Atty. Dkt. No.: 114198-0788 GENETIC ELIMINATION OF AMMONIA FROM CULTURED MAMMALIAN CELLS CROSS-REFERENCE TO RELATED APPLICATION This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63/621,072, filed January 15, 2024, the contents of which are incorporated herein by reference in their entireties. BACKGROUND OF THE DISCLOSURE Mammalian cell culture is a key source of valuable biotechnology products, but the growth of the cells is hampered by the production of ammonia. This disclosure provides methodologies for reducing or eliminating ammonia output in mammalian cells. SUMMARY OF THE DISCLOSURE Applicant’s disclosure provides methods and compositions for the knockout of both glutaminase isozymes (Gls and Gls2) and asparaginase (Aspg) in eukaryotic cells, (e.g., Chinese hamster ovary (CHO) cells) that almost entirely eliminates ammonia production before stationary phase and substantially decreases it over the course of culture, even when grown in glutamine-containing medium. This genotype and phenotype can be ‘layered’ on top of previously described engineering strategy to eliminate lactate production, resulting in cells with reduced generation of both primary mammalian cell culture inhibitory byproducts. Thus, in one aspect, Applicant provides an engineered eukaryotic cell wherein expression of endogenous genes encoding glutaminase (Gls), glutaminase 2 (Gls2) and asparaginase (Aspg) are substantially reduced or eliminated, and in one aspect reduced to substantially eliminate ammonia production by the engineered eukaryotic cell. In another aspect, the ammonia production is substantially eliminated before the stationary phase, optionally wherein the cell is grown in glutamine-containing medium. In one aspect, engineered cell is prepared by CRISPR/Cas9 knockout of the endogenous genes encoding glutaminase (Gls), glutaminase 2 (Gls2) and asparaginase (Aspg). 1 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 Also provided is an engineered eukaryotic cell wherein expression of endogenous genes encoding glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4) are substantially eliminated. In one aspect, the expression of glutaminase (Gls), glutaminase 2 (Gls2), and asparaginase (Aspg) are reduced to substantially eliminate ammonia production by the engineered eukaryotic cell. In another aspect, the ammonia production is substantially eliminated before the stationary phase, optionally wherein the cell is grown in glutamine- containing medium. In one aspect, the cell is prepared by CRISPR/Cas9 knockout of the endogenous genes expressing glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4). Also provided is a population of the cells, wherein the population is substantially homogenous for the engineered eukaryotic cells. In producing the engineered cells, genomic DNA encoding glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), and optionally lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4) are substantially eliminated, for example by targeting the endogenous genes for disruption and/or elimination, e.g., using CRISPR/Cas9 and a guide RNA that specifically hybridizes to the endogenous genes. Thus, also provided herein is a method to produce a cell having substantially reduced or eliminated ammonia production comprising substantially reducing or eliminating the production of one or more of an endogenous gene selected from glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4). In another aspect, the expression of glutaminase (Gls), glutaminase 2 (Gls2), and asparaginase (Aspg) are eliminated or substantially reduced using methods disclosed herein, e.g., by CRISPR/Cas9 knockout of an endogenous gene selected from a gene expressing glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), lactate dehydrogenase (Ldha) and/or pyruvate dehydrogense kinase 1-4 (Pdk1-4). Standard methods known by persons skill in the art can be used to determine whether a eukaryotic cell encodes or expresses the endogenous genes described herein, e.g., by measuring the ability of the polypeptide to convert pyruvate into lactate in vitro, in a cell 2 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 extract, or in vivo and/or the amount of ammonia produced by the cell as known in the art or as described herein. Exemplary in vitro cell lysate LDH detection assays include immunoassays (e.g., ELISA, Western Blots) and activity assays are demonstrated in WO 2017/192437A1. In vitro assays for detecting lactate dehydrogenase activity are known in the art and described, e.g., in Baba, et al., Antivir Chem Chemother. 2005; 16(1):33-9; Larson, J Dairy Res. (2005) 72(2):209-16; and Mori, et al., Tohoku J Exp Med. 1995 December; 177(4):315-25. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Growth profile of Gls KO cells in batch (only Gls was targeted). No drastic effect is seen in glutamine containing media. FIG. 2: NH4 concentration on day 6 (prior to death phase) for Gls KO and Ctrl cells from FIG. 1. FIG. 3: Gln concentration on day 6 (prior to death phase) for Gls KO and Ctrl cells from FIG. 1. FIG. 4: Gene expression of Gls, Gls2, and Aspg in CHO-S (reproduced from (Hefzi et al., 2016)). TPM above 1 is considered expressed. FIGS. 5A-5B: Viable cell density and viability of Aspg/Gls/Gls2 knockout clones alongside their parental cell lines in batch culture. FIG. 5A: TKO (light grey) is a clone generated from the parental WT CHO-S cell line (available from ThermoFisher Scientific). FIG. 5B: A6, B11, F2, F6, G9 (light grey in the figure legend) are clones generated from the Intermediate cell line (light grey, diamond), which cannot produce lactate. Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey, upright triangle; B11: light grey square; F2: light grey, upside down triangle; F6: light grey star; and G9: light grey, right facing triangle. All cultures grown in triplicate. FIGS. 6A-6E: Metabolite measurements of knockout cell line (TKO) derived from wildtype CHO-S cells from batch cultures depicted in FIG. 5. Media control=media containing glutamine but no cells. FIG. 6A, NH4 (mM), FIG. 6B, Glutamine (mM), FIG. 6C, Glutamate (mM), FIG. 6D, Glucose (mM), and FIG. 6E, Lactate (mM). Legend: Media 3 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 control, dotted line; Wildtype, Black circle; and TKO, light grey circle. All measurements other than the media control are from triplicate shake flasks. FIGS. 7A-E: Metabolite measurements of knockout cell lines derived from the non- lactogenic Intermediate cell line from batch cultures depicted in FIG. 5. Media control=media containing glutamine but no cells. FIG. 7A, NH4 (mM), FIG. 7B, Glutamine (mM), FIG.7C, Glutamate (mM), FIG. 7D, Glucose (mM), and FIG. 7E, Lactate (mM). Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey, upright triangle; B11: light grey square; F2: light grey, upside down triangle; F6: light grey star; and G9: light grey, right facing triangle. All measurements other than the media control are from triplicate shake flasks. FIG. 8: Viable cell density and viability of Aspg/Gls/Gls2 knockout clones alongside their parental cell lines in fed-batch culture, feeding glutamine to 6 mM on day 3 and Cell Boost 7a/7b at 3%/0.3% culture volume on day 4. Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey, upright triangle; and B11: light grey square. FIGS. 9A-9E: Metabolite measurements of knockout cell lines derived from the Intermediate cell line from fed-batch cultures depicted in FIG. 8. Media control=media containing glutamine but no cells. FIG. 9A, NH4 (mM), FIG. 9B, Glutamine (mM), FIG. 9C, Glutamate (mM), FIG. 9D, Glucose (mM), and FIG. 9E, Lactate (mM). Legend: Media Control: dotted line; Wildtype: black circles; Intermediate: light grey circles; A6: light grey, upright triangle; and B11: light grey square. FIGS. 10A-10B: Viable cell density and viability of cell lines undergoing Gs selection. Cells are selected in media without glutamine with Msx. FIG. 10A, VCD. FIG. 10B, Cell viability. Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey, upright triangle; and B11: light grey square. FIGS. 11A-11B: Viable cell density, viability and titer in shake flask batch culture of protein producing cell lines grown in media without glutamine. FIG. 11A, y axis: VCD (1e6 cells/mL). FIG. 11B, y axis: Titer (mg/mL). Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey upright triangles; and B11: light grey squares. FIGS. 12A-12E: Metabolite concentration measurements of protein producing cell lines grown in media without glutamine in batch culture in shake flasks. FIG. 12A, NH4 4 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 (mM), FIG. 12B, Glutamine (mM), FIG. 12C, Glutamate (mM), FIG.12D, Glucose (mM), and FIG. 12E, Lactate (mM). Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey, upright triangle; and B11: light grey square. FIGS. 13A-13B: Glycosylation profile of protein producing cell lines grown in medium without glutamine in 30mL flask batch culture. Glycoprofiles were analyzed from day 4 (FIG. 13A) and day 6 (FIG. 13B). FIGS. 14A-14B: Viable cell density, viability and titer in 30mL flask fed batch culture of protein producing cell lines grown in media with glutamine. FIG. 14A y axis: VCD (1e6 cells/mL). FIG. 14B, y axis: Titer (mg/mL). Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey upright triangles; and B11: light grey squares. FIG. 15: Ammonia (mM) profile for protein producing lines from FIG. 14. Legend: Wildtype: black circles; Intermediate: light grey circles; A6: light grey upright triangles; and B11: light grey squares. DETAILED DESCRIPTION OF THE DISCLOSURE Definitions As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting or separating or both limiting and separating the subject matter described. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains. The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach 5 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)). As used in the specification and claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology. “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and 6 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 concentration, including range, indicates approximations which may vary by (+) or (–) 15%, 10%, 5%, 3%, 2%, or 1 %. The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. As used herein, an amino acid (aa) or nucleotide (nt) residue position in a sequence of interest “corresponding to” an identified position in a reference sequence refers to that the residue position is aligned to the identified position in a sequence alignment between the sequence of interest and the reference sequence. Various programs are available for performing such sequence alignments, such as Clustal Omega and BLAST. In one aspect, equivalent polynucleotides, proteins and corresponding sequences can be determined using BLAST (accessible at blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed on August 1, 2021). It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, amino acid sequence, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively at least about 96 % homology or identity, or alternatively at least about 97 % homology or identity, or alternatively at least about 98 % homology or identity, or 7 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 alternatively at least about 99 % homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence. In some embodiments, a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage between the two sequences can be calculated. In further embodiments, the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence. In yet further embodiments, the identity percentage is calculated based on the full-length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence. The term “substantially reduced or eliminated” means at least 50%, or alternatively at least 60%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or at least decreased expression or activity of the endogenous gene as compared to wild type expression or the cell prior to genetic modification, i.e., the modification to eliminate or reduce expression. In one aspect the term “substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater or alternatively less than some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% greater or less than some given quantity. The term “substantially homogeneous” means that at least 50%, or alternatively at least 60%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or at least of a molecule or cell is identical or nearly identical for a given quality. In one aspect the term “substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater or alternatively less than some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% greater or less than some given quality or quantity. 