The present disclosure relates to methods and compositions for modulating the lipid metabolism pathways of a cell and engineering cells and cell lines for production of a product, e.g., a recombinant protein.

BACKGROUND

Recombinant therapeutic proteins are commonly expressed in cell expression systems, e.g., mammalian cell expression systems. In 2014, the total number of market approved biopharmaceuticals was 212, and 56% of the therapeutic products approved for market by the FDA are produced in mammalian cell lines. However, the high cost associated with production contributes to increasing global health costs.

Moreover, next generation protein biologies (NGBs) such as next generation fusion proteins, multimeric glycoproteins, or next generation antibodies often have a complex and/or non-natural structure and are proving more difficult to express than molecules such as monoclonal antibodies. Current host cell lines have not evolved pathways for the efficient synthesis and secretion of NGBs, resulting in significantly reduced growth, low productivity and often resulting in products with poor product quality attributes. Thus, these NGBs are considered difficult to express, in which the productivity and product quality do not meet clinical and market needs.

Accordingly, there is an increasing need to develop and produce recombinant biotherapeutics rapidly, efficiently, and cost-effectively while maintaining final product quality.

SUMMARY

The present disclosure is based, in part, on the discovery that modulation of lipid metabolism pathways by overexpression of a component of one or more lipid metabolism pathways increases the productivity and product quality of a cell that produces a recombinant

polypeptide product. Here, it is demonstrated that modulation of the lipid metabolism, e.g., by modulating one or more lipid metabolism pathways, can be used to engineer cells and cell-free systems that produce higher yields of products and products with improved quality. Importantly, the present disclosure features global regulation of lipid metabolism by using global regulators that modulate more than one process or pathway associated with lipid metabolism, thereby causing multiple downstream effects to achieve improved product production and quality. The methods and compositions described herein are particularly useful for improved production of recombinant products or next generation biologies (e.g., fusion proteins, bispecific or multi-format antibody molecules, multimeric proteins, and glycosylated proteins), and for development of more efficient systems for production of such products (e.g., cell lines or cell-free systems).

In one aspect, the present disclosure features a method for producing a product described herein in a cell. In an embodiment, the product is a polypeptide, e.g., a recombinant

polypeptide. In one embodiment, the method comprises providing a cell comprising a modification that modulates lipid metabolism, and culturing the cell, e.g., under conditions suitable for modulation of lipid metabolism by the modification, thereby producing the product.

In another aspect, the present disclosure features a method for producing product, e.g., a polypeptide, e.g., a recombinant polypeptide, in a cell-free system comprising: providing a cell-free system comprising a modification that modulates lipid metabolism, e.g., a cell-free system derived from a cell or cell line comprising a modification that modulates lipid metabolism, and placing the cell-free system under conditions suitable for production of the product; thereby producing the product, e.g., polypeptide, e.g., recombinant polypeptide. In one embodiment, the cell-free system is derived from a cell or cell line comprising a modification that modulates lipid metabolism. In one embodiment, the cell-free system comprises one or more components, e.g., an organelle or portion of an organelle, from a cell or cell line comprising a modification that modulates lipid metabolism. In some embodiments, the modification comprises an exogenous nucleic acid encoding a lipid metabolism modulator (LMM) and wherein the cell or cell line expresses a LMM, e.g., an LMM selected from the group consisting of SREBF1 , SREBF2, SCDl, SCD2, SCD3, SCD4, SCD5, or a functional fragment thereof. In some embodiments, the LMM alters one or more characteristics of a cell- free system selected from the group consisting of: increases the production, e.g., yield and rate of production, of the product, e.g., polypeptide, e.g., recombinant polypeptide ( GB) produced; and increases the quality, e.g., decreases

aggregation, decreases glycosylation heterogeneity, decreases fragmentation, and increases ratio of properly folded to misfolded or unfolded product, of the product.

