologous to a 3' homologous region of another DNA fragment, a region located outside the homologous region, and a 3' homologous region that is homologous to a 5' homologous region of another DNA fragment.

[0057] In certain embodiments, the two or more DNA fragments are suitable for integration into the chromosome of the Leishmania cell, optionally after the two or more DNA fragments have recombined with each other. In certain embodiments, the two or more DNA fragments are integrated into the chromosome of the Leishmania cell, optionally after the two or more DNA fragments have recombined with each other. In certain embodiments, the two or more DNA fragments are integrated tandemly into the accessory flagellar rod protein (Pfr) locus. In certain embodiments, the two or more DNA fragments are integrated into the start site (Ssu-PolI) of the 18S coding region. In certain embodiments, the two or more DNA fragments are not integrated into the chromosome of the Leishmania cell before and / or after recombination with each other.

[0058] 5.1.2 Homologous regions In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region can be 10 to 2000 nucleotides, In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region can be at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, or 800 nucleotides. , 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides.In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region is 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 800 nucleotides, 90 ...0 nucleotides, 1500 nucleotides, 2000 nucleotides, 250 nucleotides, 3000 nucleotides, 3500 nucleotides, 4000 nucleotides, 450 nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, The length of the 5' homology region and / or the 3' homology region may be 50 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides. In certain embodiments, the length of the 5' homology region and / or the 3' homology region may be greater than 200 nucleotides, 250 nucleotides, or 500 nucleotides. In certain embodiments, the 5' homology region and / or the 3' homology region may have any of the lengths described in the Examples section.

[0059] In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the first DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides. In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the first DNA fragment is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides.In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the first DNA fragment is between 10 nucleotides and 50 nucleotides, between 50 nucleotides and 100 nucleotides, between 100 nucleotides and 150 nucleotides, between 150 nucleotides and 200 nucleotides, between 200 nucleotides and 250 nucleotides, between 250 nucleotides and 300 nucleotides, between 300 nucleotides and 350 nucleotides, between 350 nucleotides and 400 nucleotides, between 400 nucleotides and 450 nucleotides, between 450 nucleotides and 500 nucleotides, between 500 nucleotides and 550 nucleotides, between 550 nucleotides and 600 nucleotides, between 600 nucleotides and 650 nucleotides, between 650 nucleotides and 700 nucleotides, between 700 nucleotides and 750 nucleotides, between 750 nucleotides and 800 nucleotides, between 800 nucleotides and 850 nucleotides, between 850 nucleotides and 900 nucleotides, between 900 nucleotides and 950 nucleotides, between 950 nucleotides and 1000 nucleotides, nucleotides to 1200 nucleotides, 1200 nucleotides to 1400 nucleotides, 1400 nucleotides to 1600 nucleotides, 1600 nucleotides to 1800 nucleotides, 1800 nucleotides to 2000 nucleotides, 2000 nucleotides to 2200 nucleotides, 2200 nucleotides to 2400 nucleotides, 2400 nucleotides to 2600 nucleotides, 2600 nucleotides to 2800 nucleotides, 2800 nucleotides to 3000 nucleotides, 3000 nucleotides to 3200 nucleotides, 3200 nucleotides to 3400 nucleotides, 3400 nucleotides to 3600 nucleotides, 3600 nucleotides to 3800 nucleotides, 3800 nucleotides to 4000 nucleotides, 4000 nucleotides to 4200 nucleotides, 4200 nucleotides to 4400 nucleotides, 4400 nucleotides to 4600 nucleotides, 4600 nucleotides to 4800 nucleotides, or 4800 nucleotides to 5000 nucleotides.

[0060] In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the second DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides. In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the second DNA fragment is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides.In certain embodiments, the length of the 5' homologous region and / or the 3' homologous region of the second DNA fragment is between 10 nucleotides and 50 nucleotides, between 50 nucleotides and 100 nucleotides, between 100 nucleotides and 150 nucleotides, between 150 nucleotides and 200 nucleotides, between 200 nucleotides and 250 nucleotides, between 250 nucleotides and 300 nucleotides, between 300 nucleotides and 350 nucleotides, between 350 nucleotides and 400 nucleotides, between 400 nucleotides and 450 nucleotides, between 450 nucleotides and 500 nucleotides, between 500 nucleotides and 550 nucleotides, between 550 nucleotides and 600 nucleotides, between 600 nucleotides and 650 nucleotides, between 650 nucleotides and 700 nucleotides, between 700 nucleotides and 750 nucleotides, between 750 nucleotides and 800 nucleotides, between 800 nucleotides and 850 nucleotides, between 850 nucleotides and 900 nucleotides, between 900 nucleotides and 950 nucleotides, between 950 nucleotides and 1000 nucleotides, nucleotides to 1200 nucleotides, 1200 nucleotides to 1400 nucleotides, 1400 nucleotides to 1600 nucleotides, 1600 nucleotides to 1800 nucleotides, 1800 nucleotides to 2000 nucleotides, 2000 nucleotides to 2200 nucleotides, 2200 nucleotides to 2400 nucleotides, 2400 nucleotides to 2600 nucleotides, 2600 nucleotides to 2800 nucleotides, 2800 nucleotides to 3000 nucleotides, 3000 nucleotides to 3200 nucleotides, 3200 nucleotides to 3400 nucleotides, 3400 nucleotides to 3600 nucleotides, 3600 nucleotides to 3800 nucleotides, 3800 nucleotides to 4000 nucleotides, 4000 nucleotides to 4200 nucleotides, 4200 nucleotides to 4400 nucleotides, 4400 nucleotides to 4600 nucleotides, 4600 nucleotides to 4800 nucleotides, or 4800 nucleotides to 5000 nucleotides.

[0061] In certain embodiments, the length of all 5' homologous regions and / or 3' homologous regions of the two or more DNA fragments is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides. In certain embodiments, the length of all 5' homologous regions and / or 3' homologous regions of the two or more DNA fragments is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides.In certain embodiments, the length of all 5' homologous regions and / or 3' homologous regions of the two or more DNA fragments is between 10 nucleotides and 50 nucleotides, between 50 nucleotides and 100 nucleotides, between 100 nucleotides and 150 nucleotides, between 150 nucleotides and 200 nucleotides, between 200 nucleotides and 250 nucleotides, between 250 nucleotides and 300 nucleotides, between 300 nucleotides and 350 nucleotides, between 350 nucleotides and 400 nucleotides, between 400 nucleotides and 450 nucleotides, between 450 nucleotides and 500 nucleotides, between 500 nucleotides and 550 nucleotides, between 550 nucleotides and 600 nucleotides, between 600 nucleotides and 650 nucleotides, between 650 nucleotides and 700 nucleotides, between 700 nucleotides and 750 nucleotides, between 750 nucleotides and 800 nucleotides, between 800 nucleotides and 850 nucleotides, between 850 nucleotides and 900 nucleotides, between 900 nucleotides and 950 nucleotides, or between 950 nucleotides and 1000 nucleotides. , 1000 nucleotides to 1200 nucleotides, 1200 nucleotides to 1400 nucleotides, 1400 nucleotides to 1600 nucleotides, 1600 nucleotides to 1800 nucleotides, 1800 nucleotides to 2000 nucleotides, 2000 nucleotides to 2200 nucleotides, 2200 nucleotides to 2400 nucleotides, 2400 nucleotides to 2600 nucleotides, 2600 nucleotides to 2800 nucleotides, 2800 nucleotides to 3000 nucleotides, 3000 nucleotides to 3200 nucleotides, 3200 nucleotides to 3400 nucleotides, 3400 nucleotides to 3600 nucleotides, 3600 nucleotides to 3800 nucleotides, 3800 nucleotides to 4000 nucleotides, 4000 nucleotides to 4200 nucleotides, 4200 nucleotides to 4400 nucleotides, 4400 nucleotides to 4600 nucleotides, 4600 nucleotides to 4800 nucleotides, or 4800 nucleotides to 5000 nucleotides.

[0062] In certain embodiments, two homologous regions that are homologous to each other have at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. In certain embodiments, two homologous regions that are homologous to each other have 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. In certain embodiments, the two homologous regions have a sufficient level of homology to allow homologous recombination of corresponding DNA fragments containing the homologous regions.

[0063] In certain embodiments, the 5' homologous region of the first DNA fragment and the 3' homologous region of the second DNA fragment share at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. In certain embodiments, the 3' homologous region of the first DNA fragment and the 5' homologous region of the second DNA fragment share at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. In certain embodiments, the 5' homologous region of the first DNA fragment and the 3' homologous region of the second DNA fragment share 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. In certain embodiments, the 3' homologous region of the first DNA fragment and the 5' homologous region of the second DNA fragment share 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity.

[0064] 5.1.3 Outside the homologous region of a DNA fragment In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 1200 nucleotides, 1500 nucleotides, 1800 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides, 3500 nucleotides, 4000 nucleotides, or 5000 nucleotides. The length of the nucleotide sequence located outside the homologous region in the DNA fragment is 10 to 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the DNA fragment is 50 to 10,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the DNA fragment is 100 to 5,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the DNA fragment is 150 to 2500 nucleotides, In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the DNA fragment is 250 to 2000 nucleotides.

[0065] In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 2000 nucleotides, 5000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, 20,000 nucleotides, 25,000 nucleotides, 30,000 nucleotides, 35,000 nucleotides, 40,000 nucleotides, 45,000 nucleotides, or 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 10 to 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 50 to 10,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 100 to 5000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 150 to 2500 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 250 to 2000 nucleotides.

[0066] In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 2000 nucleotides, 5000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, 20,000 nucleotides, 25,000 nucleotides, 30,000 nucleotides, 35,000 nucleotides, 40,000 nucleotides, 45,000 nucleotides, or 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is 10 to 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 50 to 10,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is 100 to 5000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is 150 to 2500 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is 250 to 2000 nucleotides.

[0067] In certain embodiments, the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 2000 nucleotides, 5000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, or 20,000 nucleotides, 25,000 nucleotides, 30,000 nucleotides, 35,000 nucleotides, 40,000 nucleotides, 45,000 nucleotides, or 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is 10 to 50,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is 50 to 10,000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous regions in all of the two or more DNA fragments is 100 to 5000 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous regions in the second DNA fragment is 150 to 2500 nucleotides. In certain embodiments, the length of the nucleotide sequence located outside the homologous regions in all of the two or more DNA fragments is 250 to 2000 nucleotides.

[0068] As used herein, when two nucleotide sequences are "not homologous" to each other or are "not homologous" to each other, unless otherwise specified, the sequence may be about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, about 625 nucleotides, about 650 nucleotides, or about 660 nucleotides. Two nucleotide sequences share at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% sequence identity over a region of about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides. In certain embodiments, two nucleotide sequences may have a region with 90% or greater sequence identity, where the length of such a region is at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, two nucleotide sequences may have at most 70% or 80% sequence identity over a region of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides. In certain embodiments, the level of homology between the two nucleotide sequences is insufficient to allow homologous recombination of the two nucleotide sequences. In certain embodiments, the level of homology in two nucleotide sequences may allow the number of unwanted recombination events to occur between the two nucleotide sequences to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of the nucleotide sequence over an incubation period of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0069] (i) The first DNA fragment and the second DNA fragment are not homologous to each other outside of the 5' and / or 3' homologous region(s). In certain embodiments, the nucleotide sequence of the first DNA fragment and the nucleotide sequence of the second DNA fragment are not homologous to each other outside of the homologous region(s). In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment are about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, about 625 nucleotides, about 650 nucleotides. The sequence identity over a region of about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides can be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first and second DNA fragments may have regions with 90% or more sequence identity, and the length of such regions is at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment may have at most 70% or 80% sequence identity over a region of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides. In certain embodiments, the level of homology among the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment is insufficient to allow homologous recombination of the DNA fragments at the region located outside the homologous region. In certain embodiments, the level of homology in nucleotide sequences located outside the regions of homology in the first and second DNA fragments may allow the number of unwanted recombination events occurring between the first and second DNA fragments in regions located outside the regions of homology to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of each of the first and second DNA fragments over an incubation period of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0070] (ii) all DNA fragments are not homologous to each other outside the 5' and / or 3' homologous regions(s); In certain embodiments, the nucleotide sequences of all DNA fragments are not homologous to each other outside the homologous region(s). In certain embodiments, the nucleotide sequences located outside the homologous region(s) in all DNA fragments are about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, about 625 nucleotides, about 650 nucleotides , about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides, can have at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% sequence identity. In certain embodiments, nucleotide sequences located outside the homologous region(s) in all DNA fragments may have regions with 90% or more sequence identity, with the length of such regions being at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, the nucleotide sequences located outside the homologous region(s) in all DNA fragments can have a sequence identity of at most 70% or 80% over a region of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides. In certain embodiments, the level of homology in the nucleotide sequences located outside the homologous region in all DNA fragments is insufficient to allow the homologous recombination of these DNA fragments in the region located outside the homologous region. In certain embodiments, the level of homology in nucleotide sequences located outside the homologous regions in all DNA fragments may allow the number of unwanted recombination events of those DNA fragments in regions located outside the homologous regions to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of each of those DNA fragments over an incubation period of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0071] (iii) the first DNA fragment and the second DNA fragment are not homologous to sequences in the genome of the Leishmania cell outside the 5' and / or 3' homologous region(s). In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment are not homologous to sequences in the genome of the Leishmania cell. In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment and the genome of the Leishmania cell are not homologous to sequences in the genome of the Leishmania cell that are about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, about 625 nucleotides. The sequence identity over a region of about 650 nucleotides, about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides can be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment and the genome of the Leishmania cell may have regions with 90% or more sequence identity, the length of such regions being at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, the nucleotide sequences located outside the region(s) of homology in the first and second DNA fragments and the genome of the Leishmania cell may have at most 70% or 80% sequence identity over a region of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides. In certain embodiments, the level of homology between the first and second DNA fragments outside their homologous regions and the genome of the Leishmania cell is insufficient to allow homologous recombination between the DNA fragments and the genome of the Leishmania cell in a region located outside the homologous regions. In certain embodiments, the level of homology between the first and second DNA fragments outside their homologous regions and the genome of the Leishmania cell may allow the number of unwanted recombination events between the DNA fragments and the genome of the Leishmania cell in a region located outside the homologous regions to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of each of the first and second DNA fragments over an incubation period of 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0072] (iv) All DNA fragments are not homologous to sequences in the genome of the Leishmania cell outside the 5' and / or 3' homologous region(s). In certain embodiments, the nucleotide sequences located outside the homologous region(s) in all DNA fragments are not homologous to sequences in the genome of the Leishmania cell. In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment and the genome of the Leishmania cell are not homologous to sequences in the genome of the Leishmania cell that are about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, about 625 nucleotides. The sequence identity over a region of about 650 nucleotides, about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides can be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In certain embodiments, the nucleotide sequences located outside the homologous region(s) in all DNA fragments and the genome of the Leishmania cell may have regions of 90% or more sequence identity, and the length of such regions is at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, the nucleotide sequences located outside the region(s) of homology in all DNA fragments and the genome of the Leishmania cell may have at most 70% or 80% sequence identity over a region of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides. In certain embodiments, the level of homology between the nucleotide sequences located outside the homologous regions in all DNA fragments and the genome of Leishmania cells is insufficient to allow homologous recombination between these DNA fragments and the genome of Leishmania cells in regions located outside the homologous regions.In certain embodiments, the level of homology between the nucleotide sequences located outside the homologous regions in all DNA fragments and the genome of Leishmania cells can allow the number of undesired recombination events between these DNA fragments and the genome of Leishmania cells in regions located outside the homologous regions to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of each DNA fragment in an incubation period of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0073] (v) the first DNA fragment and the second DNA fragment have no homology within each other; In certain embodiments, the nucleotide sequence of the first DNA fragment and the nucleotide sequence of the second DNA fragment do not share homology within each DNA fragment. In certain embodiments, the nucleotide sequence of the first DNA fragment and the nucleotide sequence of the second DNA fragment each contain a nucleotide sequence of about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, or about 625 nucleotides. , about 650 nucleotides, about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides, the sequence identity over the region can be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In certain embodiments, the nucleotide sequence of the first DNA fragment and the nucleotide sequence of the second DNA fragment may comprise nucleotide sequences within each DNA fragment that have 90% or more sequence identity, and the length of such region is at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, the nucleotide sequences of the first and second DNA fragments may have at most 70% or 80% sequence identity over a region of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides. In certain embodiments, the level of homology between the nucleotide sequences of the first and second DNA fragments within each DNA fragment is insufficient to allow homologous recombination of the DNA fragments within themselves. In certain embodiments, the level of homology within each DNA fragment in the nucleotide sequence of the first DNA fragment and the nucleotide sequence of the second DNA fragment may allow the number of unwanted recombination events occurring within the DNA fragment itself to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of the DNA fragment over an incubation period of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0074] (vi) All DNA fragments have no homology within each other. In certain embodiments, the nucleotide sequences of all DNA fragments are not homologous within each DNA fragment. In certain embodiments, the nucleotide sequences of all DNA fragments comprise within each DNA fragment a nucleotide sequence of about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, about 600 nucleotides, about 625 nucleotides, about 650 nucleotides. The sequence identity over a region of about 675 nucleotides, about 700 nucleotides, about 725 nucleotides, about 750 nucleotides, about 775 nucleotides, about 800 nucleotides, about 825 nucleotides, about 850 nucleotides, about 875 nucleotides, about 900 nucleotides, about 925 nucleotides, about 950 nucleotides, about 975 nucleotides, about 1000 nucleotides, about 1025 nucleotides, about 1050 nucleotides, about 1075 nucleotides, or about 2000 nucleotides can be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In certain embodiments, the nucleotide sequences of all DNA fragments may contain nucleotide sequences within each DNA fragment that have 90% or greater sequence identity, and the length of such regions is at most about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, or about 40 nucleotides.In certain embodiments, the nucleotide sequences of all DNA fragments contained within each DNA fragment may have at most 70% or 80% sequence identity over a region of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 125 nucleotides, about 150 nucleotides, about 175 nucleotides, about 200 nucleotides, about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides. In certain embodiments, the level of homology between the nucleotide sequences of all fragments within each DNA fragment is insufficient to allow homologous recombination of the DNA fragments within themselves. In certain embodiments, the level of homology within each DNA fragment in the nucleotide sequences of all DNA fragments may allow the number of unwanted recombination events occurring within the DNA fragment itself to be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per 10,000 copies of that DNA fragment over an incubation period of 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0075] In certain embodiments, the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment do not have repetitive sequences.