8 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 As used herein, the term “substantially or almost entirely eliminates ammonia production” intends a decrease of greater than 60%, or alternatively a decrease of greater than 65%, or alternatively a decrease of greater than 70 %, or alternatively a decrease of greater than 75 %, or alternatively a decrease of greater than 80 %, or alternatively a decrease of greater than 85 %, or alternatively a decrease of greater than 90 %, or alternatively a decrease of greater than 95 %, or alternatively a decrease of greater than 98%, or alternatively a decrease of greater than 99% of ammonia production as compared to production in a wild type cell or the cell prior to genetic modification. In one aspect, measurement ammonia production is determined before the stationary phase and/or over the course of culture, even when grown in glutamine-containing medium. As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals. The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non- human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. The term “culturing” refers to growing cells in a culture medium under conditions that favor expansion and proliferation of the cell. The term “culture medium” or “medium” is recognized in the art and refers generally to any substance or preparation used for the cultivation of living cells. The term “medium”, as used in reference to a cell culture, includes the components of the environment surrounding the cells. Media may be solid, liquid, gaseous or a mixture of phases and materials. Media include liquid growth media as well as liquid media that do not sustain cell growth. Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices. Exemplary gaseous media include the gaseous phase to which cells growing on a petri dish or other solid or semisolid support are exposed. 9 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 The term “medium” also refers to material that is intended for use in a cell culture, even if it has not yet been contacted with cells. In other words, a nutrient rich liquid prepared for culture is a medium. Similarly, a powder mixture that when mixed with water or other liquid becomes suitable for cell culture may be termed a “powdered medium.” A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. The term “express” refers to the production of a gene product. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. A “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated. “Under transcriptional control” is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. “Operatively linked” intends the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, this invention provides promoters operatively linked to the downstream sequences, e.g., suicide gene, VEGF, 165A VEGF, tet activator, etc. The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. A “probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a detectable label or 10 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 a means by which a label can be attached, either before or subsequent to the hybridization reaction. Alternatively, a “probe” can be a biological compound such as a polypeptide, antibody, or fragments thereof that is capable of binding to the target potentially present in a sample of interest. “Detectable labels”, “labels” or “markers” include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein. "Detectable label" or “label” as used herein refers to a molecule or a compound or a group of molecules or a group of compounds used to target of interest. In some cases, the detectable label can be detected directly. In other cases, the detectable label can be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label can be detected by various means and will depend on the nature of the detectable label. Detectable labels can be isotopes, fluorescent moieties, colored substances, and the like. Examples of means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means. Non-limiting examples include fluorescent labels such as Cy5 (see https://broadpharm.com/product-categories/fluorescent- dye/cy5-labeling?gad=1&gclid=EAIaIQobChMI-eKsm4- ogAMVwSvUAR0nIgT5EAAYASAAEgJKtPD_BwE, last access on July 242023), Non-limiting exemplary detectable labels also include a radioactive material, such as a radioisotope, a metal or a metal oxide. Radioisotopes include radionuclides emitting alpha, beta or gamma radiation. In particular embodiments, a radioisotope can be one or more of: 3H, 10B, 18F, 11C, 14C, 13N, 18O, 15O, 32P, P33, 35S, 35Cl, 45Ti, 46Sc, 47Sc, 51Cr, 52Fe,59Fe, .57Co, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 72As 76Br, 77Br, 81mKr, 82Rb, 85Sr, 89Sr, 86Y, 90Y, 95Nb, 94mTc, 99mTc, 97Ru, 103Ru, 105Rh, 109Cd, 111In, 113Sn, 113mIn, 114In, I125, I131, 140La, 141Ce, 149Pm, 153Gd, 157Gd, 153Sm, 161Tb, 166Dy, 166Ho, 169Er, 169Y, 175Yb, 177Lu, 186Re, 188Re, 201Tl, 203Pb, 211At, 212Bi or 225Ac. Additional non-limiting exemplary detectable labels include a metal or a metal oxide. In particular embodiments, a metal or metal oxide is one or more of: gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodynium, 11 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 indium, or technetium. In additional embodiments, a metal oxide includes one or more of: Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Ffe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), or Er(III). Further non-limiting exemplary detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); magnetic and paramagnetic agents (e.g., iron-oxide chelate); nanoparticles; an enzyme (horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase); a prosthetic group (e.g., streptavidin/biotin and avidin/biotin); a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); a luminescent material (e.g., luminol); or a bioluminescent material (e.g., luciferase, luciferin, aequorin). Additional non-limiting examples of tags and/or detectable labels include enzymes (horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (e.g., biotin); receptors (avidin); GST-, T7-, His-, myc-, HA- and FLAG®-tags; electron-dense reagents; energy transfer molecules; paramagnetic labels; fluorophores (Cy5, fluorescein, fluorscamine, rhodamine, phycoerthrin, phycocyanin, allophycocyanin); chromophores; chemi-luminescent (imidazole, luciferase, acridinium, oxalate); and bio-luminescent agents. As used herein, the term “vector” refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a cell, for example by a process of transformation. Vectors may be viral or non-viral. Viral vectors include retroviruses, adenoviruses, herpesvirus, bacculoviruses, modified bacculoviruses, papovirus, or otherwise modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA. 12 