Examples of products that can be produced using any of the methods or compositions described herein include recombinant products, or products in which at least one portion or moiety is a result of genetic engineering. Recombinant products described herein can be useful for diagnostic or therapeutic purposes. In one embodiment, a product comprises a polypeptide, such as an antibody molecule (e.g., a bispecific or multi-format antibody molecule), a fusion protein, or a protein-conjugate; a nucleic acid molecule (e.g., a DNA or R A molecule); or a lipid-encapsulated particle (e.g., an exosome or virus-like particle). The methods and

compositions described herein may be particularly useful for products that are difficult to produce, e.g., in high quantities or with sufficient quality for commercial or therapeutic use, such as next generation biologies (e.g., fusion proteins, bispecific or multi-format antibody molecules, multimeric proteins, and glycosylated proteins). In one embodiment, a cell as described herein, e.g., for producing the product, expresses the product. In one embodiment, the cell comprises an exogenous nucleic acid that encodes a product described herein, e.g., a polypeptide selected from Table 2 or 3. Additional examples of products are described in the section titled "Products".

The modifications disclosed herein that modulate lipid metabolism include agents or molecules that increase or decrease the expression of a lipid metabolism modulator (LMM) or increase or decrease the expression or activity of a component of a lipid metabolism pathway. In one embodiment, the modification is a nucleic acid, e.g., a nucleic acid encoding a LMM or an inhibitory nucleic acid that inhibits or decreases the expression of a LMM.

In one embodiment, the modification increases expression of a LMM, and comprises an exogenous nucleic acid encoding the LMM. In one embodiment, the method comprises forming, in the cell, an exogenous nucleic acid encoding a LMM or an exogenous LMM. In one embodiment, the forming comprises introducing an exogenous nucleic acid encoding a lipid metabolism modulator. In one embodiment, the forming comprises introducing an exogenous nucleic acid which increases the expression of an endogenous nucleic acid encoding a LMM. Examples of LMMs suitable for use in any of the methods and compositions described herein are further described in the sections titled "Modulation of Lipid Metabolism" and "Lipid Metabolism Modulators".

In one embodiment, the cell comprises one or more modifications. In one embodiment, the cell comprises one, two, three, four, five, six, seven, eight, nine or ten modifications. In some embodiments, the cell comprises more than one modification. In some embodiments, the cell comprises at least two, three, four, five, six, seven, eight, nine, or ten modifications. In one embodiment, the cell comprises a one or more second modification that modulates lipid metabolism. In one embodiment, the second modification comprises a second exogenous nucleic acid encoding a second LMM, e.g., a LMM different from the LMM of the first modification. In one embodiment, the second exogenous nucleic acid and the first exogenous nucleic acid are disposed on the same nucleic acid molecule. In one embodiment, the second exogenous nucleic acid and the first exogenous nucleic acid are disposed on different nucleic acid molecules. In one embodiment, the second modification provides increased the production or improved quality of the product, as compared to a cell not having the second modification. In one embodiment, the method comprises forming, in the cell, a second exogenous nucleic acid encoding a second LMM or a second exogenous LMM. In one embodiment, the forming comprises introducing the second exogenous nucleic acid encoding a second LMM. In one embodiment, the forming comprises introducing the second exogenous nucleic acid which increases the expression of an endogenous nucleic acid encoding a LMM.

Modulating lipid metabolism by any of the methods or compositions described herein can comprise or result in altering, e.g., increasing or decreasing, any one or more of the following: i) the expression (e.g., transcription and/or translation) of a component involved in a lipid metabolism pathway;

ii) the activity (e.g., enzymatic activity) of a component involved in a lipid metabolism pathway;

iii) the amount of lipids (e.g., phospholipids, or cholesterol) present in a cell;

iv) the amount of lipid rafts or rate of lipid raft formation;

v) the fluidity, permeability, and/or thickness of a cell membrane (e.g., a plasma membrane, a vesicle membrane, or an organelle membrane);

vi) the conversion of saturated lipids to unsaturated lipids or conversion of unsaturated lipids to saturated lipids;

vii) the amount of saturated lipids or unsaturated lipids, e.g., monounsaturated lipids;

viii) the composition of lipids in the cell to attain a favorable composition that increases ER activity;