[0076] In certain embodiments, the number of DNA fragments is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In certain embodiments, the number of DNA fragments is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0077] 5.1.4 Translation products of DNA fragments In certain embodiments, the nucleotide sequences located outside the homologous regions in the two or more DNA fragments are selected from the group consisting of intergenic regions (IRs), untranslated regions (UTRs), and open reading frames (ORFs) encoding polypeptides. In certain embodiments, the nucleotide sequences located outside the homologous regions in the DNA fragments are selected from the group consisting of intergenic regions (IRs), untranslated regions (UTRs), and open reading frames (ORFs) described in the Examples section. In certain embodiments, the IRs, UTRs, and ORFs do not share homologous sequences within themselves and / or do not share homologous sequences with each other.

[0078] In certain embodiments, the nucleotide sequence located outside the homologous regions in the DNA fragment is an ORF encoding a target polypeptide as described in Section 5.4. In certain embodiments, the nucleotide sequence located outside the homologous regions in the DNA fragment is an ORF encoding an enzyme associated with the production of the target polypeptide. Examples of enzymes can be found, but are not limited to, in International Publication No. WO2019 / 002512A2, which is incorporated herein by reference in its entirety, and the international application entitled "Glycoengineering Using Leishmania Cells," filed on the same date as the present application. In certain embodiments, the nucleotide sequence located outside the homologous regions in the DNA fragment is an ORF encoding a heterologous glycosyltransferase. In certain embodiments, the nucleotide sequence located outside the homologous regions in the DNA fragment can be transcribed into an RNA product (e.g., a ribozyme, a regulatory RNA, an ncRNA, and a crisprRNA). In certain embodiments, nucleotide sequences located outside the regions of homology in the DNA fragment may be ORFs that encode polypeptides with functions related to catalysis of metabolic reactions and DNA replication, response to stimuli, transport of molecules from one location to another, providing structure to cells and organisms, aggregation and adhesion to other cells, localization of molecules, utilization of carbon, carbohydrates, nitrogen, phosphorus, and sulfur, biomineralization, cell growth, development, and mitosis, migration, biological regulation, protein folding, and / or toxins.

[0079] In certain embodiments, the nucleotide sequences located outside the homologous region in two or more DNA fragments code for the same polypeptide.In certain embodiments, Leishmania cells have the ability to express multiple copies of the same polypeptide.In certain embodiments, the method provided herein increases the expression level of polypeptide.In certain embodiments, the use of multiple DNA fragments that code for the same polypeptide can increase the expression level of polypeptide compared to the expression level obtained by the method of using one DNA fragment that codes for polypeptide.

[0080] (i) The nucleotide sequence resulting from homologous recombination of DNA fragments In certain embodiments, the DNA fragments undergo homologous recombination to generate a nucleotide sequence having a length of 50 to 100 nucleotides, 100 to 500 nucleotides, 500 to 1000 nucleotides, 1000 to 5000 nucleotides, 5000 to 10000 nucleotides, 10000 to 15000 nucleotides, 15000 to 20000 nucleotides, 20000 to 25000 nucleotides, or 25000 to 30000 nucleotides. nucleotides, 30,000 nucleotides to 35,000 nucleotides, 35,000 nucleotides to 40,000 nucleotides, 40,000 nucleotides to 45,000 nucleotides, 45,000 nucleotides to 50,000 nucleotides, 50,000 nucleotides to 55,000 nucleotides, 55,000 nucleotides to 60,000 nucleotides, 60,000 nucleotides to 65,000 nucleotides, 65,000 nucleotides to 70,000 nucleotides, 70,000 nucleotides to 75,000 nucleotides, or 75,000 nucleotides to 80,000 nucleotides.

[0081] In certain embodiments, the DNA fragments undergo homologous recombination to produce a nucleotide sequence that contains at least 50%, 60%, 70%, 80%, 90%, or 100% of the genetic information encoded by the two or more DNA fragments, hi certain embodiments, the nucleotide sequence that results from the DNA fragments undergoing homologous recombination contains all of the genetic information encoded in the two or more DNA fragments.

[0082] 5.1.5 Undesirable deletions and / or crossovers Generally, the methods provided herein are capable of avoiding undesired genetic recombination events. In certain embodiments, undesired genetic recombination events include crossovers and deletions. In certain embodiments, undesired genetic recombination events can be single-strand annealing (SSA) or microhomology-mediated end joining (MMEJ) and non-homologous end joining (NHEJ) (Zhang (2019) Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania. doi:10.1128 / mSphere.00408-19.). In certain embodiments, undesired deletions and / or crossovers can cause deletion of genetic information from the nucleotide sequences resulting from homologous recombination of DNA fragments. In certain embodiments, undesired deletions and / or crossovers can cause deletion of genetic information from endogenous chromosomal DNA.

[0083] In certain embodiments, unwanted deletions and / or crossovers can be detected using gene sequencing techniques known in the art. In certain embodiments, unwanted deletions and / or crossovers can be detected by phenotypic testing of the resulting genetically engineered Leishmania cells, for example, by testing the activity of an enzyme encoded by one or more DNA fragments used in the methods described herein. In certain embodiments, unwanted deletions and / or crossovers can be detected using the methods described in the Assays and Examples section of this application.

[0084] In certain embodiments, the methods described herein result in low levels of unwanted deletions and / or crossovers, ie, the percentage of Leishmania cells that undergo unwanted deletions and / or crossovers over at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the percentage of Leishmania cells that undergo undesired deletions and / or crossovers over at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0085] In certain embodiments, the percentage of Leishmania cells that undergo undesired deletions and / or crossovers over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the percentage of Leishmania cells that undergo undesired deletions and / or crossovers over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0086] In certain embodiments, the proportion of Leishmania cells in which unwanted deletions and / or crossovers occur after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the proportion of Leishmania cells in which unwanted deletions and / or crossovers occur after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions is at most about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0087] In certain embodiments, the proportion of Leishmania cells that undergo undesired deletions and / or crossovers after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the proportion of Leishmania cells that undergo undesired deletions and / or crossovers after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions is at most about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0088] 5.1.6 Chromosomal integration In certain embodiments, the two or more DNA fragments are suitable for integration into the chromosome of the Leishmania cell.

[0089] In certain embodiments, two or more DNA fragments are integrated into a chromosome of the Leishmania cell. In certain embodiments, one of the DNA fragments comprises a 5' homologous region that is homologous to a region in a chromosome of the Leishmania cell and a 3' homologous region that is homologous to the 5' homologous region of another DNA fragment. In certain embodiments, one of the DNA fragments comprises a 3' homologous region that is homologous to another region in a chromosome of the Leishmania cell and a 5' homologous region that is homologous to the 3' homologous region of another DNA fragment. In certain embodiments, the homologous region that is homologous to a region in a chromosome of the Leishmania cell allows the DNA fragment to be integrated into a chromosome of the Leishmania cell. Examples of chromosomal integration described herein, without limitation, can be found in the Examples section and can be shown in at least Figures 1A, 3B, 4, 6B, and 8A.

[0090] 5.1.7 Extrachromosomal plasmids In certain embodiments, the two or more DNA fragments do not integrate into the chromosome of the Leishmania cell. In certain embodiments, homologous recombination of the two or more DNA fragments results in a circular plasmid. In certain embodiments, the circular plasmid comprises a cos site. In certain embodiments, the circular plasmid is a cosmid. In certain embodiments, the plasmid is an Escherichia coli cosmid. Examples of extrachromosomal plasmids described herein include, but are not limited to, the Escherichia coli cosmid described in Example 6 and shown at least in FIG. 11A.

[0091] 5.1.8 Introduction of DNA fragments into host cells Any method known in the art can be used to introduce a DNA fragment (eg, a gene fragment thereof) into a host cell (eg, a Leishmania cell).

[0092] In certain embodiments, the DNA fragments 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 other embodiments, the DNA fragments are introduced and site-specifically integrated into the host cell genome by homologous recombination.

[0093] In certain embodiments, the DNA fragment is introduced into the host cell described herein using a plasmid.For example, the DNA fragment is expressed in the host cell by a plasmid (e.g., an expression vector), and the plasmid is introduced into the modified host cell by transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation.In certain embodiments, the plasmid is introduced into the modified host cell by stable transfection.

[0094] In certain embodiments, the two or more DNA fragments are introduced by transfection. In certain embodiments, the two or more DNA fragments are introduced simultaneously.

[0095] 5.1.9 Cell culture method Provided herein are methods for culturing host cells (e.g., Leishmania host cells). In one embodiment, host cells are cultured using any of the standard culture techniques known in the art. For example, cells are routinely cultured in a rich medium such as brain heart infusion, Trypticase soy broth, or yeast extract (all of which contain 5 μg / ml hemin). Incubation is performed at 26°C in the dark for 2-3 days as static or shaking cultures. In some embodiments, cultures of recombinant cell lines contain an appropriate selection agent. Examples of selection agents include, but are not limited to, Table 1. In some embodiments, cultures contain biopterin at a final concentration of 10 μM to support growth. In certain embodiments, host cells can be cultured using methods described in the Assays and Examples section.

[0096] 5.2 Leishmania cells Also provided herein are genetically engineered Leishmania cells. In certain embodiments, the Leishmania cells are genetically engineered using the methods described in Section 5.1 herein. In certain embodiments, the Leishmania cells are recombinantly engineered using iterative methods described herein. In certain embodiments, the Leishmania cells described herein can be used to express the DNA fragments described in Section 5.1.1. In certain embodiments, the Leishmania cells described herein can be used as an expression system described in Section 5.3. In certain embodiments, the Leishmania cells described herein can be used to prepare polypeptides described in Section 5.4.

[0097] 5.2.1 Genetically engineered Leishmania cells In certain embodiments, the Leishmania cell is genetically engineered to be used for expression of the ORF of the DNA fragment. In certain embodiments, the DNA fragment is integrated into the chromosome of the Leishmania cell. In certain embodiments, the DNA fragment is not integrated into the chromosome of the Leishmania cell. In certain embodiments, the DNA fragment undergoes homologous recombination to circularize into an extrachromosomal plasmid. In certain embodiments, the plasmid is a cosmid. In certain embodiments, the plasmid is an E. coli cosmid.

[0098] 5.2.2 Leishmania and Kinetoplastida strains In certain embodiments, the Leishmania cell is a Leishmania tarentolae cell. In certain embodiments, the Leishmania cell is a Leishmania aethiopica cell. In certain embodiments, the Leishmania cell is part of the Leishmania aethiopica species complex. In certain embodiments, the Leishmania cell is a Leishmania aristidesi cell. In certain embodiments, the Leishmania cell is a Leishmania deanei cell. In certain embodiments, the Leishmania cell is part of the Leishmania donovani species complex. In certain embodiments, the Leishmania cell is a Leishmania donovani cell. In certain embodiments, the Leishmania cell is a Leishmania chagasi cell. In certain embodiments, the Leishmania cell is a Leishmania infantum cell. In certain embodiments, the Leishmania cell is a Leishmania hertigi cell. In certain embodiments, the Leishmania cell is part of the Leishmania major species. In certain embodiments, the Leishmania cell is a Leishmania major cell. In certain embodiments, the Leishmania cell is a Leishmania martiniquensis cell. In certain embodiments, the Leishmania cell is part of the species Leishmania mexicana. In certain embodiments, the Leishmania cell is a Leishmania mexicana cell. In certain embodiments, the Leishmania cell is a Leishmania pifanoi cell. In certain embodiments, the Leishmania cell is part of the species Leishmania tropica. In certain embodiments, the Leishmania cell is a Leishmania tropica cell.

[0099] In certain embodiments, other host cells may be genetically engineered using the methods described herein. In certain embodiments, the host cell belongs to the Bodonidae family of kinetoplasts. In certain embodiments, the host cell is a Bodo saltans cell. In certain embodiments, the host cell belongs to the Ichthyobodonidae family of kinetoplasts. In certain embodiments, the host cell belongs to the Trypanosomatidae family of kinetoplasts. In certain embodiments, the host cell belongs to the Blastocrithidia family of trypanosomatidae. In certain embodiments, the host cell belongs to the Blechomonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the Herpetomonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the Jaenimonas family of trypanosomatidae. In certain embodiments, the host cell belongs to the Lafontella family of trypanosomatidae. In certain embodiments, the host cell belongs to the Leishmaniinae family of trypanosomatidae. In certain embodiments, the host cell belongs to the Novymonas family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Paratrypanosoma family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Phytomonas family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Sergeia family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Strigomonadinae family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Trypanosoma family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Wallacemonas family of the Trypanosomatidae. In certain embodiments, the host cell belongs to the Blastocrithidia family of the Trypanosomatidae.

[0100] 5.3 Use of Leishmania cells as an expression system In certain embodiments, Leishmania cells (as described in Section 5.2) may be used as an expression system to prepare a polypeptide, hi certain embodiments, the polypeptide may be a heterologous, non-Leishmania protein, such as a therapeutic protein (e.g., an antibody).