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med.5(7):823-827. In aspects where gene transfer is mediated by a lentiviral vector, a vector construct refers to the polynucleotide comprising the lentiviral genome or part thereof, and a therapeutic gene. As used herein, “lentiviral mediated gene transfer” or “lentiviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. A “lentiviral vector” is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg. Lentiviral vectors of this invention are based on or derived from oncoretroviruses (the sub-group of retroviruses containing MLV), and lentiviruses (the sub-group of retroviruses containing HIV). Examples include ASLV, SNV and RSV all of which have been split into packaging and vector components for lentiviral vector particle production systems. The lentiviral vector particle according to the invention may be based on a genetically or otherwise (e.g. by specific choice of packaging cell system) altered version of a particular retrovirus. That the vector particle according to the invention is "based on" a particular retrovirus means that the vector is derived from that particular retrovirus. The genome of the vector 13 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 particle comprises components from that retrovirus as a backbone. The vector particle contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include gag and pol proteins derived from the particular retrovirus. Thus, the majority of the structural components of the vector particle will normally be derived from that retrovirus, although they may have been altered genetically or otherwise so as to provide desired useful properties. However, certain structural components and in particular the env proteins, may originate from a different virus. The vector host range and cell types infected or transduced can be altered by using different env genes in the vector particle production system to give the vector particle a different specificity. The term “promoter” refers to a region of DNA that initiates transcription of a particular gene. The promoter includes the core promoter, which is the minimal portion of the promoter required to properly initiate transcription and can also include regulatory elements such as transcription factor binding sites. The regulatory elements may promote transcription or inhibit transcription. Regulatory elements in the promoter can be binding sites for transcriptional activators or transcriptional repressors. A promoter can be constitutive or inducible. A constitutive promoter refers to one that is always active and/or constantly directs transcription of a gene above a basal level of transcription. Non-limiting examples of such include the phosphoglycerate kinase 1 (PGK) promoter; SSFV, CMV, MNDU3, SV40, Ef1a, UBC and CAGG. An inducible promoter is one which is capable of being induced by a molecule or a factor added to the cell or expressed in the cell. An inducible promoter may still produce a basal level of transcription in the absence of induction, but induction typically leads to significantly more production of the protein. Promoters can also be tissue specific. A tissue specific promoter allows for the production of a protein in a certain population of cells that have the appropriate transcriptional factors to activate the promoter. An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter/enhancer" is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer/promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer/promoter is one which is 14 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter. A “composition” as used herein, refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active. The carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra- oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general 15 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Provided herein in the experimental examples are effective amounts determined in a murine or rat animal model, which can be converted to an effective dose by converting the reported µg of particle per g of the mouse. A typical adult mouse is from about 15 to about 35g for a female mouse and about 20g to about 30g for a male mouse. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. The terms "oligonucleotide" or "polynucleotide" or "portion," or "segment" thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules. The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence comprising, or consisting essentially of, or yet further consisting of hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must 16 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration. In some embodiments, contacting in vitro can also be described as administering. The polynucleotides used in the present disclosure, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant polynucleotide. In some cases, a polynucleotide can encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for detection or purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. A tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide. Recombinant DNA technology can be employed wherein a polynucleotide which encodes a polypeptide of the disclosure is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. In some embodiments, the cell as disclosed herein is a eukaryotic cell or a prokaryotic cell. 17 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The host cell can be a prokaryotic or a eukaryotic cell. “Eukaryotic cells” comprise all of the life kingdoms except Monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human. The cells can be from an established cell line that can be commercially available, or from a tissue biopsy and therefore a primary cultured cell. Non-limiting of mammalian cells include Chinese Hamster Ovary (CHO) cells, human embryonic kidney cells 293 (HEK293 cells, e.g., CRL 1573, American Type Culture Collection (ATCC)), and human induced pluripotent cell lines (iPSCs). “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called an episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 μm in diameter and 10 μm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.’ Lactate dehydrogenase A (LDHA) converts pyruvate into lactate. The accession numbers of exemplary LDHA polynucleotides and polypeptides include, but are not limited to, NM_001244050.1→NP_001230979.1 (Chinese Hamster LDHA), NM_005566.3→NP_005557.1 (human LDHA isoform 1), NM_001135239.1→NP_001128711.1 (human LDHA isoform 2), NM_001165414.1→NP_001158886.1 (human LDHA isoform 3), 18 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 NM_001165415.1→NP_001158887.1 (human LDHA isoform 4), NM_001165416.1→NP_001158888.1 (human LDHA isoform 5), NM_010699.2→NP_034829.1 (mouse LDHA isoform 1), NM_001136069.2→NP_001129541.2 (mouse LDHA isoform 2), and NM_017025.1→NP_058721.1 (rat LDHA). Lactate dehydrogenase B (LDHB) converts pyruvate into lactate. The accession numbers of exemplary LDHB polynucleotides and polypeptides include, but are not limited to human isoforms NM_001174097.2→NP_001167568.1; NM_001315537.1→NP_001302466.1; and NM_002300.7→NP_002291.1. Polynucleotides and polypeptides for Chinese Hamster LDHB include, e.g., XM_007643790.1→XP_007641980.1 and XM_007624678.1→XP_007622868.1. Polynucleotides and polypeptides for mouse LDHB include, e.g., NM_001302765.1→NP_001289694.1; NM_001316322.1→NP_001303251.1; NM_008492.3→NP_032518.1. Polynucleotides and polypeptides for rat LDHB include, e.g., NM_001316333.1→NP_001303262.1; NM_001316334.1→NP_001303263.1; and NM_012595.2→NP_036727.1. Pyruvate dehydrogense kinase (PDK) inhibits the conversion of pyruvate into acetyl- CoA. The accession numbers of exemplary PDK1 polypeptides and nucleic acids include, but are not limited to, NM_172665.5→NP_766253.2 (mouse PDK1), L42450 (human), and BC089783 (rat). The accession numbers of exemplary PDK2 polypeptides and nucleic acids include, but are not limited to, NM_002611.4→NP_002602.2 (human PDK2 isoform 1), NM_001199898.1→NP_001186827.1 (human PDK2 isoform 2), NM_001199899.1→NP_001186828.1 (human PDK2 isoform 2), NM_001199900.1→NP_001186829.1 (human PDK2 isoform 3), NM_030872 (rat), and NM_133667 (mouse). The accession numbers of exemplary PDK3 polypeptides and nucleic acids include, but are not limited to, NM_001142386.2→NP_001135858.1 (human PDK3 isoform 1), NM_005391.4→NP_005382.1 (human PDK3 isoform 2), BC169078 (rat PDK3), and NM_145630 (mouse PDK3). The accession numbers of exemplary PDK4 polypeptides and nucleic acids include, but are not limited to, NM_002612 (human PDK4), NM_053551 (rat PDK4), and NM_013743.2→NP_038771.1 (mouse PDK4). Polynucleotides and polypeptides for Chinese Hamster PDK include, e.g., gene ID 100774056, Refseq 19 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 XM_007645579.2→XP_007643769.1 or XM_007645572.2→XP_007643762.1 or XM_007611707.1→XP_007609897.1 and XM_007611708.2→XP_007609898.1. Modes For Carrying Out the Disclosure Applicant provides herein engineered cell lines that are useful in the biopharmaceutical industry as ammonia and lactate negatively impact growth, production, and product quality. Without being bound by theory, ammonia generated in later stages of culture for the TKO lines originates from and potentially address that via additional genetic engineering strategies to fully–or at least further–decrease the secretion of ammonia. Applicant’s knockout cell lines can have beneficial uses in the cultured meat industry. Cultured meat has in recent years gained traction (and substantial funding) as an environmentally friendly alternative to conventional meat production. However, concerns have been raised around the economic feasibility. One of the concerns addresses the waste products lactate and ammonia, and their inhibition of cell growth in large-scale cell production. Culturing a cell line with minimal lactate and ammonia secretion could circumvent these issues. Ammonia is a toxic metabolite that accumulates during cell culture with negative impacts on cell growth, production, and product quality (Thorens and Vassalli, 1986; Borys, Linzer and Papoutsakis, 1994; Yang and Butler, 2000). While it is generated from multiple sources, most secreted ammonia comes from amino acid catabolism. Deamination of glutamine and asparagine (often the most abundant amino acids in media) is mediated by glutaminase (Gls or Gls2) and asparaginase (Aspg), followed by transamination to alpha-ketoglutarate and oxaloacetate, respectively. These amino acids are taken up by the cells in excess of biomass requirements (Zielinski et al., 2017) and appear to replenish TCA cycle intermediates. It has thus been expected that knockout of these genes would be deleterious for cell proliferation. Applicant performed a metabolic CRISPR knockout screen in media with and without glutamine (Karottki, 2021) and found that Gls knockout was enriched under glutamine depletion. Later characterization of the knockout in batch culture showed little difference in growth between KO and control cells in glutamine-containing medium (FIG. 1) but 20 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 significantly decreased ammonia levels (FIG. 2) likely due to decreased glutamine uptake (FIG. 3). Given the Gls KO’s lack of effect on growth rate combined with the severe drop in glutamine utilization, Applicant hypothesize that metabolic rewiring towards increased asparagine uptake has occurred, explaining the only modest decrease in ammonia secretion. Furthermore, Applicant previously explored knocking out Aspg for the purpose of generating a double selection system in concert with the well-established glutamine synthetase (Gs) selection system that relies upon the inhibition of the Glul gene product (Ha et al., 2022). Aspg KO cells grew similarly to wildtype in medium containing glutamine but secreted less ammonia and consumed more glutamine (data not shown). As all three genes are expressed in CHO-S (FIG. 4), Applicant hypothesized that triple KO of Gls (Entrez ID: 100689202), Gls2 (Entrez ID: 100774486), and Aspg (Entrez ID: 100750655) should eliminate the majority of ammonia secretion regardless of any metabolic rewiring that may occur. Applicant’s disclosure provides methods and compositions for the knockout of both glutaminase isozymes (Gls and Gls2) and asparaginase (Aspg) in eukaryotic cells, (e.g., Chinese hamster ovary (CHO) cells) that almost entirely eliminates ammonia production before stationary phase and substantially decreases it over the course of culture, even when grown in glutamine-containing medium. This genotype and phenotype can be ‘layered’ on top of previously described engineering strategy to eliminate lactate production, resulting in cells with reduced generation of both primary mammalian cell culture inhibitory byproducts. Thus, in one aspect, Applicant provides an engineered eukaryotic cell wherein expression of endogenous genes encoding glutaminase (Gls), glutaminase 2 (Gls2) and asparaginase (Aspg) are substantially reduced or eliminated, and in one aspect reduced to substantially