ix) the expansion of the ER (e.g., size of the ER, the ER membrane surface, or the amounts of the proteins and lipids that constitute and/or reside within the ER); x) the expansion of the Golgi (e.g., the number and size of the Golgi, the Golgi surface, or the number or amounts of proteins and molecules that reside within the Golgi);

xi) the amount of secretory vesicles or the formation of secretory vesicles;

xii) the amount or rate of secretion of the product;

xiii) the proliferation capacity, e.g., the proliferation rate;

xiv) culture viability or cell survival;

xv) activation of membrane receptors;

xvi) the unfolded protein response (UPR);

xvii) the yield or rate of production of the product;

xviii) the product quality (e.g., aggregation, glycosylation heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling); and /or

xix) cell growth proliferation or cell specific growth rate.

In such embodiments, the increase or decrease of any of the aforementioned characteristics of the cell can be determined by comparison with a cell not having a modification.

The methods and compositions described herein result in increased production of the product as compared to a cell not having the modification. An increase in production can be characterized by increased amounts, yields, or quantities of product produced by the cell and/or increased rate of production, where the rate of production is equivalent to the amount of product over time. In one embodiment, production of the product, e.g., a recombinant polypeptide, is increased by 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 85%, or 100%, or more e.g., as compared to the production of by a cell without modulation of the lipid metabolism; or 1-fold, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold, 100-fold, e.g., as compared to the production of by a cell without modulation of the lipid metabolism.

The methods and compositions described herein can also result in improved quality of the product (i.e. product quality) as compared to a cell not having the modification. Improvements in the quality of the product (i.e. product quality) can be characterized by one or more of:

aggregation (e.g., a decrease in aggregates or aggregation); proper folding or assembly (e.g., a decrease in misfolded or unfolded products; or partially assembled or disassembled products); post-translation modification (e.g., increase or decrease in glycosylation heterogeneity, higher percentage of desired or predetermined post-translational modifications); fragmentation (e.g., a decrease in fragmentation); disulfide bond scrambling (e.g., a decrease in undesired isoforms or structures due to disulfide bond scrambling). In one embodiment, the quality of the product, e.g., recombinant polypeptide, is increased, e.g., 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 85%, or 100%, e.g., as compared to the production of by a cell without modulation of the lipid metabolism; or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, e.g., as compared to the quality of product produced by a cell without modulation of the lipid metabolism.

In embodiments, the method for producing a product as described herein can comprise one or more additional steps, which include, but are not limited to: introducing a modification to the cell that improves ER processing capacity (ER expansion) or secretion; obtaining the product from the cell, or a descendent of the cell, or from the medium conditioned by the cell, or a descendent of the cell; separating the product from at least one cellular or medium component; and/or analyzing the product, e.g., for activity or for the presence of a structural moiety. In one embodiment, the method further comprises a step for improving ER processing capacity (or ER expansion) by introducing a nucleic acid encoding PDI, BiP, ERO, or XBP1. In one

embodiment, the method further comprises an additional step for improving secretory capacity or rate of secretion by modulating SNARE machinery or other machinery involved in the secretory pathway, e.g., by introducing a nucleic acid encoding a SNARE component.

Modulation of Lipid Metabolism

The present disclosure features methods and compositions for modulating lipid metabolism.

In one embodiment, the modification results in modulating, e.g., increasing, one or more lipid metabolism pathways, which include, but are not limited to: de novo lipogenesis, fatty acid re-esterification, fatty acid saturation or desaturation, fatty acid elongation, and phospholipid biosynthesis.