[0101] 5.3.1 Compositions Comprising Host Cells In one aspect, provided herein are compositions comprising the host cells described herein, such as compositions comprising Leishmania cells described in Section 5.2. Such compositions can be used in methods for producing target polypeptides described in Section 5.4. In certain embodiments, compositions comprising host cells can be cultured under conditions suitable for producing the polypeptide. Such polypeptides can then be isolated from the host cell-containing compositions using methods known in the art.

[0102] Compositions comprising the host cells provided herein can contain additional components suitable for the maintenance and survival of the host cells described herein, and can further contain additional components necessary or advantageous for the production of a polypeptide by the host cell, such as inducers for inducible promoters (such as arabinose, IPTG, tetracycline, and doxycycline).

[0103] In certain embodiments, provided herein are kits comprising one or more containers and instructions for use, wherein the one or more containers comprise Leishmania cells as described herein.

[0104] 5.3.2 Methods for Producing Target Polypeptides In one aspect, provided herein are methods for preparing a target polypeptide described in Section 5.4. In one embodiment, provided herein are methods for in vivo production of a target polypeptide described in Section 5.4 using a host cell described herein. In certain embodiments, provided herein are methods for producing a target polypeptide, the methods comprising: (i) culturing a host cell provided herein under conditions suitable for polypeptide production; and (ii) isolating the target polypeptide. In certain embodiments, the host cell comprises (a) a recombinant nucleic acid encoding the target polypeptide, and (b) a recombinant nucleic acid encoding one or more heterologous glycosyltransferases. In certain embodiments, the heterologous glycosyltransferase is an N-acetylglucosamine transferase, or a heterologous galactosyltransferase, or a heterologous sialyltransferase. In certain embodiments, the host cell is a Leishmania cell.

[0105]

[0013] In one aspect, provided herein is a method for preparing a polypeptide described in Section 5.4, the method comprising: (a) culturing a Leishmania cell described in Section 5.2 herein under conditions suitable for producing the polypeptide; and (b) isolating the polypeptide. In certain embodiments, the method further comprises introducing a nucleotide sequence encoding the polypeptide.

[0106] In certain embodiments, the target polypeptide produced by the provided host cells is a therapeutic polypeptide, i.e., a polypeptide used to treat a disease or disorder. For example, the target polypeptide produced by the host cells provided herein can be an enzyme, cytokine, or antibody. Section 5.4 provides a list of exemplary, but non-limiting, target polypeptides.

[0107] 5.4 Target Polypeptide In one aspect, provided herein are polypeptides produced by the methods described in Section 5.3. In certain embodiments, the target polypeptides produced by the provided Leishmania cells are therapeutic polypeptides, i.e., polypeptides used to treat a disease or disorder. For example, the target polypeptides produced by the host cells provided herein can be enzymes, cytokines, or antibodies. In certain embodiments, the target polypeptide is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO).

[0108] Any polypeptide known in the art (or a peptide / polypeptide corresponding to such a polypeptide) can be used as a target polypeptide according to the methods described herein. Those skilled in the art will understand that the nucleic acid sequences of known polypeptides as well as newly identified polypeptides can be easily deduced using methods known in the art. Therefore, it is assumed that it is well within the capabilities of those skilled in the art to introduce a nucleic acid encoding any polypeptide of interest into the host cells provided herein (e.g., via an expression vector (e.g., a plasmid), for example, by site-specific integration by homologous recombination).

[0109] In certain embodiments, the target polypeptide is glycosylated (e.g., sialylated). Those skilled in the art will further recognize that the methods described herein can be used to glycosylate a target polypeptide (wherein the glycosylation is performed in vivo, e.g., using a host cell provided herein, or in vitro), and that such glycosylated target polypeptides have a therapeutic effect (e.g., due to improved pharmacokinetics) and can be used to treat subjects with diseases / disorders that would benefit from treatment with such glycosylated (e.g., polysialylated) target polypeptides.

[0110] In certain embodiments, the target polypeptide is human interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor necrosis factor alpha (TNF-α), 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 morphogenesis The target polypeptide may comprise the amino acid sequence of human bone marrow protein 2 (hBMP2), human bone morphogenetic protein 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-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), the extracellular domain of CTLA4 (e.g., Fc fusion), or the extracellular domain of a TNF receptor (e.g., Fc fusion). In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is an enzyme or inhibitor.Examples of enzymes and inhibitors that can be used as target polypeptides include, but are not limited to, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, antithrombin III (AT-III), polypeptide C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, alglucosidase-α, laronidase (α-L-iduronidase), idursulfase (iduronidase), and the like. These include protonate-2-sulfatase, galsulfase, agalsidase-β (human α-galactosidase A), botulinum toxin, collagenase, human DNAse-I, hyaluronidase, papain, L-asparaginase, uricase (urate oxidase), glutamic acid carboxypeptidase (glucarpidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0111] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is a cytokine. Examples of cytokines that can be used as target polypeptides include, but are not limited to, interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), and tumor necrosis factor alpha (TNF-α).

[0112] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is a hormone or growth factor. Examples of hormones and growth factors that can be used as target polypeptides include, but are not limited to, 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 morphogenetic polypeptide 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (GnRH), and thyroid-stimulating hormone (TSH). ), 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-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0113] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is a receptor. Examples of receptors that can be used as target polypeptides include, but are not limited to, the extracellular domain of human CTLA4 (e.g., fused to Fc) and soluble TNF receptors (e.g., fused to Fc).

[0114] In other embodiments, the target polypeptide is a therapeutic polypeptide. In other embodiments, the target polypeptide is an approved biologic. In another embodiment, the therapeutic polypeptide is abatacept (e.g., Orencia), aflibercept (e.g., Eylea), agalsidase beta (e.g., Fabrazyme), albiglutide (e.g., Eperzan), aldesleukin (e.g., Proleukin), alefacept (e.g., Amevive), alglucerase (e.g., Ceredase), alglucosidase alfa (e.g., Lumizyme), aliskiren (e.g., Tekturna), alpha-1- Polypeptidase inhibitors (e.g., Aralast), alteplase (e.g., Activase), anakinra (e.g., Kineret), anistreplase (e.g., Eminase), human anthrax immune globulin (e.g., ANTHRASIL), antihemophilic factor (e.g., Advate), antiinhibitor blood coagulation complex (e.g., FeibaNf), antithrombin alpha, human antithrombin III, antithymocyte globulin (e.g., antithymocyte globulin), antithymocyte globulin (U), Antithymocyte globulin (rabbit) (e.g., ATG-Fresenius), aprotinin (e.g., Trasylol), asfotase alfa, asparaginase (e.g., Elspar), asparaginase Erwinia chrysanthemum (e.g., Erwinaze), becaplermin (e.g., Regranex), belatacept (e.g., Nulojix), belactant, bivalirudin (e.g., Angiomax), botulinum toxin type A (e.g., BOTOXE), type B botulinum toxin (e.g., Myobloc), brentuximab vedotin (e.g., Adcetris), buserelin (e.g., Suprecur), C1 esterase inhibitor (human), C1 esterase inhibitor (recombinant) (e.g., Ruconest), certolizumab pegol (e.g., Cimzia), chorionic gonadotropin alpha (e.g., chorionic gonadotropin alpha), chorionic gonadotropin (human) (e.g., Ovidrel), chorionic gonadotropin (recombinant) (e.g., Ovitrelle),Coagulation factor IX (e.g., Alprolix), coagulation factor VIIa (e.g., NovoSeven), coagulation factor X human (e.g., Coagadex), coagulation factor XIII A-subunit (recombinant), collagenase (e.g., Cordase), conestat alfa, corticotropin (e.g., HPActhar), cosyntropin (e.g., Cortrosyn), darbepoetin alfa (e.g., Aranesp), defibrotide (e.g., Noravid), denileukin diftitox (e.g., Ontak), desirudin, digoxin immune Fab (ovine) (e.g., DIGIBIND), dornase alfa (e.g., Pulmozyme), drotrecogin alfa (e.g., Xigr), is), dulaglutide, efmoloctocog alfa (e.g., ELOCTA), elosulfase alfa, enfuvirtide (e.g., FUZEON), epoetin alfa (e.g., Binocrit), epoetin zeta (e.g., Retacrit), eptifibatide (e.g., INTEGRILIN), etanercept (e.g., Enbrel), exenatide (e.g., Byetta), factor IX complex (human) (e.g., AlphaNine), fibrinolysin (also known as plasmin) (e.g., Elase), filgrastim (e.g., NA), filgrastim-sndz, follitropin alfa (e.g., Gonal-F), follitropin beta (e.g., Follistim AQ), galsulfase (e.g., Naglazyme), gastric intrinsic factor, gemtuzumab ozogamicin (e.g., Mylotarg), glatiramer acetate (e.g., Copaxone), recombinant glucagon (e.g., GlucaGen), glucarpidase (e.g., Voraxaze), gramicidin D (e.g., Neosporin), hepatitis B immune globulin, human calcitonin, human clostridium tetani toxin 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), human varicella-zoster immune globulin (e.g.,Varizig), hyaluronidase (e.g., HYLENEX), hyaluronidase (human recombinant), ibritumomab tiuxetan (e.g., Zevalin), idursulfase (e.g., Elaprase), imiglucerase (e.g., Cerezyme), human immunoglobulin, insulin aspart (e.g., NovoLog), bovine insulin, insulin degludec (e.g., Tresiba), insulin detemir (e.g., LEVEMIR), insulin glargine (e.g., Lantus), insulin glulisine (e.g., APIDRA), insulin lispro (e.g., Humalog), porcine insulin (e.g., Iletin II), regular insulin (e.g., Humulin R), porcine insulin (e.g., vetsulin), isophane insulin (e.g., Novolin N), recombinant interferon alpha-2a (e.g., Roferon A), interferon alpha-2b (e.g., INTRON A), interferon alfacon-1 (e.g., INFERGEN), interferon alfa-n1 (e.g., Wellferon), interferon alfa-n3 (e.g., Alferon), interferon beta-1a (e.g., Avonex), interferon beta-1b (e.g., Betaseron), interferon gamma-1b (e.g., Actimmune), intravenous immunoglobulin (e.g., Civacir), laronidase (e.g., Aldurazyme), lenograstim (e.g., Granocyte), lepirudin (e.g., Refludan), leuprolide ( e.g., Eligard), liraglutide (e.g., Saxenda), lucinactant (e.g., Surfaxin), lutropin alfa (e.g., Luveris), mecasermin (e.g., NA), menotropin (e.g., Menopur), methoxypolyethylene glycol-epoetin beta (e.g., Mircera), metreleptin (e.g., Myalept), natural alpha interferon or multiferon (e.g., Intron / Roferon-A), nesiritide (e.g., NATRECOR), ocriplasmin (e.g., Jetrea), oprelvekin (e.g.,Neumega), OspA lipopolypeptide (e.g., Lymerix), oxytocin (e.g., Pitocin), palifermin (e.g., Kepivance), pancrelipase (e.g., Pancrecarb), bovine pegademase (e.g., Adagen), pegaspargase (e.g., Oncaspar), pegfilgrastim (e.g., Neulasta), peginterferon alfa-2a (e.g., Pegasys), peginterferon alfa-2b (e.g., PEG-Intron), peginterferon beta-glucan (e.g., PEG-10), Antithrombin-1a (e.g., Plegridy), pegloticase (e.g., Krystexxa), pegvisomant (e.g., SOMAVERT), poractant alfa (e.g., Curosurf), pramlintide (e.g., Symlin), Preotact (e.g., Preotact E), protamine sulfate (e.g., Protamine Sulfate Injection, USP), human polypeptide S (e.g., human polypeptide S), prothrombin (e.g., FeibaNf), prothrombin complex (e.g., Cofact), prothrombin complex concentrate (e.g., Kcentra), rasburicase (e.g., Elitek), reteplase (e.g., Retavase), rilonacept (e.g., Arcalyst), romiplostim (e.g., Nplate), sacrosidase (e.g., Sucraid), salmon calcitonin (e.g., Calcimar), sargramostim (e.g., Leucomax), satumomab pendetide (e.g., OncoScint), sebelipase alfa (e.g., Kanuma), secretin (e.g., SecreFlo), sermorelin (e.g., sermorelin acetate), hemoglobin (hemoglobin), serum albumin (e.g., Albunex), iodinated serum albumin (e.g., Megatope), simoctocog alfa (e.g., Nuwiq), sipuleucel-T (e.g., Provenge), recombinant somatotropin (e.g., Nutropin AQ), recombinant somatropin (e.g., BioTropin), streptokinase (e.g., Streptase), sucoctocog alfa (e.g., Obizur), taliglucerase alfa (e.g., Elelyso), teduglutide (e.g., Gattex), tenecteplase (e.g.,TNKase), teriparatide (e.g., Forteo), tesamorelin (e.g., Egrifta), thrombomodulin alpha (e.g., Recomodulin), thymalfasin (e.g., Zadaxin), thyroglobulin, thyrotropin alpha (e.g., Thyrogen), purified polypeptide derivative tuberculin (e.g., Aplisol), turoctocog alpha (e.g., Zonovate), urofollitropin (e.g., BRAVELLE), urokinase (e.g., Kinl ytic), vasopressin (e.g., Pitressin), velaglucerase alfa (e.g., Vpriv), abciximab (e.g., ReoPro), adalimumab (e.g., Humira), alemtuzumab (e.g., CAMPATH), alirocumab (e.g., Praluent), arcitumomab (e.g., CEA-Scan), atezolizumab (e.g., Tecentriq), basiliximab (e.g., Simulect), belimumab (e.g., Benlysta), bevacizumab (e.g., , Avastin), blinatumomab (e.g., Blincyto), brodalumab (e.g., Siliq), canakinumab (e.g., ILARISE), canakinumab (e.g., Ilaris), capromab (e.g., ProstaScint), cetuximab (e.g., Erbitux), daclizumab (e.g., Zenapax), daratumumab (e.g., DARZALEX), denosumab (e.g., Xgeva), dinutuximab (e.g., unituxin), eculizumab (e.g., Soliris ), efalizumab (e.g., RAPTIVA), elotuzumab (e.g., EMPLICITI), evolocumab (e.g., Repatha), golimumab (e.g., Simponi injection), ibritumomab (e.g., Zevalin), idarucizumab (e.g., Praxbind), infliximab (e.g., REMICADE), ipilimumab (e.g., YERVOY), ixekizumab (e.g., Taltz), mepolizumab (e.g., Nucala), muromonab (e.g., ORTHOCLONE OKT3), natalizumab (e.g., Tysabri), necitumumab (e.g., Portrazza), nivolumab (e.g., Opdivo), obiltoxaximab (e.g.,Anthim), obinutuzumab (e.g., Gazyva), ofatumumab (e.g., Arzerra), omalizumab (e.g., Xolair), palivizumab (e.g., Synagis), panitumumab (e.g., Vectibix), pembrolizumab (e.g., Keytruda), pertuzumab (e.g., , Perjeta), ramucirumab (e.g., Cyramza), ranibizumab (e.g., Lucentis), raxibacumab (e.g., RAXIBACUMAB), rituximab (e.g., Rituxan), secukinumab (e.g., Cosentyx), siltuximab (e.g., Sylvant), tocilizumab (e.g., Actemra), tositumomab (e.g., Bexxar), trastuzumab (e.g., Herceptin), ustekinumab (e.g., Stelara), or vedolizumab (e.g., Entyvio).

[0115] In another embodiment, the target polypeptide is an antibody. In another embodiment, the antibody is selected from the group consisting of adalimumab (Humira), Remicade (infliximab), ReoPro (abciximab), Rituxan (rituximab), Simulect (basiliximab), Synagis (palivizumab), Herceptin (trastuzumab), Mylotarg (gemtuzumab ozogamicin), Campath (alemtuzumab), Zevalin (ibritumomab tiuxetan), Xolair (omalizumab), Bexxar (tositumomab-I-131), Erbitux (cetuximab), Avastin (bevacizumab), Tysabri (natalizumab), Actemra (tocilizumab), Vecti bix (panitumumab), Lucentis (ranibizumab), Soliris (eculizumab), Cimzia (certolizumab pegol), Simponi (golimumab), Ilaris (canakinumab), Stelara (ustekinumab), Arzerra (ofatumumab), Prolia (denosumab), Numax (motavizumab), ABThrax (raxibacumab), Benlysta (belimumab), Yervoy (ipilimumab), Adcetris (brentuximab vedotin), Perjeta (pertuzumab), Kadcyla (Ado-trastuzumab emtansine), or Gazyva (obinutuzumab).