eliminate ammonia production by the engineered eukaryotic cell. In another aspect, the ammonia production is substantially eliminated before the stationary phase, optionally wherein the cell is grown in glutamine-containing medium. In one aspect, the cell is a mammalian cell, e.g., a CHO cell. 21 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 In one aspect, engineered cell is prepared by CRISPR/Cas9 knockout of the endogenous genes encoding glutaminase (Gls), glutaminase 2 (Gls2) and asparaginase (Aspg). Also provided is an engineered eukaryotic cell wherein expression of endogenous genes encoding glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4) are substantially reduced or eliminated. In one aspect, the expression of glutaminase (Gls), glutaminase 2 (Gls2), and asparaginase (Aspg), and optionally lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4) are reduced to substantially eliminate ammonia production by the engineered eukaryotic cell. In another aspect, the ammonia production is substantially reduced or eliminated before the stationary phase, optionally wherein the cell is grown in glutamine-containing medium. In one aspect, the cell is prepared by CRISPR/Cas9 knockout of the endogenous genes expressing glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), and optionally additionally lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4). In one aspect, the engineered cells are mammalian cells, e.g., CHO cells. The cells can be detectably labeled or further comprises a transgene for recombinant production. Also provided is a population of the cells, wherein the population is substantially homogenous for the engineered eukaryotic cells. The eukaryotic cells can be mammalian cells and of the same or different type in the population. Also provided are compositions comprising the engineered cells or population of cells, and a carrier, such as cell culture medium and optionally, a cryopreservative (e.g., ethylene glycol, dimethyl sulfoxide (DMSO), and glyceral) to protect the cells for freezing and transport. Further optionally agents include antibiotics or antimicrobials. In producing the engineered cells, genomic DNA encoding glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), and optionally lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4) are substantially reduced or eliminated, for example by targeting the endogenous genes for disruption and/or elimination, e.g., using CRISPR/Cas9 and a guide RNA that specifically hybridizes to the endogenous genes. Thus, 22 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 also provided herein is a method to produce a cell having substantially reduced or eliminated ammonia production comprising substantially reducing or eliminating the production of one or more of an endogenous gene selected from glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), and optionally in addition lactate dehydrogenase (Ldha) and pyruvate dehydrogense kinase 1-4 (Pdk1-4). In another aspect, the expression of glutaminase (Gls), glutaminase 2 (Gls2), and asparaginase (Aspg) are eliminated or substantially reduced using methods disclosed herein, e.g., by CRISPR/Cas9 knockout of an endogenous gene selected from a gene expressing glutaminase (Gls), glutaminase 2 (Gls2), asparaginase (Aspg), and optionally in addition lactate dehydrogenase (Ldha) and/or pyruvate dehydrogense kinase 1- 4 (Pdk1-4). Standard methods known by persons skill in the art can be used to determine whether a eukaryotic cell encodes or expresses the endogenous genes described herein, e.g., by measuring the ability of the polypeptide to convert pyruvate into lactate in vitro, in a cell extract, or in vivo and/or the amount of ammonia produced by the cell as known in the art or as described herein. Exemplary in vitro cell lysate LDH detection assays include immunoassays (e.g., ELISA, Western Blots) and activity assays are demonstrated in WO 2017/192437A1. In vitro assays for detecting lactate dehydrogenase activity are known in the art and described, e.g., in Baba, et al., Antivir Chem Chemother. 2005; 16(1):33-9; Larson, J Dairy Res. (2005) 72(2):209-16; and Mori, et al., Tohoku J Exp Med. 1995 December; 177(4):315-25. The following methods are intended to illustrate but not limit the disclosure as described herein. Materials and Methods Transfection CHO cells were transfected using either FreeStyle MAX reagent (Gibco Cat. # 16447100). The day prior to transfection, viable cell density was adjusted to 8x105 cells/mL in transfection medium: CD CHO medium (Gibco Cat. #10743-029) supplemented with 8 mM L-glutamine (Lonza Cat. # BE17-605F). On the day of transfection, viable cell density was adjusted to 1x106 cells/mL in an MD6 plate (Falcon Cat. # 351146) containing 3 mL transfection medium per well. For each transfection, 1.9 µg Cas9-2A-GFP plasmid DNA and 23 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 1.9 µg gRNA plasmid DNA (divided evenly between gRNAs if multiple used) was diluted in 60 µL OptiPro SFM (Gibco Cat. # 12309019). Separately, 3.8 µL FreeStyle MAX reagent was diluted in 60 µL OptiPro SFM and the two mixtures were incubated for 5 minutes at room temperature. After incubation, the plasmid DNA/OptiPro SFM mixture was added to the FreeStyle MAX/OptiPro SFM mixture and incubated at room temperature for an additional 20 minutes. The resultant 120 µL DNA/lipid mixture was added dropwise to the cells in one well. Plasmids were constructed using the uracil-specific excision reagent (USER) cloning method as described previously (https://doi.org/10.1002/bit.25233), with the sgRNA1_C plasmid as a backbone. Single cell sorting and expansion Transfected cells were single cell sorted 48 hours post transfection, using the FACSJazz, based on green fluorescence with gating determined by comparison to non- transfected cells. Sorting was done into MD384 plates (Corning Cat. # 3542) containing CD CHO medium (Gibco Cat. # 10743-029) supplemented with 8 mM L-glutamine (Lonza Cat. # BE17-605F), 1% antibiotic-antimycotic (Gibco Cat. # 15240-062), and 1.5% HEPES buffer (Gibco Cat. # 15630-056). After 15 days, colonies were transferred to an MD96F plate (Falcon Cat. # 351172) containing CD CHO medium supplemented with 8 mM L-glutamine, 2 mL/L anti-clumping agent (Gibco Cat. # 0010057AE), and 1% antibiotic-antimycotic. Clone genotyping After two days, 50 uL cell suspension from each well was transferred to a MicroAmp Fast 96 well reaction plate (Thermo Cat. # 4346907), along with 5x105 wildtype cells as a control. The plate was centrifuged at 1000 x g for 10 minutes, then the supernatant was removed via rapid inversion. 