The modifications described herein suitable for modulating lipid metabolism include introduction of an exogenous nucleic acid that increase or decreases the expression or activity of a component of a lipid metabolism pathway or a LMM, a LMM polypeptide, or other molecule that increases or decreases the expression or activity of a component of the lipid metabolism pathway. The present disclosure features the use of lipid metabolism modulators to modulate lipid metabolism, e.g., by increasing or decreasing expression or activity of a component associated with lipid metabolism. In an embodiment, the LMM is a global regulator described herein

In one embodiment, the modification that modulates lipid metabolism results in the global regulation of lipid metabolism, e.g., by increasing or decreasing the expression or activity of a global regulator. Such global regulators are molecules that are sufficiently upstream in one or more pathways, such that it can influence multiple downstream effects, for example, increasing the expression or activity of more than one, e.g., two, three, four, five, or more, components of different lipid metabolism processes or pathways. A component of a lipid metabolism process or pathway can include, but is not limited to, an enzyme, a cofactor, or other molecule that is involved in the synthesis, degradation, elongation, or structural conformation of lipid molecules.

In one embodiment, the global regulator described herein is a transcription factor that upregulates, e.g., increases the expression, of a component of the lipid metabolism, e.g., a lipid metabolism gene product selected from Table 1. By way of example, a global regulator increases the expression of two or more lipid-associated gene products, e.g., an enzyme involved in lipid biosynthesis and an enzyme involved in the saturation level of a lipid molecule.

In any of the methods or compositions described herein, the LMM comprises any of the following: a global regulator of lipid metabolism, e.g., a transcription factor that upregulates lipid metabolism genes, or a component (e.g., an enzyme, a cofactor, or a molecule) that plays a role in the de novo lipogenesis, fatty acid re-esterification, fatty acid saturation or desaturation, fatty acid elongation, or phospholipid biosynthesis pathways.

In one embodiment, the lipid metabolism modulator comprises a transcription regulator, e.g., a transcription factor, that mediates, e.g., upregulates, the expression of a lipid metabolism gene product. Examples of lipid metabolism gene products include, but are not limited to, those provided in Table 1. a global regulator of lipid metabolism, e.g., a transcription factor that upregulates lipid metabolism genes.

In one embodiment, the LMM comprises SREBF1, or SREBF2, or a functional fragment or analog thereof. In one embodiment, the lipid metabolism modulator comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with the amino acid sequence of SREBF1; e.g., SEQ ID NOs:l or 34, or a functional fragment thereof, e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 36; or differs by 1 , 2, or 3 or more amino acid residues but no more than 50, 40, 30, 20, 15, or 10 amino acid residues from the amino acid sequence of SREBF1, e.g., SEQ ID NOs: 1 or 34, or a functional fragment thereof, e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 36. In one embodiment, the nucleic acid encoding the lipid metabolism modulator comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with any of the nucleic acid sequences selected from SEQ ID NOs: 2 or 32, or the nucleic acids encoding SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 36.

In one embodiment, the LMM comprises SCD1 , SCD2, SCD3, SCD4, or SCD5, or a functional fragment or analog thereof. In one embodiment, the lipid metabolism modulator comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with the amino acid sequence of SCD1; e.g., SEQ ID NO:3, or a functional fragment thereof; or differs by 1 , 2, or 3 or more amino acid residues but no more than 50, 40, 30, 20, 15, or 10 amino acid residues from the amino acid sequence of SCD1, e.g., SEQ ID NO: 3, or a functional fragment thereof. In one embodiment, the nucleic acid encoding the lipid metabolism modulator comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with any of the nucleic acid sequences selected from SEQ ID NOs: 4.

In one embodiment, the LMM comprises any of the components provided in Table 1 or a functional fragment thereof. In one embodiment, the LMM comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with the amino acid sequence of any of the components provided in Table 1 or a functional fragment thereof; or differs by 1 , 2, or 3 or more amino acid residues but no more than 50, 40, 30, 20, 15, or 10 amino acid residues from the amino acid sequence of any of the components provided in Table 1 or a functional fragment thereof. In one embodiment, the nucleic acid encoding the lipid metabolism modulator comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with a nucleic acid sequence encoding any of the components provided in Table 1 or a functional fragment thereof.

In one embodiment, the modification comprises a cis or trans regulatory element that increases the expression of a nucleic acid that encodes a lipid metabolism gene product, e.g., a lipid metabolism gene product selected from Table 1.

In one embodiment, the nucleic acid encoding the lipid metabolism modulator comprises a plasmid or a vector.