[0116] In other embodiments, the antibody is a full-length antibody, Fab, F(ab')2, Scfv, or sdAb. In other embodiments, the target polypeptide comprises the amino acid sequence of an enzyme or its inhibitor. In further embodiments, the target polypeptide is Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, antithrombin III (AT-III), polypeptide C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, alglucosidase-α, laronidase (α-L-iduronidase), idursulfase (iduronate-2-sulfatase), or a combination thereof. The amino acid sequences include those of: human α-galactosidase A, galsulfase, agalsidase-β (human α-galactosidase A), botulinum toxin, collagenase, human DNAse-I, hyaluronidase, papain, L-asparaginase, uricase (urate oxidase), glutamic acid carboxypeptidase (glucarpidase), α-1 protease inhibitor (α-1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0117] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is a receptor. Examples of receptors that can be used as target polypeptides include, but are not limited to, the extracellular domain of human CTLA4 (e.g., fused to Fc) and soluble TNF receptors (e.g., fused to Fc).

[0118] In another embodiment, the target polypeptide is secreted into the culture medium. In certain embodiments, the target polypeptide is purified from the culture medium. In another embodiment, the target polypeptide is purified from the culture medium by affinity purification or ion exchange chromatography. In another embodiment, the target polypeptide comprises an Fc domain and is affinity purified from the culture medium by polypeptide-A. In another embodiment, the target polypeptide comprises an affinity tag and is affinity purified.

[0119] In certain embodiments, the target polypeptide used along with the methods and host cells described herein can be a full-length polypeptide, a truncation thereof, a polypeptide domain, region, motif, or peptide.

[0120] In certain embodiments, the target polypeptide is an FC fusion polypeptide.

[0121] In certain embodiments, the target polypeptide is a biologic that comprises the Fc domain of IgG.

[0122] In certain embodiments, the target polypeptide may be modified. In another embodiment, the target polypeptide is engineered to include a signal sequence from Leishmania. In other embodiments, the signal sequence is processed and removed from the target polypeptide. In another embodiment, the target polypeptide is engineered to include one or more tag(s). In other embodiments, the tag(s) is processed and removed from the target polypeptide.

[0123] 5.4.1 Compositions and / or Formulations Comprising Polypeptides In another aspect, provided herein are compositions (e.g., pharmaceutical compositions) comprising one or more of the target polypeptides described herein. The compositions described herein are useful for treating and / or preventing diseases / disorders in a subject (e.g., a human subject) (see Section 5.4.2).

[0124] In certain embodiments, compositions (e.g., pharmaceutical compositions) described herein comprise a pharmaceutically acceptable carrier in addition to a target polypeptide described herein. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, for use in humans. As used herein in the context of a pharmaceutically acceptable carrier, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used in administering a pharmaceutical composition. Saline solutions and aqueous dextrose and glycerol solutions can also be used 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, dry milk fat, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0125] 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 can be formulated to be suitable for subcutaneous administration, parenteral administration, oral administration, intradermal administration, transdermal administration, colorectal administration, intraperitoneal administration, and rectal administration. In certain embodiments, the pharmaceutical compositions can be formulated for intravenous administration, oral administration, intraperitoneal administration, intranasal administration, intratracheal administration, subcutaneous administration, intramuscular administration, topical administration, intradermal administration, transdermal administration, or pulmonary administration.

[0126] In certain embodiments, the compositions described herein may further comprise one or more buffering agents. Such buffering agents include, for example, phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the compositions described herein do not comprise a buffering agent.

[0127] In certain embodiments, the compositions described herein further comprise one or more salts. Such salts include, for example, sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixtures of such aluminum salts). In other embodiments, the compositions described herein do not comprise a salt.

[0128] The compositions described herein can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0129] The compositions described herein can be stored until use. For example, the compositions can be stored frozen (e.g., at about -20°C or about -70°C), stored under refrigerated conditions (e.g., at about 4°C), or stored at room temperature.

[0130] 5.4.2 Prophylactic and therapeutic uses In one aspect, provided herein is a method for preventing or treating a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or composition thereof described herein. Further provided herein is a method for preventing a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or composition thereof described herein.

[0131] In one aspect, provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or composition thereof described herein. In another aspect, provided herein is a method for preventing a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or composition thereof described herein. In certain embodiments, provided herein is a method for preventing or treating a disease or disorder in a subject, the method comprising administering to the subject a poly-sialylated target polypeptide produced according to the methods described herein.

[0132] In certain embodiments, a disease or disorder may be caused by the presence in a subject of a defective version of a target polypeptide, the absence of a target polypeptide in a subject, or reduced expression of a target polypeptide in a subject, and may be treated or prevented using a target polypeptide produced using the methods described herein. In certain embodiments, the disease or disorder may be mediated by a receptor to which a target polypeptide produced using the methods described herein binds, or by a ligand to which a target polypeptide produced using the methods described herein binds (e.g., the target polypeptide is a receptor for a ligand).

[0133] In certain embodiments, a method for preventing or treating a disease or disorder in a subject comprises administering to the subject an effective amount of a target polypeptide or composition thereof described herein, in certain embodiments, an effective amount is a therapeutic amount having prophylactic and / or therapeutic effect(s). In certain embodiments, an "effective amount" refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of a disease / disorder or symptoms associated therewith, (ii) reducing the duration of a disease / disorder or symptoms associated therewith, (iii) preventing the progression of a disease / disorder or symptoms associated therewith, (iv) inducing regression of a disease / disorder or symptoms associated therewith, (v) preventing the onset or occurrence of a disease / disorder or symptoms associated therewith, (vi) preventing the recurrence of a disease / disorder or symptoms associated therewith, (vii) reducing organ failure associated with a disease / disorder, (viii) reducing the number of hospitalizations of a subject with a disease / disorder, (ix) reducing the length of hospitalization of a subject with a disease / disorder, (x) increasing the survival time of a subject with a disease / disorder, (xi) eliminating the disease / disorder in a subject, and / or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another treatment.

[0134] 5.5 Assay 5.5.1 Strains, propagation methods, and genetic methods Provided herein are methods for culturing host cells. Host cells are cultured using any of the standard culture techniques known in the art. For example, cells are routinely cultured in a rich medium such as brain heart infusion, Trypticase soy broth, or yeast extract (all containing 5 μg / ml hemin). Incubation is performed at 26°C in the dark for 2-3 days as static or shaking cultures. In some embodiments, cultures of recombinant cell lines contain an appropriate selection agent.

[0135] Table 1 provides an exemplary, non-limiting list of selected agents. [Table 2]

[0136] 5.5.2 Plasmids Plasmids were derived from a pUC57 vector backbone for propagation in E. coli and contained ampicillin or kanamycin selection markers. Expression cassettes were flanked by restriction enzyme recognition sites suitable for excision. The cassette composition depended on the intended use and was described in the respective methods and examples. The gene of interest was included as an ORF whose codon usage was optimized for L. tarentolae by back-translating the protein sequence into a nucleotide sequence using a custom Python3 script that stochastically selected codons based on codon usage in L. tarentolae while excluding low-frequency codons (frequency <10%). Codon usage was calculated using cusp (Rice, et al. (2000) Trends in genetics: TIG 16(6), pp. 276-277) for all annotated L. tarentolae nucleotide coding sequences. Optimized sequences were manually curated to avoid restriction enzyme recognition sites and remove repetitive or homopolymeric stretches.

[0137] To select new intergenic regions for artificial polycistron generation, we searched for homologs of the L. major gene (Rastrojo, et al. (2013) BMC Genomics 14, p. 223), which has been shown to have high relative transcript levels, in the genomes of L. mexicana, L. donovani, and L. infantum. We then used blastn (Camacho, et al. (2009) BMC Bioinformatics 10, p. 421) to further screen for related 3' intergenic regions (Murray, et al. (2007) Molecular and Biochemical Parasitology 153(2), pp. 125-132) using cd-hit (Li, et al. (2006) Bioinformatics 22(13), pp. 1658-1659) were used) 3' intergenic regions that were more than 80% identical to each other or intervals longer than 30 bp identical to the L. tarentolae genome were excluded.

[0138] For long integration constructs, the constructs were divided into several smaller pieces, usually less than 2500 bp, which contained regions at their ends for homologous recombination with each other (usually 200 bp) or with the chromosomal integration locus (usually 500 bp), allowing assembly by the Leishmania tarentolae homologous recombination system.

[0139] Plasmid generation and sequencing was performed by a gene synthesis provider. Plasmids and descriptions are shown in the sequence listing.

[0140] (i) Transfection Method (A) DNA preparation Restriction enzyme digestion (12 μg of DNA in a total volume of 240 μL) was performed using standard restriction enzymes (ThermoFisher, preferably FastDigest) according to the manufacturer's instructions. Restriction enzyme digestion was carried out at 30°C until completion or overnight, and DNA purification was performed by EtOH precipitation. For this EtOH precipitation, 1 volume of digested DNA was added with 2 volumes of 100% ice-cold EtOH, incubated on ice for 30 minutes, and centrifuged at 17,500 × g for 30 minutes at 4°C. The pellet was washed with 70% EtOH, then dried for up to 15 minutes before being resuspended in ddH2O. To optimally remove circular plasmids, one or two restriction enzymes with recognition sites in the vector backbone were selected, digested for 1 hour at 37°C, and purified with EtOH as described above. Analysis of the digests was performed by agarose gel electrophoresis in 0.7-2% agarose gels (TAE buffer). Optionally, gel extraction was performed using NucleoSpin® gels, and undigested plasmid was removed from the preparations using a PCR Clean-up kit (Macherey & Nagel) according to the manufacturer's instructions.

[0141] (B) DNA preparation for transfection Linear DNA fragments for integration were mixed in the required combination for transfection at 1 μg per fragment. The volume of this mixture was reduced in a vacuum concentrator at 30°C to approximately 2 μl per transfection. For episomal transfection of plasmids, 0.1–1 μg of plasmid DNA was used directly for transfection.

[0142] (C) Transfection using Nucleofector One day before transfection, a densely grown culture of the parent strain was diluted 1:10 into fresh medium (Brain Heart Infusion + Hemin (BHIH) or Yeast Extract + Hemin (YEH)) containing all antibiotics corresponding to the previously integrated selectable markers and grown overnight at 26°C.

[0143] Transfections were performed using the 4D-Nucleofector™ Core X with the P3 Primary Cell 4D-Nucleofector™ X kit (Lonza). For this, DNA prepared as described above was mixed with 16.4 μl of P3 Primary Cell solution and 3.6 μl of supplement solution. 7 An equal volume of culture containing 100 cells (OD approximately 0.3-1.0 / ml, cell shape should be round to droplet-like) was pelleted by centrifugation at 1800 g for 5 minutes, and the supernatant was removed. The cell pellet was resuspended in the above DNA mixture and transfected using 16-well electroporation strips with a pulse of FI-158 (in some cases, FP167, CM150, EO115, DN100, FP158, or FB158 were used as alternative pulses). As a negative control, an additional culture was transfected with ddH2O only.

[0144] 80 μl of fresh medium (BHIH or YEH + parental cell line selection marker) was added to each well, and 45 μl of the mixture (in duplicate) was transferred to individual wells of a 96-well culture plate pre-filled with 200 μl of fresh medium. After 24 hours of incubation (recovery) at 26°C in the dark, a new selection marker was added at a 50% concentration (pre-selection, see Table 2). After an additional 1-2 days of incubation, the selection marker was replenished to 100% (main selection, see Table), and several dilutions (1:2 to 1:10) were performed in a 96-well format (final volume 250 μl). The 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 for 10 minutes at room temperature), and the cultures were again incubated for up to 7 days. This step was repeated as necessary. Prior to analysis, growth cultures were expanded by dilutions ranging from 1:5 to 1:20 to increase the culture volume.

[0145] (D) Transfection using Gene Pulser Xcell™ (Biorad) Leishmania cultures for transfection (static, 26°C) were prepared by diluting 1:10 a high-density culture in BHIH or YEH the day before transfection. The OD at 600 nm was measured in a disposable cuvette using a photometer. To optimize efficiency, the OD was adjusted to 0.4–1.0 (4–6 × 10). *The cells were grown in logarithmic growth phase, indicated by the presence of a mixed population of round and droplet-shaped cells. A higher percentage of round cells was preferred. A 10 ml culture was used per transfection, and one culture was always electroporated with ddH2O as a negative control for each selection marker. For transfection, the culture was spun down at 1,800 × g for 5 minutes at room temperature. The supernatant was removed, and the pellet was resuspended in 5 ml of transfection buffer (200 mM Hepes (pH 7.0), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM anhydrous dextrose (glucose), filter-sterilized (0.22 μm pore size). The cells were centrifuged again, and the pellet was resuspended in 400 μl of transfection buffer. 400 μl of cells were added to the DNA, transferred to a cuvette, and incubated on ice for 10 minutes. Electroporation was performed using a Gene Pulser Xcell™ (Biorad) using a low-voltage protocol (μ exponential decay wave: 450 V, 450 μF, 5–6 ms, cuvette: d = 2 mm) and immediately placed on ice for exactly 10 minutes. The entire contents of the cuvette were transferred to 10 ml of BHIH or YEH without any selection marker, and the cells were grown in static culture at 26°C for 20–24 hours with aeration in the dark. For selection of polyclonal cell lines, half the concentration of the selection marker was added, and the culture was incubated at 26°C for 1–2 days before being passaged at a 1:10 ratio in 10 ml of BHIH or YEH containing the full concentration of the selection marker. The cells were further grown in the dark at 26°C. If the culture becomes cloudy after 7 days, the cells will be spun down at 1,800 x g for 5 minutes at room temperature, and the pellet will be resuspended in fresh BHIH or YEH medium containing the full concentration of the selection marker.

[0146] (ii) Clonal selection For clonal selection, cells were streaked onto BHIH or YEH plates (containing 1.4% agar and the appropriate 100% selective drug) as soon as the liquid culture became cloudy. Plates were covered with parafilm and incubated upside down in the dark at 26°C for 7–10 days. Single colonies (1–2 mm in size) were transferred to 24-well plates containing 1 ml of BHIH or YEH, covered with parafilm, and incubated in the dark at 26°C for approximately 7–10 days. Subsequently, 1 ml of culture was transferred from the 24-well plate to a flask containing 10 ml of BHI or YEH and further grown in static culture as usual.

[0147] (iii) Extraction of genomic DNA by gravity flow for long-read sequencing Genomic DNA was extracted from a 10 ml high-density culture of Leishmania tarentolae (grown for 3 days (OD ∼2)) using Macherey Nagel NucleoBond CB100 Kit #740508 (Nucleobond Buffer set IV #740604 + AXG100 columns). For this, cells were pelleted at 1600 g for 15 minutes and washed twice with 10 ml of PBS. The cell pellet was then resuspended in 1 ml of PBS and subjected to the extraction protocol according to the manufacturer's instructions.

[0148] (iv) Nanopore sequencing Verification of the construction of strains St16834, St17311, St17212, and St17180 was performed by long-read Nanopore sequencing. Library preparation was performed according to the manufacturer's instructions (Oxford Nanopore Technologies, Oxford, UK). Nanopore sequencing was performed on a GridION X5 instrument (Oxford Nanopore Technologies) capable of real-time base calling. Sequencing runs were terminated after 48 hours. Assembly of raw reads was performed using the Canu hierarchical assembler (version 1.8) (Koren, et al. (2017) Genome research 27(5), pp. 722-736). The assembled contigs were compared to the target in silico reference sequences using BLAST (Camacho, et al. (2009) BMC Bioinformatics 10, p. 421) and the Artemis Comparison Tool (Carver, et al. (2005 Bioinformatics 21(16), pp. 3422-3423).