20 µL of QuickExtract DNA Extraction Solution (Epicentre Cat. # QE09050) (prewarmed to 65°C) was added to each well and mixed via pipetting. The plate was then processed in the thermocycler (65°C for 15 minutes followed by 95°C for 5 minutes). Amplicons were generated for each gene of interest per well using Phusion Hot Start II DNA Polymerase (Thermo Cat. # F549L) and verified to be present visually on a 2% agarose gel. Amplicons from each well had unique barcodes, allowing them to be pooled and purified using AMPure XP beads (Beckman Coulter Cat. # A63881) according to 24 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 manufacturer’s protocol, except using 80% ethanol for washing steps and 40 uL beads for 50 uL sample. Samples were indexed using the Nextera XT Index kit attached using 2x KAPA HiFi Hot Start Ready mix (Fisher Scientific Cat. # KK2602). AMPure XP beads were used to purify the resulting PCR products. DNA concentrations were determined with the Qubit 2.0 Fluorometer and used to pool all indices to an equimolar value and diluted to a final concentration of 10 nM using 10mM Tris pH 8.5, 0.1% Tween 20. The average size of the final library was verified with the Bioanalyzer 2100. The amplicon library was then sequenced on an Illumina MiSeq. Insertions and deletions were identified by comparison of expected vs. actual amplicon size. Two rounds of engineering were carried out. In the first round of engineering a clone with frameshift insertions/deletions in all alleles of Gls and Gls2 was generated. The second round of engineering retargeted Aspg and isolated a clone with frameshift mutations in all alleles of Aspg while maintaining the frameshift mutations in Gls and Gls2. Sequences used:
Cell line generation Knockout cell lines were established in CHO-S (WT cells) (Life Technologies, Carlsbad, CA) using CRISPR/Cas9 as previously described (Grav et al., 2015). Ldha, Pdk1, Pdk2, Pdk3 and Pdk4 ## were targeted, using ## gRNAs and ## primers, to generate the 25 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 Intermediate cell line. In addition, Aspg, Gls1 and Gls2 were targeted, using ##gRNAs and ##primers, to generate the TKO cell line. Cell lines were cultured in CD-CHO Medium (cat. No 10743029, Thermo Fisher Scientific, Waltham, MA) with 8 mM glutamine and 0.2 % Anti-Clumping Agent (cat. no. 0010057AE, Gibco, Waltham, MA) unless otherwise stated. Viability and viable cell density (VCD) were measured using NucleoCounter® NC-200™ or NucleoCounter® NC-250™ (ChemoMetec, Allerod, Denmark). Batch culture Cells were seeded at an initial cell density of 3 x 105 cells/mL in 125 mL shake flasks (Corning) containing 30 mL medium in media containing 8 mM glutamine. All cultures were incubated at 80% humidity, 5% CO2, 37ºC and were shaken at 120 rpm. Fed batch culture Cells were grown as described in batch culture, except glutamine was fed on day 3 to reach a concentration of 6 mM and Cell Boost 7a/7b (Cytiva HyClone, Thermo Fisher Scientific, Waltham, MA) were fed at 3% and 0.3% culture volume, respectively, on day 4. Protein quantification The protein concentration was measured using an Octet RED96 (Pall, Menlo Park, CA, USA), as described previously (Kallehauge et al., 2017). Purification and N-glycan analysis of mAb The supernatant taken from the cultures was centrifuged and filtrated to remove the cells and cell debris, after which purification and N-glycan analysis were performed as described previously (Grav et al., 2015). Briefly, mAb was purified by protein A affinity chromatography (recombinant protein A agarose, Pierce, Rockford, IL). GlykoPrep Rapid N- Glycan kit (ProZyme, Hayward, CA) was used for the fluorescent label of mAb. N-glycan was measured by LC-MS system using a Thermo Ultimate 3000 HPLC equipped with a fluorescence detector and a Thermo Velos Pro Iontrap MS. Cell Lines Applicant generated triple knockout (Aspg, Gls, Gls2) cell lines using CRISPR/Cas9, verifying knockouts using amplicon sequencing to confirm a frameshift insertion or deletion 26 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 in all alleles of targeted genes. One triple knockout cell line (TKO) was generated from a wildtype CHO-S cell (Thermo Fisher), five cell lines (B11, A6, F6, G9, and F2) were generated from an earlier knockout cell line (referred to as Intermediate), which lacked the ability to produce lactate, the other major inhibitory byproduct generated in cell culture, due to the knockout of Ldha and Pdk1-4 (Hefzi and Lewis, 2022). Batch culture in shake flasks All cell lines were grown in shake flasks in batch culture in media with glutamine. The TKO cell line generated from wildtype CHO-S cells grew slightly slower than the wildtype (FIG. 5A) while the knockout cell lines generated from the non-lactogenic Intermediate cell line grew as well or better than their parental line (FIG. 5B). Applicant also measured metabolite concentrations in these clones along with a plain media control (media containing glutamine cultured without cells to quantify spontaneous deamidation of glutamine over time as well as potential instrument drift). All knockout cell lines secreted negligible ammonia through day 3 (equivalent to ammonia generated in the media control, and significantly less than the Intermediate or wildtype cells). For the TKO (FIG. 6A), ammonia secretion remained ~0 until day 4, after which there was some ammonia generation. For knockouts generated from the Intermediate cell line (FIG. 7A), after day 3 there was a period of ammonia consumption followed by ammonia generation (similar to the Intermediate cell line). Knockout lines also showed a significant decrease in glutamine consumption, consistent with the knockout of the primary glutamine catabolic pathway (Gls/Gls2) (FIG. 6B, FIG.7B). Ammonia levels in all knockout lines were universally lower than in the parental lines, markedly so during the exponential and early stationary phase (e.g., >60% reduction on day 6 for wildtype derived knockout cells and >40% reduction on day 5 for the Intermediate cell line derived knockout cells). Fed-batch culture in shake flasks As the TKO cell line grew slightly worse than its parental line (potentially due to clonal variability), Applicant chose to do further experiments on the knockouts derived from the Intermediate cell line. To test the robustness of decreased ammonia production in