In one embodiment, the nucleic acid encoding the lipid metabolism modulator is introduced into the cell by transfection (e.g., electroporation), transduction, or any other delivery method described herein.

In one embodiment, the nucleic acid encoding the lipid metabolism modulator is integrated into the chromosomal genome of the cell. In one embodiment, the LMM is stably expressed.

In one embodiment, the nucleic acid encoding the lipid metabolism modulator is not integrated into the chromosomal genome of the cell. In one embodiment, the LMM is transiently expressed.

Products

Products described herein include polypeptides, e.g., recombinant proteins; nucleic acid molecules, e.g., DNA or R A molecules; multimeric proteins or complexes; lipid-encapsulated particles, e.g., virus-like particles, vesicles, or exosomes; or other molecules, e.g., lipids. In an embodiment, the product is a polypeptide, e.g., a recombinant polypeptide. For example, the recombinant polypeptide can be a difficult to express protein or a protein having complex and/or non-natural structures, such as a next generation biologic, e.g., a bispecific antibody molecule, a fusion protein, or a glycosylated protein.

In any of the methods described herein, the method for producing a product further comprises introducing to the cell an exogenous nucleic acid encoding the product, e.g., polypeptide, e.g., recombinant polypeptide.

In one embodiment, the exogenous nucleic acid encoding the recombinant polypeptide is introduced after providing a cell comprising a modification that modulates lipid metabolism. In another embodiment, the exogenous nucleic acid encoding the recombinant polypeptide is introduced after culturing the cell, e.g., under conditions suitable for modulation of lipid metabolism by the modification.

In one embodiment, the exogenous nucleic acid encoding the product, e.g., recombinant polypeptide, is introduced prior to providing a cell comprising a modification that modulates lipid metabolism. In another embodiment, the exogenous nucleic acid encoding the recombinant polypeptide is introduced prior to culturing the cell, e.g., under conditions suitable for modulation of lipid metabolism by the modification.

In any of the compositions, preparations, or methods described herein, the product, e.g., recombinant polypeptide, is a therapeutic polypeptide or an antibody molecule, e.g., an antibody or an antibody fragment thereof. In one embodiment, the antibody molecule is a monoclonal antibody. In one embodiment, the antibody molecule is a bispecific antibody molecule, e.g., a BiTE (Bispecific T cell Engager), a DART (Dual Affinity Re-Targeting or Redirected T cell).

In one embodiment, the product, e.g., recombinant polypeptide, is selected from Table 2 or 3.

In embodiments, the product is stably expressed by the cell. In one embodiment, the exogenous nucleic acid encoding the product, e.g., recombinant polypeptide, is integrated into the chromosomal genome of the cell. Alternatively, the product is transiently expressed by the cell. In one embodiment, the exogenous nucleic acid encoding the product, e.g., the recombinant polypeptide, is not integrated into the chromosomal genome of the cell.

Host Cells

Provided herein are cells for producing the products described herein and methods of engineering such cells.

In any of the compositions, preparations, or methods described herein, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell, a yeast cell, an insect cell, an algae cell, or a plant cell. In one embodiment, the cell is a rodent cell. In one embodiment, the cell is a Chinese hamster ovary (CHO) cell. Examples of CHO cells include, but are not limited to, CHO-K1 , CHOK1SV, Potelligent CHOK1 SV (FUT8-KO), CHO GS-KO, Exceed

(CHOK1SV GS-KO), CHO-S, CHO DG44, CHO DXB11, CHOZN, or a CHO-derived cell.

In any of the compositions, preparations, or methods described herein, the cell is selected from the group consisting of HeLa, HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK, VERO, SP2/0, NSO, YB2/0, EB66, C127, L cell, COS, e.g., COS1 and COS7, QCl-3, CHO-K1 , CHOK1 SV, Potelligent CHOK1 SV (FUT8-KO), CHO GS-KO, Exceed

(CHOK1 SV GS-KO), CHO-S, CHO DG44, CHO DXB 11 , and CHOZN.