[0149] (v) PacBio sequencing PacBio long-read genome sequencing was performed on two PacBio SMRT cells (v2.1 chemistry) for St15448 and one PacBio sequel SMRT cell for St17527, with libraries prepared according to the manufacturer's specifications.

[0150] Assembly of PacBio raw reads into long contigs was performed using HGAP [https: / / github.com / PacificBiosciences / Bioinformatics-Training / wiki / HGAP-in-SMRT-Analysis], and error correction of PacBio raw reads was performed by applying Arrow twice [https: / / github.com / PacificBiosciences / GenomicConsensus].

[0151] (vi) Illumina sequencing Genomic DNA from St17527 was further subjected to sequencing on an Illumina NextSeq (2 × 150 bp paired-end sequencing; TruSeq libraries were prepared according to the manufacturer's instructions). The resulting quality-trimmed data consisted of approximately 20M paired reads per strain. These reads were aligned to reference sequences using BWA-MEM (Li and Heng (2013) Aligning sequence reads, clone sequences, and assembly contigs with BWA-MEM. Available online at http: / / arxiv.org / pdf / 1303.3997v2).

[0152] 5.5.3 Expression analysis (i) Sample preparation from Leishmania tarentolae Cells were grown in static culture at 26°C for 2–3 days (e.g., 3 ml of growth culture was performed in a 6-well plate). Analysis of whole cell extracts (WCE) and corresponding cell-free culture supernatants was performed by Western blot. For supernatant analysis, growth cultures were centrifuged at 1800 g for 5 minutes at room temperature, and the 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 1x PBS, centrifuged again at 1800 g for 5 minutes at room temperature, and frozen at -80°C for a minimum of 30 minutes. After thawing again at room temperature, the pellet was dissolved in Laemmli (reducing) buffer, boiled again at 95°C for 10 minutes, and vortexed vigorously.

[0153] (ii) Expression analysis by Western blot Samples were subjected to 4-12% Bis-Tris SDS PAGE at 200V for 60 minutes using MOPS running buffer. Gels were transferred to PVDF membranes using an Iblot apparatus for 7 minutes. The membranes were blocked in 10% milk at room temperature for at least 30 minutes. Primary antibodies (i.e., goat anti-human IgG-HRP (A6029, Sigma) at a dilution of 1:2000, mouse anti-human kappa light chain (K4377, Sigma) at a dilution of 1:5000, or rabbit anti-S. pneumoniae serotype 1 polysaccharide (SSI, #16744) at a dilution of 1:100) diluted in 1x PBST containing 1% milk were incubated overnight at 4°C. The blots were then washed three times for 5 minutes with 1x PBST, followed by detection with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse polyvalent-HRP (A0412, Sigma) at a dilution of 1:2000 or anti-rabbit-HRP conjugate (Jackson ImmunoResearch #111-035-008) at a dilution of 1:2000) in 1x PBST containing 1% milk for 3 hours at 30°C with rotation. The blots were then washed three times for 5 minutes with 1x PBST and stained with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (TMBM-1000-01, Surmodics) for colorimetric detection.

[0154] 5.5.4 Small-scale expression and purification of adalimumab Host cells were routinely grown for 48 hours at 26°C in 50 ml cultures in BHIH or YEH with shaking at 140 rpm. Cultures were harvested and centrifuged at 1800 × g for 10 minutes at room temperature. Culture supernatants were filtered through 0.22 μm pore-size filters (Steriflip, SCGP00525), and a 1:100 dilution of EDTA (0.5 M pH 8) was added to each load. Culture supernatants from each strain were incubated in batches with shaking for 4 hours at room temperature with 100 μl of Protein A resin (Protein A-Sepharose 4B Fast Flow, Sigma-Aldrich, P9424) per Falcon tube. After treatment with Protein A resin, samples were centrifuged at 500 × g for 5 minutes, the unbound portion was discarded, and the resin was transferred to a spin column. 100 μl of resin was washed three times with 500 μl of Buffer A (20 mM NaHPO, pH 7.2, 150 mM NaCl, pH adjusted to 7.20 with HCl) (5 CV), with centrifugation at 1000 × g for 1 min at room temperature between each wash step. Elution was performed (over several CVs) using 100 μl of buffer B (0.1 M acetic acid, 100 mM NaCl, pH adjusted to 3.20 with 1 M NaOH) per 100 μl of resin. This elution (e.g., three times with 1 CV and one time with 0.5 CV) included centrifugation at 1000 × g for 1 min at room temperature between each step. The eluted fractions were pooled and quickly neutralized by adding 100 mM Tris-HCl (1 M pH 8). The pooled eluate was then buffer exchanged into PBS (pH 6) using a 7K ZebaSpin desalting column (2 ml) and, optionally, concentrated using an Amicon 30K concentrator (0.5 ml).

[0155] 5.5.5 HILIC-UPLC-MS Analysis of Purified Proteins and Cell Surface-Released N-Glycans Enzymatic release of N-glycans from purified proteins was performed using Rapid PNGase F (New England Biolabs) as recommended by the supplier. 8 μl of sample (15 μg of protein) was mixed with 2 μl of Rapid buffer and 1 μl of Rapid PNGase F. The mixture was incubated at 50°C for 10 min, followed by incubation at 90°C for 1 min.

[0156] Enzymatic release of N-glycans from the cell surface was performed using PNGase F (New England Biolabs). Cells (grown at 26°C for 48 or 72 hours with shaking at 140 rpm) were harvested and washed with PBS by centrifugation at 1800 × g for 10 minutes at room temperature. 50 mg of cell pellets were resuspended in Glyco Buffer 2 and incubated with 1 μl of PNGase F at 37°C and 650 rpm for 1 hour. Cells were again pelleted by centrifugation, and 75 μl of the supernatant was dried in a SpeedVac concentrator. Glycans were resuspended in 10 μl of water. After glycan release, glycans were directly labeled with procainamide as previously reported (Behrens, et al. (2018) Glycobiology 28(11), pp. 825-831). Briefly, released glycans were mixed with 1 μl of acetic acid, 8 μl of procainamide stock solution (550 mg / ml in DMSO), and 12 μl of sodium cyanoborohydride stock solution (200 mg / ml in HO). Samples were incubated at 65°C for 60 min and purified using LC-PROC-96 purification plates (Ludger Ltd) according to the manufacturer's instructions.

[0157] Analysis of procainamide-labeled N-glycans was performed by hydrophilic interaction chromatography-ultra-performance liquid chromatography-mass spectrometry (HILIC-UPLC-MS) using an Acquity UPLC System (Waters) with fluorescence detection coupled to a Synapt G2-Si mass spectrometer (Waters). Glycan separation was performed using an Acquity BEH amide column (130 Å, 1.7 μm, 2.1 mM × 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% to 55% solvent B over 40 min at 0.5 ml / min. Fluorescence detection was performed at an excitation wavelength of 310 nm and a detection wavelength of 370 nm. A Synapt G2-Si mass spectrometer equipped with a Zspray electrospray ion source was used for mass detection in positive resolution mode. The following parameters were used for this detection: scan range: m / z 300–3500, scan time: 1 s, capillary voltage: 2.2 kV, ion source temperature: 120 °C, and sampling cone voltage: 75 V. Data acquisition was performed using MassLynx 4.2 (Waters). Data processing and analysis were performed using Unifi 1.9.4.053 (Waters). Glucose units were assigned using a fifth-order polynomial distribution curve based on the retention time of a procainamide-labeled dextran ladder (Ludger Ltd). Glycan structures were assigned based on their m / z values and retention times and subjected to matching against a previously constructed N-glycan library. For each sample, a UPLC was coupled to a Synapt HDMS mass spectrometer using equivalent settings.

[0158] For a small number of samples, use the Waters RapiFluor labeling kit (Waters instructions:<Quality control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation> The glycans were labeled with α- and β-glucan (mostly used in accordance with the above) and analyzed using the same instrument as for the procainamide-labeled glycans (RF-MS).

[0159] 5.5.6 DMB labeling of Neu5Ac and CMP-Neu5Ac We employed a highly sensitive strategy for quantifying the concentration of nucleotide-activated sialic acids by combining reduction with fluorescent labeling using the fluorophore 1,2-diamino-4,5-methylenedioxybenzene (DMB). Labeling with DMB requires the free keto and carboxyl groups of the sialic acid molecule. Reducing the keto group prior to the labeling process prevents labeling of non-activated sialic acids (Neu5Ac). Nucleotide-activated sialic acids remain labelable even after reduction because their keto groups are protected from reduction by the CMP substitution. Subsequent DMB-high-performance liquid chromatography (HPLC) analysis allows for the identification and quantification of both total Neu5Ac and modified CMP-sialic acids in the femtomole range (Galuska, et al. (2010) Anal Chem 82(11), pp. 4591-4598).

[0160] The MeOH / chloroform extraction procedure for L. tarentolae cell pellets was performed on each sample with an OD of 4 (collected by centrifugation, washed twice with 1x PBS, and frozen). For extraction, the pellets were thawed, resuspended in 480 μl of MeOH, 20 μl of water added, and sonicated in a water bath for 15 minutes at room temperature. The samples were centrifuged at 18,000 g in a tabletop centrifuge for 10 minutes at 4°C. The supernatant was transferred to a glass vial, and 268 μl of chloroform was added and vortexed. Next, 500 μl of HO (MS grade) was added, and the sample was vortexed again. This MeOH / chloroform / HO (1 / 0.54 / 1) mixture was centrifuged at 2,200 g for 20 minutes at room temperature to remove proteins, lipids, and DNA into the CHCl3 phase. Approximately half of the upper MeOH / HO phase (525 μl) was collected and transferred to an Eppendorf tube. This amount corresponds to the extract from a pellet with an OD of 2. This sample was dried in a speed-vac, resuspended in 16 μl of HO, and divided into two 8 μl samples. These samples were subjected to DMB labeling under reducing and non-reducing conditions, respectively. As a control, HO containing Neu5Ac was dried in a SpeedVac, and the dried material was diluted with HO and divided into two portions for both labeling procedures. To one set of samples, 10 μl of ice-cold 0.4 M sodium borate buffer (pH 6.8) and 2 μl of freshly thawed 2 M borohydride in 0.5 M NaOH were added (final = 0.2 M borohydride in 0.2 M sodium borate buffer (pH 8.8)) and incubated at room temperature for 2 h (reduced sample). For the second set of samples, 10 μl of ice-cold 0.4 M sodium borate buffer (pH 6.8) was added along with 2 μl of 0.5 M NaOH (final = 0.2 M sodium borate buffer (pH 8.8)) and incubated at room temperature for 2 hours (non-reduced samples). The samples were then dried in a speedVac, resuspended in 3 μl of HO, and subjected to standard DMB labeling using the Takara labeling kit (#4400) according to the manufacturer's instructions. Finally, samples were analyzed in duplicate by RP-C18-LC.Quantification was performed using a defined standard curve obtained by incubating standard solutions in sodium borate buffer (non-reducing) and labeling with DMB similar to the procedure described above for the non-reduced samples. [Example]

[0161] 6. Working Example 6.1 Example 1 To analyze the ability of Leishmania tarentolae to assemble chromosomal integration constructs from multiple DNA fragments by homologous recombination, parallel transfections of the same construct (for expressing a monoclonal antibody (rituximab)) were attempted with one- and two-fragment versions.

[0162] The single-fragment version (pLMTB5026) contains the coding sequences for the light chain, heavy chain, and selectable marker (nourseothricin (ntc)), flanked by and interposed intergenic regions. These intergenic regions are used as spacers (intergenic regions (IR)) in the construction of the synthetic polycistron because they are central elements ensuring proper splicing of pre-mRNA in natural polycistronic gene clusters in Leishmania and are thought to affect gene expression by regulating transcript stability. Furthermore, to facilitate genome integration of the construct, the ends of the DNA fragment contain regions (600-1000 bp) homologous to the L. tarentolae rDNA locus (ssu) (see Figure 34 of International Publication No. WO 2019 / 002512 A2, which is incorporated by reference in its entirety).

[0163] The two-fragment version contains the same genetic elements, but these elements are distributed across two DNA fragments. Fragment P1 (pLMTB5024) contains the coding sequences for the light and heavy chains, as well as an intergenic region located upstream of these CDSs. The 5' end of this fragment contains a homologous recombination site for integration into the ssu locus. The last 250 bp of the heavy chain CDS (the 3' end of the P1 construct) share a sequence identical to the first 250 bp of the second fragment (P2; pLMTB5025), allowing homologous recombination between these two fragments via these identical sequences. In addition, P2 contains an intergenic region (CamIR) located downstream of the heavy chain, a selectable marker (ntc), another intergenic region (3'UTR = dhfr-ts) located downstream of ntc, and a 3' homologous recombination site for integration into the ssu locus (Figure 1A).

[0164] These different constructs (either pLMTB5026 linearized with SwaI or pLMTB5024 + pLMTB5025 linearized with SwaI) were transfected into L. tarentolae (St10569) (Biorad system). Multiple viable clones were obtained for both versions, and Western blot analysis of clones obtained with the two-fragment version also showed significant monoclonal antibody expression, detected with light- or heavy-chain-specific antibodies (Figure 1B). This demonstrates the feasibility of generating expression constructs from several DNA fragments.

[0165] 6.2 Example 2 To generate cell lines expressing four different glycosyltransferases for the conversion of endogenous Man3 to G2 N-glycans (two functionally redundant enzymes (SfGnt1 and drMGAT1) responsible for the initial glycosylation step, and rnMGAT2 and hsB4GalT1 for further elongation to obtain the N-glycan "G2") (International Publication No. WO 2019 / 002512 A2, which is incorporated herein by reference in its entirety), we transfected wild-type L. tarentolae (St10569) with expression constructs formed by homologous recombination of 10 DNA fragments. These DNA fragments were designed similarly to previous transfected constructs, using intergenic regions to intersperse coding sequences in the resulting synthetic polycistron assembly and a Pol I promoter region derived from a well-established ribosomal DNA locus to support high-level expression of the reverse integration construct. Typically, in Leishmania, most protein-coding genes are transcribed by Pol II, making it difficult to identify promoter regions specific to such genes, and this is also the case for the aquaporin locus. Homologous recombination between the fragments and between the fragments and the genomic aquaporin locus (AQP) was enabled by 200-bp and 500-bp homologous regions, respectively (Figure 2, top). Furthermore, these overlaps were designed in a way that allowed modular exchange of individual enzymes or intergenic regions by combining linear fragments from different donor plasmids.

[0166] To improve the transfection efficiency of multi-fragment homologous recombination, a new transfection system (Nucleofector) was tested alongside an older system (BioRad). Linear DNA fragments derived from the plasmids (pLMTB6855, pLMTB6952, pLMTB6958, pLMTB6807, pLMTB6848, pLMTB6852, pLMTB6811, pLMTB6860, pLMTB6906, and pLMTB6861) were transfected into wild-type L. tarentolae (St10569) by either transfection method 1 using the Biorad system or transfection method 2 using the Nucleofector system. Multiple viable clones were obtained for both methods, suggesting successful recombination of the split selectable marker. The engineered N-glycans of the resulting clones were analyzed by RF-MS at the whole-cell protein level (an example for St15257 is shown in Figure 2), demonstrating successful glycosylation modification up to G2 (16%). This suggests that expression constructs covering at least three of the enzymes were assembled by L. tarentolae. This demonstrates the general feasibility of incorporating multi-fragment assemblies into L. tarentolae for glycosylation modification. Both transfection methods yielded clones with similar properties, demonstrating the feasibility of transfection regardless of the transfection method applied. Meanwhile, the growth rate of clones obtained by Nucleofector transfection was slightly faster than that obtained from the BioRad system, suggesting good cell viability after transfection and therefore potentially good transfection efficiency.