knockouts to additional nutrient feeding, Applicant grew two Intermediate derived knockout cell lines (A6 and B11), as well as the Intermediate cell line and wildtype CHO-S, in an 27 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 identical experiment as described above, except glutamine was fed on day 3 to reach a concentration of 6 mM and Cell Boost 7a/7b (Cytiva HyClone) were fed at 3% and 0.3% culture volume, respectively, on day 4. Again, knockouts cells grew similarly to the Intermediate and wildtype cell lines (FIG. 8). Metabolite trends were measured and were similar to batch culture (FIG. 9). Applicant again highlight that ammonia secretion was ~0 for knockout cells through day 5 of culture, while both the Intermediate and wildtype cell lines produced appreciable ammonia. Protein production test in batch culture in media without glutamine To examine the performance of the two knockout cell lines (A6 and B11) in a protein production setting, Applicant generated Enbrel-producing cells using the Gs/Msx selection system and proceeded to grow the resulting pools in shake flask batch culture without glutamine. Interestingly, the selective pressure was much stronger in the knockout cell lines (viability dropped below 50% compared to the intermediate and wildtype cell lines when undergoing selection (FIG. 10B)), likely owing to the Aspg KO in these lines. When the selected cell lines were grown in media without glutamine (FIG. 11) the knockout cell lines grew to a markedly lower viable cell density compared to the Intermediate and wildtype cell lines. However, they maintained a similar titer until day 7 after which the titer of the knockout cell lines increased greatly compared to the parental lines. The knockout cell lines remained low secretors of ammonia (FIG. 12A) and the glycosylation profile was similar to that of both the wildtype and Intermediate cell line (FIG. 13). Protein production in fed-batch culture in glutamine containing medium Applicant finally carried out a fed-batch culture in medium with glutamine. The knockout cell lines grew similarly to the intermediate and wildtype cell lines but reached significantly higher product titer (FIG. 14) while producing significantly less ammonia (FIG. 15). Experimental Summary Ammonia is a toxic metabolite that accumulates during cell culture with negative impacts on cell growth, production, and product quality (Borys et al., 1994; Thorens and 28 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 Vassalli, 1986; Yang and Butler, 2000a, 2000b). While it is generated from multiple sources, by-and-large ammonia is a result of amino acid catabolism. Specifically, glutamine and asparagine (the most abundant amino acids in media) are taken up by the cells in excess of biomass requirements (Zielinski et al., 2017) and appear to replenish TCA cycle intermediates. Their uptake by the cell is followed by deamination (i.e., ammonia generation) and further catabolism to alpha-ketoglutarate and oxaloacetate, respectively. Simultaneous knockout of Gls, Gls2, and Aspg is sufficient to largely eliminate ammonia production (FIG. 6A) with only a minimal (~18%) decrease in cell proliferation (FIG. 5A). This observation is unexpected in light of the fact that Gls KO is compensated by Aspg activity and vice versa (with respect to ammonia generation), e.g., more than the sum of their parts. Gls is a therapeutic target for many cancers so KO would be predicted to be lethal, i.e., thus the prior literature teaches away from its knockout. Aspg in mammalian cells has been stated to not be functional at biological asparagine levels (https://www.ncbi.nlm.nih.gov/pubmed/29337136). Thus, the prior art literature also art teaches away from its knockout. For example, WO 2018/033542 shows minimal reduction of ammonia levels (best knockout is ~20% reduction in specific ammonia secretion). Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr- cells decreases accumulation of ammonium ion in culture media (https://www.ncbi.nlm.nih.gov/pubmed/10989172) and Reduction of Ammonia Accumulation and Improvement of Cell Viability by Expression of Urea Cycle Enzymes in Chinese Hamster Ovary Cells (http://www.jmb.or.kr/journal/download.php?Filedir=../submission/Journal/013/&num=1575 and KR100499824B1) reduce ammonia accumulation in media but do not eliminate it at any point (basal glutamine level in MEM-alpha is 2 mM so spontaneous ammonia generation from nonenzymatic glutamine degradation is lower than what is observed in FIG. 6A). Equivalents 29 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed. Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology. It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were 30 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 incorporated by reference individually. In case of conflict, the present specification, including definitions, will control. 31 4920-8577-7422.2
Atty. Dkt. No.: 114198-0788 REFERENCES: 1. Borys, M.C., Linzer, D.I.H. and Papoutsakis, E.T. (1994) ‘Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by chinese hamster ovary cells in a pH-dependent manner’, Biotechnology and Bioengineering, pp.505– 514. Available at: https://doi.org/10.1002/bit.260430611. 2. Grav, L.M. et al. (2015) ‘One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment’, Biotechnology journal, 10(9), pp.1446– 1456. 3. Ha, T. et al. (2022) ‘Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium’. Available at: https://doi.org/10.22541/au.165606842.29880741/v1. 4. Hefzi, H. et al. (2016) ‘A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism’, Cell systems, 3(5), pp.434–443.e8. 5. Hefzi, H. and Lewis, N.E. (2022) ‘Mammalian cells devoid of lactate dehydrogenase activity’, US Patent. Available at: https://patentimages.storage.googleapis.com/b6/0c/a8/b7a19529eaeb38/US11242510. pdf (Accessed: 13 June 2023). 6. Kallehauge, T.B. et al. (2017) ‘Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion’, Scientific reports, 7, p.40388. 7. Thorens, B. and Vassalli, P. (1986) ‘Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion’, Nature, 321(6070), pp.618–620. 8. Yang, M. and Butler, M. (2000) ‘Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation’, Biotechnology and Bioengineering, pp. 370–380. Available at: https://doi.org/10.1002/(sici)1097- 0290(20000520)68:4<370::aid-bit2>3.0.co;2-k. 9. Zielinski, D.C. et al. (2017) ‘Systems biology analysis of drivers underlying hallmarks of cancer cell metabolism’, Scientific reports, 7, p.41241. 32 4920-8577-7422.2