In one embodiment, the cell is a eukaryotic cell other than a mammalian cell, e.g., an insect, a plant, a yeast, or an algae cell. In one embodiment, the cell is a prokaryotic cell.

In one aspect, the present disclosure features a method of engineering a cell having increased production capacity and/or improved quality of production (e.g., producing product with one or more improved product quality) comprising introducing to the cell or forming in the cell an exogenous nucleic acid encoding a lipid metabolism modulator, thereby engineering a cell having increased production capacity and/or improved quality of production . In an embodiment, the exogenous nucleic acid encoding a lipid metabolism modulator is introduced to the cell by transfection, transduction, e.g., viral transduction, electroporation, nucleofection, or lipofection. In an embodiment, the exogenous nucleic acid encoding a lipid metabolism modulator is integrated into the chromosomal genome of the cell. In an embodiment, the method further comprises introducing to the cell an exogenous nucleic acid encoding a recombinant polypeptide. In an embodiment, the exogenous nucleic acid encoding a recombinant polypeptide is introduced prior to introducing the exogenous nucleic acid encoding the LMM. In an embodiment, the exogenous nucleic acid encoding a recombinant polypeptide is introduced after introducing the exogenous nucleic acid encoding the LMM.

In one aspect, the present disclosure features a cell produced by providing a cell and introducing to the cell a LMM described herein, e.g., introducing an exogenous nucleic acid encoding a LMM.

In one aspect, the present disclosure features a cell comprising an exogenous nucleic acid encoding a LMM described herein

In one aspect, the present disclosure features a cell engineered to produce a LMM, wherein the LMM modulates the expression of a product, e.g., a next generation biologic ( GB) described herein. In one embodiment, the cell is a CHO cell.

In one aspect, the present disclosure features a CHO cell engineered to produce a LMM, wherein the LMM modulates the expression of a product, e.g., a Next generation biologic (NGB) described herein.

In one aspect, the present disclosure features a CHO cell engineered to express an LMM and a NGB, wherein the population has been selected for high level expression of the NGB.

In one aspect, the present disclosure features a CHO cell engineered to express an LMM, wherein the LMM modulates one or more characteristics of the CHO cell, wherein the engineered CHO cell is selected based on modulation of one or more characteristics selected from the group consisting of

i) the expression (e.g., transcription and/or translation) of a component involved in a lipid metabolism pathway;

ii) the activity (e.g., enzymatic activity) of a component involved in a lipid metabolism pathway;

iii) the amount of lipids (e.g., phospholipids, or cholesterol) present in a cell;

iv) the amount of lipid rafts or rate of lipid raft formation;

v) the fluidity, permeability, and/or thickness of a cell membrane (e.g., a plasma membrane, a vesicle membrane, or an organelle membrane);

vi) the conversion of saturated lipids to unsaturated lipids or conversion of unsaturated lipids to saturated lipids;

vii) the amount of saturated lipids or unsaturated lipids, e.g., monounsaturated lipids; viii) the composition of lipids in the cell to attain a favorable composition that increases ER activity;

ix) the expansion of the ER (e.g., size of the ER, the ER membrane surface, or the amounts of the proteins and lipids that constitute and/or reside within the ER); x) the expansion of the Golgi (e.g., the number and size of the Golgi, the Golgi surface, or the number or amounts of proteins and molecules that reside within the

Golgi);

xi) the amount of secretory vesicles or the formation of secretory vesicles;

xii) the amount or rate of secretion of the product;

xiii) the proliferation capacity, e.g., the proliferation rate;

xiv) culture viability or cell survival;

xv) activation of membrane receptors;

xvi) the unfolded protein response (UPR);

xvii) the yield or rate of production of the product;

xviii) the product quality (e.g., aggregation, glycosylation heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling); and /or

xix) cell growth proliferation or cell specific growth rate.

In any of the methods or cells, e.g., engineered cells, described herein, the cell expresses or comprises the LMM is selected from a group consisting of SREBF1 , SREBF2, SCD1, SCD2, SCD3, SCD4, and SCD5, or a functional fragment thereof.