[0167] 6.3 Example 3 To achieve conversion of endogenous Man3 to G2 N-glycans in strains previously transfected with the rituximab expression construct, a second expression construct formed by homologous recombination of 10 DNA fragments was transfected into St12427. This construct encodes the expression of four different glycosyltransferases: two functionally redundant enzymes (SfGnt1 and drMGAT1) to add the initial GlcNAc, and rnMGAT2 and hsB4GalT1 to achieve further elongation into G2.

[0168] DNA fragments from the plasmids (pLMTB6950, pLMTB6956, pLMTB6808, pLMTB6849, pLMTB6852, pLMTB6811, pLMTB6816, pLMTB6873, pLMTB6855, and pLMTB6861) were transfected into L. tarentolae St12427 by transfection method 2 using the Nucleofector system. Multiple viable clones were obtained, suggesting successful recombination of the split selectable marker.

[0169] The resulting clones were analyzed by RF-MS at the whole-cell protein level, demonstrating that some strains (e.g., St15368) had glycosylation modifications up to G2 (4%). This suggests that expression constructs covering at least three of the enzymes were assembled by L. tarentolae. This confirms the general feasibility of integrating multi-fragment assemblies into L. tarentolae for glycosylation modifications. In contrast, other clones (e.g., St15448) only progressed up to G0-N, suggesting incomplete integration of the construct (Figure 3A).

[0170] To assess the genetic composition of strain St15448, gDNA was prepared (Macherey & Nagel NucleoBond® CB100) and subjected to PacBio long-read genome sequencing (v2.1 chemistry, library preparation according to the manufacturer's instructions) in two PacBio SMRT cells. Several of the long subreads indicated incomplete integration due to the integration of constructs containing only the coding sequences for glycosyltransferase drMGAT1 and glycosyltransferase SfGnt1 (both of which catalyze the addition of the first GlcNAc to Man3). Therefore, the resulting sequencing data are consistent with the observed N-glycan profile phenotype. These data further support the idea that while the 3' end of the construct integrated correctly into the AQP locus on chromosome 31, instead of the 5' end integrating into AQP, the integration was incomplete due to recombination of the intergenic region Pfr IR (approximately 2 kb of native L. tarentolae sequence) with the endogenous Pfr expression locus on chromosome 29. This created a chromosomal crossover (Figure 3B). Additionally, native chromosomes 29 and 31 were also detected, most likely in diploid form (Figure 3C shows a schematic representation of the data).

[0171] These sequencing data indicate that erroneous homologous recombination occurred preferentially at the intended integration locus, which may have been facilitated by the fact that the Pfr intergenic region is long (approximately 2 kb) and 100% identical to the native locus, and that the AQP locus is very close to the chromosomal telomere.

[0172] Another example demonstrated with a construct supports the hypothesis that homologous stretches between fragments should be avoided. In this example, hsB4GalT1-Strep, hsMAGT1-3xHA, and rnMGAT2-3xHA were transfected into the wild-type background of L. tarentolae (St10569 + pLMTB6946, pLMTB6951, pLMTB8080, pLMTB8081, pLMTB8082, pLMTB8083, pLMTB8085, pLMTB6924). In the resulting strain St16834, hsMGAT1 activity was nearly undetectable, and MGAT2 activity was completely absent (M3 78%, G0-N 11%, and G1-N 11%). Long-read sequencing using Nanopore technology revealed that the two fragments carrying rnMGAT2-3xHA were not integrated into the genomic locus because the two adjacent glycosyltransferases both carried the 3xHA tag and the nucleotide sequences of the tag regions were 100% identical, demonstrating that a 93-bp interval was sufficient for homologous recombination to occur (Fig. 4).

[0173] 6.4 Example 4 Previous examples suggest that the use of homologous sequences between different integration fragments and between such fragments and the L. tarentolae genome can lead to undesired homologous recombination. On the one hand, this indicates the need to use codon-diversified variants or homologs of glycosyltransferases when increasing the number of genes used for a specific N-glycan conversion step. On the other hand, this finding also prohibits the repeated use of previously successfully tested intergenic regions. Because data on the precise signals for splicing and mRNA stability in L. tarentolae are unavailable, designing synthetic intergenic regions is currently not feasible. To overcome this limitation, we designed a new set of DNA fragments using intergenic regions from other Leishmania species, as well as various codon usage variants and different homologs of different glycosyltransferases.

[0174] By searching for homologs of L. major genes that have been shown to have high relative transcript levels (Rastrojo, et al. (2013) BMC Genomics 14, p. 223), we identified genes from L. mexicana, L. donovani, and L. infantum that are likely to be highly expressed. The 3' untranslated regions (UTRs) of these genes are likely to support high protein expression levels (Murray, et al. (2007) Molecular and Biochemical Parasitology 153(2), pp. 125-132). To minimize the possibility of unwanted homologous recombination, sequences with greater than 80% identity to each other (using cd-hit (Li, et al. (2006) Bioinformatics 22(13), pp. 1658-1659)) and intervals longer than 30 bp identical to the L. tarentolae genome (using blastn (Camacho, et al. (2009) BMC Bioinformatics 10, p. 421)) were excluded.

[0175] Protein sequences were back-translated into nucleotide sequences using a custom Python script that probabilistically selected codons based on codon usage in L. tarentolae while excluding low-frequency codons (frequency <10%). Codon usage was calculated using cusp (Rice, et al. (2000) Trends in Genetics: TIG 16(6), pp. 276-277) for all annotated L. tarentolae nucleotide coding sequences.

[0176] As with previous multi-fragment homologous recombination constructs, we again included a 200-bp overlap between the fragments and a 500-bp homology region to the predicted integration site to allow assembly of the fragments in Leishmania. New integration loci were designed and used in both "tandem integration" strategies (Figure 5, bottom). In this strategy, the new construct is integrated between the coding sequence and 5' UTR of a highly expressed or multicopy gene (e.g., alpha-tubulin (aTub)). In this case, no additional promoter region (PolI) is included in the construct, so PolII-mediated transcription of the first coding sequence of the integration construct is controlled by the endogenous IR of the target locus. Consequently, the integration construct must terminate at its 3' end in an intergenic region, which provides spacing between the final CDS of the construct and the endogenous gene of the target locus. Alternatively, the new loci are used in a "disruptive integration" strategy (Figure 5, top). In this method, the CDS of the target gene is replaced with an integration construct. To take advantage of the high transcription efficiency of RNA PolI in Leishmania, this integration method can be combined with the use of PolI promoter regions from L. tarentolae and reverse integration. Reverse integration avoids unbalanced transcription of neighboring genes that are normally transcribed by PolII (Figure 5).

[0177] To test the ability of IRs from different species to support glycosyltransferase expression, a series of four different transfections was performed using the Nucleofector method. Here, the glycosyltransferase, selectable marker, and integration locus (the GP63 locus is targeted in the example shown) were kept constant, and only the intergenic region was varied. To determine whether compatibility was limited to specific species (L. major, L. donovani, L. infantum, L. mexicana), four different IRs from the same species were combined in each transfection construct. For St17212, these fragments were derived from pLMTB8234, pLMTB8235, pLMTB8250, pLMTB8295, pLMTB8297, pLMTB8301, pLMTB8302, pLMTB8303, and pLMTB6933. For St17311, these fragments were derived from pLMTB8234, pLMTB8235, pLMTB8250, pLMTB8306, pLMTB8307, pLMTB8310, pLMTB8311, pLMTB8312, and pLMTB6933. For St17176, these fragments were derived from pLMTB8250, pLMTB8334, pLMTB8234, pLMTB8335, pLMTB8235, pLMTB8336, pLMTB8328, pLMTB8330, and pLMTB6933. For St17180, these fragments were obtained from pLMTB8250, pLMTB8322, pLMTB8234, pLMTB8323, pLMTB8235, pLMTB8324, pLMTB8316, pLMTB8318, and pLMTB6933.

[0178] All transfections yielded viable clones that grew in 50 ml shake flask cultures. These clones were subjected to N-glycan analysis of the surface protein fractions of L. tarentolae (St17212 = LmIR, St17311 = LdIR, St17176 = LiIR, St17180 = LmxIR; Figure 6A). These N-glycan profiles demonstrated that all four transfections were largely successful. This was determined by the fact that the variants containing the L. major-, L. donovani-, and L. mexicana-derived IRs progressed up to G2, whereas the variant containing the L. infantum-derived IR progressed at least to G1-N (Figure 6A). This demonstrates that intergenic regions from all four Leishmania species can be used to support the expression of recombinant glycosyltransferases in L. tarentolae, enabling complete customization of host cell function. The detectable activity of most GTs suggests that the majority of IRs used were functional, and only a few IRs, namely those supporting MGAT2 expression in St17176, were inefficient. Based on the available data, it is not possible to clearly distinguish the contributions of the 5'UTR and 3'UTR, and therefore it is not possible to unambiguously identify "non-functional" IRs. Furthermore, heterologous coding sequences may contribute to sequence-mediated mRNA stability.

[0179] On the other hand, the observed N-glycan profiles varied greatly among the analyzed strains, further supporting the hypothesis that intergenic regions do indeed affect the expression levels of their neighboring genes.

[0180] To verify the correctness of integration, Nanopore sequencing was performed on St17311, St17212, and St17180, and the correct integration of all test constructs was confirmed (Fig. 6B).

[0181] Next, we evaluated whether combining multiple such integrations within a single strain could improve glycosylation activity and enhance uniformity of N-glycan conversion. To this end, we created St17238 by transfecting linearized DNA fragments from plasmids (pLMTB8253, pLMTB8313, pLMTB8314, pLMTB8236, pLMTB8315, pLMTB8255, pLMTB8259, pLMTB6940, and pLMTB8379) to target the alpha-tubulin locus of the adalimumab-expressing strain (St15449). Next, in the second transfection, a nine-fragment construct from plasmids (pLMTB8389, pLMTB8301, pLMTB8234, pLMTB8302, pLMTB8235, pLMTB8303, pLMTB8295, pLMTB8297, and pLMTB8392) was integrated into the pfr locus to generate St17294. Finally, DNA fragments from plasmids (pLMTB8247, pLMTB8285, pLMTB8237, pLMTB8286, pLMTB8238, pLMTB8287, pLMTB8383, pLMTB8282, and pLMTB6936) were transfected into St17294 to integrate the third GT expression construct into the GP63 locus. Comparison of the resulting St17826 strain with its predecessor strains by surface protein N-glycan analysis showed that the stepwise increase in GT activity resulted in a nearly uniform G2 N-glycan content (88%) in the final strain (Figure 7). This confirms the utility of this multi-fragment integration method for achieving the integration of multiple enzyme copies into the Leishmania genome and supports the feasibility of increasing glycosyltransferase copy number by using codon-diversifying enzymes and homologs from different species (here: hs, Homo sapiens; rn, Rattus norvegicus; dr, Danio rerio; gj, Gekko japonicus; ag, Anopheles gambiae).

[0182] In summary, heterologous intergenic regions from other Leishmania species were used to successfully engineer host cells to allow multiple homologous recombination events to occur correctly in Leishmania tarentolae, and these heterologous intergenic regions containing regulatory elements were sufficient to direct splicing and expression of heterologous coding sequences.

[0183] 6.5 Example 5 Having successfully used non-identical heterologous sequences to successfully generate multiple recombination events with up to 10 DNA fragments, we tested generating even larger chromosomal integration clusters by using genetic elements derived from 13 donor fragments. Importantly, we subsequently created a more engineered strain by transfecting the existing galactosylation-competent strain St17311 (described in Example 4 and Figure 6A) with an expression construct conferring the sialic acid production capability required for N-glycan sialylation engineering (International Publication No. WO2019 / 002512A2, which is incorporated herein by reference in its entirety). DNA elements were used to target the alpha-tubulin locus using a tandem insertion strategy. NeuC 3×Myc , IrLiH, CgNal, IrLiI, NeuB 3×HA , IrLmR, 3×HA mmST6, IrLiKNeuA 3×HA , IrLiL, hsCST 3×mycThe desired expression cassette, including IrLiM, SM(pac), and the 3'UTR downstream of SM, was inserted into host cells by transfecting 13 donor fragments excised from pLMTB8443, pLMTB8528, pLMTB8448, pLMTB8529, pLMTB8509, pLMTB8507, pLMTB8505, pLMTB8531, pLMTB8449, pLMTB8532, pLMTB8517, pLMTB8520, and pLMTB6939 into St17311 (Figure 8A). The resulting St17527 strain was further analyzed for 1) its phenotype by labeling the produced Neu5Ac and CMP-Neu5Ac with DMB (Figure 8B), and 2) its engineered N-glycans (Figure 8C). Based on calculations based on a standard curve (not shown), the Neu5Ac production concentration of St17527 was 0.49 nmol / OD and the CMP-Neu5Ac production concentration was 0.17 nmol / OD. Furthermore, a significant amount of sialylated N-glycans was observed in the protein-bound N-glycans, with a total of 11.6% being sialylated glycoforms (Figure 8C). These results demonstrate that the glycosylation pathway is fully functional, indicating that the genetic information was fully inserted into the L. tarentolae host cells. This was finally confirmed by PacBio sequencing, clearly demonstrating that fully functional customized L. tarentolae host cells were successfully generated from the applied genetic system.

[0184] 6.6 Example 6 As previously shown in Example 3, integration into the natural expression site of the paraflagellar rod protein 1D (Pfr) was undesired, but its occurrence appeared to support high expression of the integrated construct. Therefore, we assessed whether intentional targeting of this locus in a non-disruptive "in-tandem" integration manner would also result in high-level expression (compared to Example 4) and how comparable this was to expression from the L. tarentolaer DNA locus ("ssu"). Additionally, we tested a different type of integration into the rDNA locus. In this integration, the 5' integration site was shifted 141 bp toward the transcription start site of the rDNA locus, to the start of the 18S (ssu) coding sequence. For integration into this site ("Ssu-PolI"), the protein expression construct was equipped with an artificial splice leader acceptor site to ensure correct processing (Figure 9A). The 3' integration site remained the same as in the "ssu" locus, thus also disrupting one of the ssu expression regions.

[0185] pLMTB8389, pLMTB8301, pLMTB8234, pLMTB8629, pLMTB8238, pLMTB8287, pLMTB8383, pLMTB8282, pLMTB8822 (for St18332), pLMTB9299, pLMTB8301, pLMTB8234, pLMTB8629, pLMTB8238, pLMTB8287, pLMTB8383, pLMTB8384, pLMTB8994 (for St18621), or pLMTB8223, pLMTB8381, pLMTB8301, pLMTB823 Glycosylation-engineered constructs encoding three MGAT1 orthologs (drMGAT1, gjMGAT1, and agMGAT1) and one MGAT2 ortholog (drMGAT2) for converting N-glycans to G0 glycoforms were transfected into three different loci of a wild-type L. tarentolae strain by transfection with either pLMTB8629, pLMTB8238, pLMTB8287, pLMTB8383, pLMTB8281, or pLMTB9304. The integration efficiencies were compared by comparing the profiles of N-glycans released from Leishmania surface glycoproteins. All three integrations resulted in high levels of G0 conversion. Integration into the two ssu locus variants achieved an indistinguishable G0 rate of 99%, while integration into the novel "Pfr" locus resulted in a G0 N-glycan rate of 92% (Figure 9B). No significant differences were observed in the viability, proliferation, or productivity of the resulting cell lines. Thus, the "Pfr" locus is a novel, highly expressed locus in Leishmania.

[0186] To further evaluate whether differences exist between the two integration variants ("Ssu" vs. "Ssu-PolI" (see Figure 9A)) at the ssu locus, another G0 glycosylation-engineered construct consisting of two functional homologs of MGAT1 (sfGNT1, drMGAT1B) and two codon usage variants of rat MGAT2 was integrated into each locus and combined with the target protein expression construct. Because the Fc N-glycans of this highly expressed monoclonal antibody are more sterically restricted, the conversion of this Fc N-glycan provides a more stringent measure of glycosylation efficiency.

[0187] Linearized fragments from the plasmids (pLMTB9301, pLMTB9070, pLMTB8568, pLMTB9072, pLMTB9080, pLMTB9082, pLMTB9083, pLMTB8461, and pLMTB8994) were transfected into St18344 to integrate them into the "Ssu" locus, and the resulting strain St18625 was then transfected with adalimumab expression constructs (pLMTB6737, pLMTB8698, pLMTB7084, pLMTB6681, pLMTB6683) to obtain strain St18703. For the generation of strain St19042, linearized fragments from plasmids (pLMTB8223, pLMTB8564, pLMTB9070, pLMTB8568, pLMTB9072, pLMTB9080, pLMTB9082, pLMTB9083, pLMTB8461, and pLMTB8994) were transfected into the adalimumab-expressing strain St18607 to integrate the G0 construct into the “Ssu-PolI” locus.

[0188] Comparison of the Fc N-glycan profiles of adalimumab purified from these two strains clearly demonstrated differences between the integration variants, supporting the superiority of the "Ssu-PolI" integration variant (the "Ssu-PolI" integration variant achieved a G0 rate of 87%, while the G0 rate obtained in St18703 was only 68%) (Figure 9C). Therefore, to achieve high-level conversion, the "Ssu-PolI" locus is the most suitable integration locus, but balanced high-level expression can also be achieved by targeting the "Pfr" locus or by using alternative "Ssu" integration variants.

[0189] 6.7 Example 7 To further explore opportunities to expand the multiple homologous recombination method, we attempted to transfect genetic elements assembled from 25 different donor fragments. This genetic module combined expression constructs for enzymes in the sialic acid biosynthesis pathway (NeuC3xMyc, 3xflagcgNal, NeuB3xHA, NeuA3xHA, and three Spinv-Δ88ST6 codon usage variants) with glycosyltransferases. To optimize glycosylation up to G2, we combined three copies of MGAT1, three copies of MGAT2, and two copies of hsB4GalT. This combination was achieved by using codon usage variants for hsB4GalT1 and orthologs from different organisms for MGAT1 and MGAT2. Furthermore, this construct utilized 15 different intergenic regions from the four previously described Leishmania species to avoid repeated use of the same sequence. Finally, the construct contained a selectable marker (pac), a 3'UTR, and flanking sequences for tandem homologous integration into the Pfr locus. Notably, in this case, the selectable marker was not placed at the end of the construct but between the glycosylation modification clusters.

[0190] For integration of this construct, plasmids (pLMTB8389, pLMTB8310, pLMTB8234, pLMTB8311, pLMTB8235, pLMTB8312, pLMTB8254, pLMTB9220, pLMTB8528, pLMTB8448, pLMTB8529, pLMTB8509, pLMTB9131, pLMTB9132, pLMTB9133, pLMTB9134, pLMTB9135, pLMTB9136, pLMTB9137, pLMTB9138, pLMTB9139 ... Twenty-five donor fragments excised from pLMTB8449, pLMTB9339, pLMTB9340, pLMTB8333, pLMTB8636, pLMTB8313, pLMTB8236, pLMTB8314, pLMTB8379, pLMTB8315, and pLMTB9320) were transfected into wild-type L. tarentolae (St18344) ( Figure 10A ).

[0191] The phenotype of the resulting strain, St18700, was analyzed by N-glycan profiling of its surface glycoproteins. This strain demonstrated a highly efficient N-glycan conversion capacity, up to G2S2, with 90% galactosylated N-glycan species and a total of 43% sialylated N-glycans (Fig. 10A). This suggests a fully functional glycosylation pathway, as previously, achieving surface glycan galactosylation rates approaching 90% was only achievable by combining two galactosyltransferase-containing modules at different expression loci (compare Fig. 7). Long-read sequencing of the resulting cell line, St19384 (see below), confirmed the complete and correct integration of the 30-kbp construct assembled from 25 individual fragments in L. tarentolae. This further demonstrates the high potential of the novel method for recombinant engineering of Leishmania cells of the present invention, which involves homologous recombination of multiple DNA fragments.

[0192] The resulting strain was then further modified by incorporating two additional glycosylation modification constructs to improve G2S2 conversion. First, linearized inserts from plasmids (pLMTB8391, pLMTB8285, pLMTB8237, pLMTB8286, pLMTB8238, pLMTB8287, pLMTB8383, pLMTB8281, and pLMTB8821) were transfected into St18700. These linearized inserts constituted another glycosylation modification module containing different hsB4GalT1 codon usage variants, rnMGAT2 codon usage variants, and two additional MGAT1 orthologs from different organisms. This modification significantly increased the G2 content of the surface N-glycan profile of the resulting St19084 strain (Figure 10B).

[0193] To improve sialylation of these N-glycan species, St19084 was transfected with an additional glycosylation modification module containing sfGNT1 to enhance the conversion of Man3 to highly modified N-glycan variants, a functional MGAT1 homolog, and additional MGAT1 codon usage variants from zebrafish and MGAT2 codon usage variants from rat. Furthermore, this module also contained another orthologue of sialyltransferase ST6 to improve sialic acid activation and transfer to protein acceptors. strepThe St19384 strain was also transfected with linearized fragments from plasmids (pLMTB8223, pLMTB8564, pLMTB8567, pLMTB8568, pLMTB8823, pLMTB8599, pLMTB9486, pLMTB8488, pLMTB8447, pLMTB8490, and pLMTB9205). In this case, two plasmids (8223 and 9205) required digestion with alternative restriction enzymes (HindIII + SmiI or BglII + SmiI) to create the desired overhangs for homologous recombination. This transfection resulted in a high sialylation rate of N-glycans released from surface glycoproteins, with a G2S2 rate of 74% (Figure 10B).

[0194] To confirm whether all of the glycosylation modification modules described above were correctly integrated into this highly modified strain, high-molecular-weight gDNA was prepared from St19384 and subjected to Nanopore sequencing, which confirmed that the three glycosylation modification modules were correctly integrated into the target loci.

[0195] This example conclusively demonstrates the high potential of the genetic modification of L. tarentolae using the techniques described herein to generate multiple homologous recombination events in each subsequent series of manipulations. A total of 45 linearized fragments were transfected into cells to establish three different glycosylation modification modules consisting of seven orthologs or functional homologs of MGAT1, five orthologs or codon usage variants of MGAT2, three codon usage variants of hsB4GalT1, four codon usage variants or orthologs of ST6, and six enzymes of the sialic acid biosynthetic pathway. In this way, by avoiding the repetition of identical sequences in the coding as well as non-coding regions of the constructs, as detailed in the previous examples, it is possible to extensively modify Leishmania cells without undesired recombination.

[0196] Confirmation of the reproducibility of extensive strain engineering such as those described above was obtained with strains St20157, St20208, and St20224, which each contain three glycosylation-engineered constructs as well as an O-glycosylation knockout (see the co-filed international application entitled "Glycoengineering Using Leishmania Cells") and were derived from the common parent strain St19084 (Figure 10C).

[0197] 6.8 Example 8 Assembly of a hybrid prokaryotic gene cluster on an Escherichia coli cosmid in Leishmania tarentolae Ten foreign genes are required for recombinant expression of Streptococcus pneumoniae serotype 1 capsular polysaccharide as lipid-linked glycans (LLO) in E. coli. Seven genes are present as a cluster in S. pneumoniae, and three genes are present elsewhere in the genome. These three genes were chosen from Plesiomonas shigelloides O17 (a closely related species to E. coli) because their orthologs are widely present in prokaryotes. This choice was made because other studies have shown that these genes function well when recombinantly expressed in E. coli.

[0198] The goal of this experiment was to obtain functional hybrid clusters cloned into the E. coli-compatible cosmid pLAFR1 (Vanbleu, E. et al. (2004) DNA Seq 15(3):225-227) by exploiting the ability of the L. tarentolae recombination machinery to assemble DNA fragments that share homology between their ends. pLAFR1 contains tetracycline resistance for its selection and a broad-range origin of replication for Enterobacteriaceae. pGVXN775 is a derivative of pLAFR1 that incorporates a multiple cloning site, the constitutive promoter J23114 (Anderson collection), and a transcription terminator. Linearization of pGVXN775 with AsiSI and XhoI allowed for the insertion of DNA fragments between the constitutive promoter and the terminator.

[0199] Eleven fragments were designed as shown in Figure 11A. Synthesis of these fragments was performed by GENEWIZ Germany GmbH. The following aspects were taken into consideration in the design: a) fragment length should not exceed 2000 bp to improve synthesis rate, and b) overlap between fragments and between fragments and vector should be 200 bp to optimize homologous recombination efficiency.

[0200] The final construct was designed to contain a selectable marker (compatible with L. tarentolae) to be inserted 3' into the gene cluster along with the necessary 5' and 3' regulatory elements. The selectable marker gene, streptothricin acetyltransferase (sat) (conferring resistance to nourseothricin (NTC)), is split into two fragments so that it is only intact upon recombination. The selectable marker cassette is flanked by BsiWI restriction enzyme recognition sites for its excision.

[0201] Two recombination sets were performed. In one set (designated the "pLAFR_Sp1" set), the 10 genes required for the biosynthetic pathway and the selection marker cassette were split into nine fragments and recombined into pGVXN775. The total insert size was 14,789 bp. The product was capable of transforming E. coli into an S. pneumoniae serotype 1 LLO-producing strain. The second set (designated the "pLAFR_SM" set) was a control strategy in which the selection marker cassette was split into two fragments and recombined into pGVXN775. The total insert size was 2,956 bp.

[0202] Three different transfections were performed in Leishmania tarentolae St10569. Table 2 shows a summary of these transfections. For all transfections, the Biorad transfection method was followed. In transfection #1, cells were co-transfected with AsiSI-XhoI linearized pGVXN775 and the nine fragments required for "pLAFR_Sp1". Transfection #2 differs from transfection #1 in that the target vector was not linearized. In transfection #3, non-linearized pGVXN775 was co-transfected with pGVXN775. 5 is co-transfected with the two fragments required for the "pLAFR_SM" set.

[0203] Growth cultures were analyzed by colony PCR using DreamTaq DNA polymerase (Thermo Fisher Scientific) according to the manufacturer's instructions. PCR A used oligonucleotides o4949 and o4978 and served as a positive control for lysis. PCR B used oligonucleotides o229 and o6775 to amplify the intersection between pGVXN775 and the 5' portion of the inserted "pLAFR_Sp1" set. PCR C used oligonucleotides o228 and o6045 to amplify the intersection between the 3' portion of the inserted "pLAFR_Sp1" set and pGVXN775. PCR D used oligonucleotides o6517 and o6521 to amplify an internal sequence of the "pLAFR_Sp1" set. PCR E used oligonucleotides o5976 and o6776 to amplify the intersection between the 3' portion of the inserted "pLAFR_SM" set and pGVXN775. PCR A, PCR B, PCR C, and PCR D were applied to cells obtained from transfections #1 and #2, and PCR A and PCR E were applied to cells obtained from transfection #3. A polyclonal was defined as positive if all applied PCRs gave the expected product band. Table 2 reports the number of positive polyclonals per transfection.

[0204] DNA was isolated from eight PCR-positive L. tarentolae polyclones from transfection #1 (polyclone 1.1, polyclone 1.2, polyclone 1.3, polyclone 1.4, polyclone 1.5, polyclone 1.6, polyclone 1.7, and polyclone 1.8), three PCR-positive polyclones from transfection #2 (polyclone 2.1, polyclone 2.2, and polyclone 2.3), and two PCR-positive polyclones from transfection #3 (polyclone 3.1 and polyclone 3.2) using a Macherey-Nagel NucleoSpin plasmid Miniprep kit according to the manufacturer's instructions for isolating low-copy-number E. coli plasmids. The eluted material potentially contained episomal and chromosomal DNA. This DNA was used to transform chemically competent E. coli DH5α by heat shock. Transformed colonies were plated onto LB-agar tetracycline plates. The growing colonies were capable of expressing the tetracycline resistance cassette encoded in pGVXN775. One single colony per clone was plated in liquid LB tetracycline and plasmid DNA was isolated using the Macherey Nagel NucleoSpin plasmid kit according to the manufacturer's instructions.

[0205] E. coli DH5α cells were transformed with all plasmids from the multiple clones obtained from transfections #1 and #2, and the transformed E. coli DH5α cells were evaluated for their ability to express S. pneumoniae serotype 1 polysaccharide as lipid-linked glycans (LLO). Five-mL LB tetracycline cultures were grown overnight at 37°C in shaking culture tubes. A volume equivalent to an OD of 2 was centrifuged, and the pellet was resuspended in Lammli buffer, incubated at 95°C for 10 minutes, cooled, and 2 μL of Tritirachium album proteinase K (Sigma-Aldrich P4850) at 800 units / mL or higher was added. The mixture was incubated at 55°C for 1 hour and then at 70°C for 10 minutes. Ten μL (equivalent to an OD of 0.1) was loaded onto a 4-12% Bis-Tris polyacrylamide gel for SDS page. After electrophoresis, the gel material was transferred to a membrane and probed with an antibody specific for S. pneumoniae serotype 1 polysaccharide. Figure 11B shows Western blots for some strains. As shown in Table 2, transfection #1 showed production of S. pneumoniae type 1 polysaccharide in eight of eight clones, and transfection #2 showed production of S. pneumoniae type 1 polysaccharide in one of three clones. The production of this glycan indicates that assembly had occurred correctly.

[0206] To confirm the correctness of the assembly and to identify non-producers, restriction enzyme digestion analysis was performed using standard restriction enzymes (Thermo Fisher Scientific). Plasmids obtained from polyclone 1.1, polyclone 1.7, polyclone 2.1, and polyclone 2.2 were individually digested with BstBI or BsiWI. The LLO-positive clones tested exhibited the expected restriction enzyme digestion pattern. The non-producer (polyclone 2.2) obtained from transfection #2 also exhibited the correct pattern, whereas non-producer 2.1 exhibited a negative pattern (Figure 11C). Polyclone 3.1 and polyclone 3.2 obtained from transfection #3 were digested with SacI. The expected pattern for the selectable marker cassette insertion was observed (Figure 11D). This expected pattern was inferred from comparison with the same plasmid (pLMTB6412) obtained by conventional cloning.

[0207] Plasmids obtained from polyclone 1.1 and polyclone 2.2 were further examined by primer-walking Sanger sequencing of the entire cosmid. Polyclone 1.1 showed 100% sequence identity with the expected 35,038 bp construct. Polyclone 2.2 showed the correct restriction enzyme digestion pattern but no activity. Sequencing showed 99% identity, with the deletion of GG causing a frameshift in wbzG, thereby inactivating polysaccharide production. Analysis of the inserted selectable marker cassette in the plasmid obtained from polyclone 3.2 and its intersection with pGVXN775 by Sanger sequencing confirmed 100% identity with the expected sequence. The plasmid obtained from polyclone 1.1 was digested with BsiWI to remove the selection marker cassette and religated. The resulting plasmid still possessed S. pneumoniae serotype 1 glycan production activity.