In any of the methods or cells, e.g., engineered cells, described herein, the cell expresses or comprises a product, e.g., a recombinant product, e.g., a next generation biologic selected from a group consisting of a bispecific antibody, a fusion protein, or a glycosylated protein.

In any of the methods or cells, e.g., engineered cells described herein, the cell is a CHO cell selected from the group consisting of CHO-K1 , CHOKISV, Potelligent CHOKISV (FUT8-KO), CHO GS-KO, Exceed (CHOKI SV GS-KO), CHO-S, CHO DG44, CHO DXBl 1 , CHOZN, or a CHO-derived cell.

Compositions and Preparations

In one aspect, the present disclosure also features a preparation of a product described herein made by a method described herein. In one embodiment, at least 70, 80, 90, 95, 98 or 99

%, by weight or number, of the products in the preparation are properly folded or assembled. In one embodiment, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, by weight or number, of the products in the preparation are aggregated. In one embodiment, less than 50%, 40%, 30%,

25%, 20%, 15%, 10%, or 5%, by weight or number, of the products in the preparation are fragments of the product.

In some embodiments, the present disclosure features a preparation of a polypeptide, e.g., a polypeptide of Table 2 or Table 3, made by a method described herein. In some embodiments, the cell used in the method is a CHO cell selected from the group consisting of CHOK1 ,

CHOKISV, Potelligent CHOKI SV, CHO GS knockout, CHOKI SV GS-KO, CHOS, CHO

DG44, CHO DXBl 1 , CHOZN, or a CHO-derived cell.

In one aspect, the present disclosure features a mixture comprising a cell described herein, e.g., a cell comprising a modification that modulates lipid metabolism, and a product produced by the cell. In one embodiment, the mixture comprises the product at a higher

concentration, e.g., at least, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, or 30% higher concentration, by weight or number, of product than would be seen without the modification. In one embodiment, at least 70%, 80%, 90%, 95%, 98 %or 99%, by weight or number, of the products in the mixture are properly folded or assembled. In one embodiment, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, by weight or number, of the products in the mixture are aggregated. In one embodiment, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, by weight or number, of the products in the mixture are fragments of the product. In some embodiments, the product is a recombinant polypeptide, e.g., a recombinant polypeptide of Table 2 or Table 3.

In one aspect, the present disclosure features a preparation of medium conditioned by culture of a cell described herein, wherein the cell comprises a modification that modulates lipid metabolism. In one embodiment, the product is present in the preparation at a higher concentration, e.g., at least, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, or 30% higher concentration, by weight or number, than would be seen without the modification. In one embodiment, at least 70%, 80%, 90%, 95%, 98% or 99 %, by weight or number, of the product in the preparation are properly folded or assembled. In one embodiment, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, by weight or number, of the products in the preparation are aggregated. In one embodiment, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, by weight or number, of the products in the preparation are fragments of the product. In some embodiments, the product is a recombinant polypeptide, e.g., a recombinant polypeptide of Table 2 or Table 3.

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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Headings, sub-headings or numbered or lettered elements, e.g., (a), (b), (i) etc., are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a series of immunofluorescent images obtained of Flp-In CHO engineered cell pools, separately transfected with either a control expression vector (Ctrl), or ones encoding SCDl fused at its C-terminus to a V5 epitope tag (SCD1-V5) or SREBFl fused at its C-terminus to a V5 epitope tag (SREBFl -V5). The pools were imaged with an anti-V5 primary antibody and a secondary anti-mouse FITC antibody (middle images) as well as DAPI (left images) and an overlay of both the left and middle images (right hand column) is shown. Images were generated using a Leica Confocal Microscope.

Figure 2 shows a series of immunofluorescent images obtained of CHOK1 SV glutamine synthetase knock-out (GS-KO) cell pools, transfected with either a control expression vector (Ctrl), or ones encoding SCD1 -V5 or SREBFl -V5. The pools were imaged with an anti-V5 primary and anti-mouse secondary TRTTC antibody (middle images) as well as DAPI (left images) and an overlay of both the left and middle images (right hand column) is also shown. Images were generated using a Leica Confocal Microscope.