[0208] Cotransfection of the nine fragments and the linearized vector (transfection #1) achieved correct gene assembly in 100% of the analyzed plasmids. Although assembly efficiency appears to be lower with circular vectors, the pLAFR_Sp1 set (transfection #2) yielded 1 phenotype out of 3, and the pLAFR_SM set (transfection #3) yielded 2 phenotypes out of 2, demonstrating that circular vectors remain a viable option when suitable restriction enzyme sites are not available. [Table 3]

[0209] 7. Equivalents The scope of the viruses, nucleic acids, methods, host cells, and compositions disclosed herein is not limited by the specific embodiments described herein. Indeed, various modifications of such 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 be within the scope of the appended claims. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for recombinantly engineering Leishmania cells, said method comprising: (a) introducing two or more DNA fragments into said Leishmania cells; and (b) incubating the Leishmania cells to allow homologous recombination of the DNA fragments; Including, a first DNA fragment of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region, and the 5' homologous region is homologous to a 3' homologous region of a second DNA fragment of the two or more DNA fragments, or the 3' homologous region of the first DNA fragment is homologous to a 5' homologous region of the second DNA fragment; The recombinant engineering method, wherein the nucleotide sequences located outside the homologous region(s) in the first DNA fragment and the second DNA fragment are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or have no homology within each DNA fragment. (Aspect 2) 2. The method of embodiment 1, wherein each of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region, and the 5' homologous region of each of the two or more DNA fragments is homologous to the 3' homologous region of another of the two or more DNA fragments, or the 3' homologous region of each of the two or more DNA fragments is homologous to the 5' homologous region of another of the two or more DNA fragments, and the nucleotide sequences located outside the homologous regions in each DNA fragment are not homologous to each other, are not homologous to sequences in the genome of the Leishmania cell, and / or have no homology within each DNA fragment. (Aspect 3) 3. The method of any one of aspects 1-2, wherein said two or more DNA fragments are suitable for integration into a chromosome of said Leishmania cell, optionally after said two or more DNA fragments have recombined with one another. (Aspect 4) 4. The method of embodiment 3, wherein the two or more DNA fragments are integrated into the chromosome of the Leishmania cell, optionally after the two or more DNA fragments have recombined with one another. (Aspect 5) 5. The method of embodiment 4, wherein the two or more DNA fragments are integrated tandemly into the paraflagellar rod protein (Pfr) gene locus. (Aspect 6) 5. The method of embodiment 4, wherein the two or more DNA fragments are integrated at the start site (Ssu-PolI) of the 18S coding region. (Aspect 7) 3. The method according to any one of aspects 1 to 2, wherein the two or more DNA fragments are not integrated into the chromosome of the Leishmania cell before and / or after recombination with one another. (Aspect 8) The method of embodiment 7, wherein said two or more DNA fragments undergo homologous recombination to generate a circular plasmid. (Aspect 9) Aspects 1 to 8. The method of any one of aspects 1 to 8, wherein the length of the nucleotide sequence located outside the homologous region in the first DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 2000 nucleotides, 5000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, or 20,000 nucleotides, 25,000 nucleotides, 30,000 nucleotides, 35,000 nucleotides, 40,000 nucleotides, 45,000 nucleotides, or 50,000 nucleotides. (Aspect 10) Aspects 1 to 9. The method of any one of aspects 1 to 9, wherein the length of the nucleotide sequence located outside the homologous region in the second DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 2000 nucleotides, 5000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, or 20,000 nucleotides, 25,000 nucleotides, 30,000 nucleotides, 35,000 nucleotides, 40,000 nucleotides, 45,000 nucleotides, or 50,000 nucleotides. (Aspect 11) The method according to any one of aspects 1 to 10, wherein the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 2000 nucleotides, 5000 nucleotides, 10,000 nucleotides, 15,000 nucleotides, or 20,000 nucleotides, 25,000 nucleotides, 30,000 nucleotides, 35,000 nucleotides, 40,000 nucleotides, 45,000 nucleotides, or 50,000 nucleotides. (Aspect 12) The DNA fragments undergo the homologous recombination to generate a nucleotide sequence, and the length of the nucleotide sequence is 50 nucleotides to 100 nucleotides, 100 nucleotides to 500 nucleotides, 500 nucleotides to 1000 nucleotides, 1000 nucleotides to 5000 nucleotides, 5000 nucleotides to 10000 nucleotides, 10000 nucleotides to 15000 nucleotides, 15000 nucleotides to 20000 nucleotides, 20000 nucleotides to 25000 nucleotides, 25000 nucleotides to 30000 nucleotides, 30000 nucleotides, or 12. The method of any one of aspects 1 to 11, wherein the length of the nucleotide is between 35,000 nucleotides, between 35,000 nucleotides and 40,000 nucleotides, between 40,000 nucleotides and 45,000 nucleotides, between 45,000 nucleotides and 50,000 nucleotides, between 50,000 nucleotides and 55,000 nucleotides, between 55,000 nucleotides and 60,000 nucleotides, between 60,000 nucleotides and 65,000 nucleotides, between 65,000 nucleotides and 70,000 nucleotides, between 70,000 nucleotides and 75,000 nucleotides and 80,000 nucleotides. (Aspect 13) 13. The method according to any one of aspects 1 to 12, wherein the length of the 5′ homologous region and / or the 3′ homologous region of the first DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides. (Aspect 14) 14. The method according to any one of aspects 1 to 13, wherein the length of the 5′ homologous region and / or the 3′ homologous region of the second DNA fragment is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides. (Aspect 15) Aspect 15. The method according to any one of Aspects 1 to 14, wherein the length of all of the 5' homologous regions and / or the 3' homologous regions of the two or more DNA fragments is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450 nucleotides, or 500 nucleotides. (Aspect 16) 16. The method according to any one of aspects 1 to 15, wherein the length of the 5' homologous region and / or the 3' homologous region of the first DNA fragment is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides. (Aspect 17) 17. The method according to any one of aspects 1 to 16, wherein the length of the 5′ homologous region and / or the 3′ homologous region of the second DNA fragment is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides. (Aspect 18) Aspect 18. The method according to any one of aspects 1 to 17, wherein the length of all of the 5' homologous regions and / or the 3' homologous regions of the two or more DNA fragments is at most 500 nucleotides, 550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2200 nucleotides, 2400 nucleotides, 2600 nucleotides, 2800 nucleotides, 3000 nucleotides, 3200 nucleotides, 3400 nucleotides, 3600 nucleotides, 3800 nucleotides, 4000 nucleotides, 4200 nucleotides, 4400 nucleotides, 4600 nucleotides, 4800 nucleotides, or 5000 nucleotides. (Aspect 19) 19. The method of any one of aspects 1 to 18, wherein the 5′ homologous region of the first DNA fragment and the 3′ homologous region of the second DNA fragment have at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. (Aspect 20) 19. The method of any one of aspects 1 to 18, wherein the 3′ homologous region of the first DNA fragment and the 5′ homologous region of the second DNA fragment have at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity. (Aspect 21) 21. The method according to any one of aspects 1 to 20, wherein the two or more DNA fragments are introduced by transfection. (Aspect 22) 21. The method according to any one of aspects 1 to 20, wherein the two or more DNA fragments are introduced simultaneously. (Aspect 23) 23. The method of any one of aspects 1 to 22, wherein the number of DNA fragments is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. (Aspect 24) 24. The method according to any one of aspects 1 to 23, wherein the nucleotide sequences located outside the homologous regions in the two or more DNA fragments are selected from the group consisting of an intergenic region (IR), an untranslated region (UTR), and an open reading frame (ORF) encoding a polypeptide. (Aspect 25) 25. The method of embodiment 24, wherein the IR, the UTR, and the ORF have no homologous sequences within themselves and / or to each other. (Aspect 26) Aspect 26. The method according to any one of aspects 1 to 25, wherein the nucleotide sequences located outside the homologous regions in the two or more DNA fragments encode the same polypeptide. (Aspect 27) 27. The method of embodiment 26, wherein the Leishmania cells are capable of expressing multiple copies of the same polypeptide. (Aspect 28) 28. The method according to any one of embodiments 26 and 27, wherein the method increases the expression level of the polypeptide. (Aspect 29) 27. The method of any one of aspects 1 to 26, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence comprising at least 50%, 60%, 70%, 80%, 90%, or 100% of the genetic information encoded by the two or more DNA fragments. (Aspect 30) 30. The method of any one of aspects 1 to 29, wherein the percentage of Leishmania cells undergoing unwanted deletions and / or crossovers over at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. (Aspect 31) 30. The method of any one of aspects 1 to 29, wherein the proportion of Leishmania cells in which unwanted deletions and / or crossovers occur after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions is at most 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. (Aspect 32) 32. The method of any one of aspects 1 to 31, wherein the Leishmania cell is Leishmania tarentolae. (Aspect 33) 33. A Leishmania cell recombinantly engineered using the method of any one of embodiments 1 to 32. (Aspect 34) 34. The Leishmania cell of embodiment 33, wherein said Leishmania cell is recombinantly engineered using said method iteratively. (Aspect 35) 35. The Leishmania cell according to any one of aspects 33 to 34, wherein the Leishmania cell is Leishmania tarentolae. (Aspect 36) 36. A kit comprising one or more containers and instructions for use, wherein said one or more containers comprise a Leishmania cell according to any one of aspects 33 to 35. (Aspect 37) 36. A method for preparing a polypeptide, the method comprising (a) culturing a Leishmania cell according to any one of aspects 33 to 35 under conditions suitable for polypeptide production, and (b) isolating the polypeptide. (Aspect 38) 38. The method of embodiment 37, wherein the method further comprises introducing a nucleotide sequence encoding the polypeptide. (Aspect 39) A polypeptide produced by the method according to any one of embodiments 37 to 38.

[0210] Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties. [Table 4] 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Claims

1. A method for recombinantly manipulating Leishmania cells, said method comprising: (a) introducing two or more DNA fragments into said Leishmania cells; and (b) incubating the Leishmania cells to allow homologous recombination of the DNA fragments; Including, a first DNA fragment of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region; the 5' homologous region has at least 90% sequence identity with the 3' homologous region of a second DNA fragment of the two or more DNA fragments, or the 3' homologous region of the first DNA fragment has at least 90% sequence identity with the 5' homologous region of the second DNA fragment; and the nucleotide sequences located outside the 5' homologous region and / or the 3' homologous region in the first DNA fragment and the second DNA fragment have sequence identity (i) with each other, (ii) with any sequence in the genome of the Leishmania cell, and / or (iii) within each of the DNA fragments, of at most 80% over a region of 20 nucleotides; The recombinant engineering method.

2. each of the two or more DNA fragments comprises a 5' homologous region and / or a 3' homologous region; the 5' homologous region of each of the two or more DNA fragments has at least 90% sequence identity with the 3' homologous region of another one of the two or more DNA fragments, or the 3' homologous region of each of the two or more DNA fragments has at least 90% sequence identity with the 5' homologous region of another one of the two or more DNA fragments; and 2. The method of claim 1, wherein the nucleotide sequences located outside the homologous regions in each DNA fragment have sequence identity (i) with each other, (ii) with any sequence in the genome of the Leishmania cell, and / or (iii) within each DNA fragment, of at most 80% over a region of 20 nucleotides.

3. 3. The method of claim 1 or 2, wherein the two or more DNA fragments are suitable for integration into the chromosome of the Leishmania cell.

4. 4. The method of claim 3, wherein the two or more DNA fragments are integrated into the chromosome of the Leishmania cell.

5. The method described in claim 4, wherein the two or more DNA fragments are integrated into the chromosome of the Leishmania cell after the two or more DNA fragments have undergone recombination with each other.

6. The two or more DNA fragments (i) tandemly integrated into the paraflagellar rod protein (Pfr) gene locus; or (ii) The method according to claim 4 or 5, wherein the DNA is integrated into the start site (Ssu-PolI) of the 18S coding region.

7. 3. The method of claim 1 or 2, wherein the two or more DNA fragments are not integrated into the chromosome of the Leishmania cell.

8. The method described in claim 7, wherein the homologous recombination of the two or more DNA fragments produces a circular plasmid.

9. (i) located outside the homologous region in the first DNA fragment; (ii) located outside the region of homology in the second DNA fragment; and / or (iii) the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is 10 to 50,000 nucleotides; The method according to any one of claims 1 to 8.

10. The method described in claim 9, wherein the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is 50 to 10,000 nucleotides.

11. The method described in claim 9, wherein the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is 150 to 2,500 nucleotides.

12. The method described in claim 9, wherein the length of the nucleotide sequence located outside the homologous region in all of the two or more DNA fragments is 250 to 2000 nucleotides.

13. The method according to any one of claims 1 to 12, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence, and the length of the nucleotide sequence is 100 to 500 nucleotides.

14. A method according to any one of claims 1 to 12, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence, and the length of the nucleotide sequence is 500 to 1000 nucleotides.

15. A method according to any one of claims 1 to 12, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence, and the length of the nucleotide sequence is between 1,000 and 5,000 nucleotides.

16. A method according to any one of claims 1 to 12, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence, and the length of the nucleotide sequence is between 5,000 and 10,000 nucleotides.

17. (i) the first DNA fragment, (ii) of said second DNA fragment, and / or (iii) all of the two or more DNA fragments; The method according to any one of claims 1 to 16, wherein the length of the 5' homologous region and / or the 3' homologous region is at least 90 nucleotides.

18. the 5' homologous region of the first DNA fragment and the 3' homologous region of the second DNA fragment have at least 95% or 100% sequence identity; or 18. The method of any one of claims 1 to 17, wherein the 3' homologous region of the first DNA fragment and the 5' homologous region of the second DNA fragment have at least 95% or 100% sequence identity.

19. The method according to any one of claims 1 to 18, wherein the two or more DNA fragments are introduced by transfection and / or simultaneously.

20. The method according to any one of claims 1 to 19, wherein the number of DNA fragments is at least four.

21. the nucleotide sequences located outside the homologous regions in the two or more DNA fragments are (i) is selected from the group consisting of an intergenic region (IR), an untranslated region (UTR), and an open reading frame (ORF) encoding a polypeptide; and / or (ii) encoding the same polypeptide,

22. The method described in claim 21, wherein the IR, the UTR, and the ORF do not have homologous sequences within themselves and / or do not have homologous sequences with each other.

23. A method described in claim 21 or 22, wherein the Leishmania cells have the ability to express multiple copies of the same polypeptide.

24. A method described in any one of claims 21 to 23, which increases the expression level of the polypeptide.

25. The method according to any one of claims 1 to 24, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence comprising at least 50% of the genetic information encoded by the two or more DNA fragments.

26. A method described in any one of claims 1 to 24, wherein the DNA fragments undergo homologous recombination to produce a nucleotide sequence containing at least 60% of the genetic information encoded by the two or more DNA fragments.

27. A method described in any one of claims 1 to 24, wherein the DNA fragments undergo homologous recombination to produce a nucleotide sequence containing at least 70% of the genetic information encoded by the two or more DNA fragments.

28. A method described in any one of claims 1 to 24, wherein the DNA fragments undergo homologous recombination to produce a nucleotide sequence containing at least 80% of the genetic information encoded by the two or more DNA fragments.

29. A method described in any one of claims 1 to 24, wherein the DNA fragments undergo homologous recombination to produce a nucleotide sequence containing at least 90% of the genetic information encoded by the two or more DNA fragments.

30. A method described in any one of claims 1 to 24, wherein the DNA fragments undergo homologous recombination to generate a nucleotide sequence containing 100% of the genetic information encoded by the two or more DNA fragments.

31. The method of any one of claims 1 to 30, wherein the Leishmania cell is Leishmania tarentolae.

32. A Leishmania cell comprising two or more DNA fragments, wherein the DNA fragments are any one of claims 1, 2, 9-12, 17, 18, 20, and 21-24.

33. The Leishmania cell described in claim 32, wherein the Leishmania cell is Leishmania tarentolae.

34. 34. A kit comprising one or more containers and instructions for use, wherein the one or more containers contain the Leishmania cells of claim 32 or 33.

35. 1. A method for preparing a polypeptide, comprising: (a) culturing the Leishmania cell of claim 32 or 33 under conditions suitable for producing a polypeptide, wherein the nucleotide sequences of the two or more DNA fragments located outside the regions of homology comprise an open reading frame (ORF) encoding the polypeptide; and (b) isolating the polypeptide.

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