Figures 3 A, 3B, and 3C show the determination of exogenous SCD1-V5 and SREBFl -V5 expressed in CHO Flp-In™ cell pools following transient transfection with a plasmid encoding a difficult to express recombinant Fc fusion protein (also referred to as Fc fusion protein or FP) (Fig. 3A) or eGFP (Fig. 3B). Fig 3C shows determination of exogenous V5-tagged SCDl and SREBFl expressed in untransfected stably expressing CHO Flp-In™ cell pools. Western blot analysis was performed on cell lysates obtained 96 hours following electroporation with the Fc fusion protein, as well as the cell pool solely expressing the indicated V5-tagged lipid metabolism modulator (LMM), SCDl or SREBFl . Anti-V5 primary antibody and anti-mouse HRP conjugated secondary antibody was used to detect expression of the V5-tagged LMM and anti- -actin or anti L7a (as indicated) followed by exposure with anti-mouse and anti-rabbit HRP conjugated secondary antibodies respectively were used as loading controls for LMM detection.

Figure 4 shows the viable cell concentration, as determined using a ViCell cell counter, of the CHO Flp-In cell pools engineered to stably overexpress the LMM SCD1-V5 and

SREBF1-V5 post transfection with eGFP-containing construct JB3.3 (n=2).

Figures 5 A and 5B show the cell culture concentration and culture viability at 24, 48, 72, and 96 hours after transfection of control, SCD1-V5, SREBF1 -V5 and SREBF410-V5 over-expressing CHOK1 SV GS-KO cell pools with an eGFP containing plasmid. Figure 5 A shows cell concentration. The lower columns represent viable cell concentration whilst the whole column represents the total concentration of cells; lower error bars represent the standard deviation of viable cells whilst upper error bars represent that of the total cell concentration. Figure 5B shows culture viability based on the data outlined in Fig. 5A. Error bars represent standard deviation. Statistical significance was calculated using two-tailed T-test compared to the control values of the particular time points: *Viable cell concentration significance using two-tailed T-test [p<0.05]. +Total cell concentration significance using two-tailed T-tests \p<0.05] (n=3).

Figures 6A, 6B and 6C show flow cytometry generated data using a FACSCalibur instrument (BD Biosciences). Median (Fig. 6A), geometric mean (Fig. 6B) and arithmetic mean (Fig. 6C) values were acquired at 24, 48, 72 and 96 hours post transfection with an eGFP containing plasmid where samples were taken from control, SCD1-V5 or SREBF1-V5 overexpressing Flp-In CHO cell pools (n=2).

Figures 7A, 7B and 7C show flow cytometry generated data using a FACSCalibur instrument (BD Biosciences). Median (Fig. 7A), geometric mean (Fig. 7B) values were acquired at 24, 48, 72 and 96 hours post transfection with an eGFP containing plasmid where samples were taken from control, SCD1-V5, SREBF1 -V5 or SREBF410-V5 overexpressing CHOK1 SV GS-KO derived cells. Figure 7C shows the total fluorescence per ml of culture as calculated by multiplying the measured arithmetic mean fluorescence by total cell concentration (xl06/ml). Error bars indicate standard deviation. Statistical significance was calculated using a two-tailed T-test compared to the control values of the particular time points (n=3). ^Indicates statistically significant values [p<0.05]. Data was generated using FACSCalibur (BD Biosciences).

Figures 8A and 8B show antibody A production in CHO Flp-In cells stably

overexpressing SCD1-V5 and SREBF1-V5 after transient transfection of a nucleic acid construct encoding antibody A heavy and light chains. Fig. 8A is a western blot showing bands

corresponding to antibody A, as detected by using an anti-heavy chain primary antibody and an anti-rabbit HRP conjugated secondary antibody. Fig. 8B shows the average fold change in antibody production in the LMM engineered cell pools compared to values generated from the control cell pool as determined by Protein A HPLC.

Figures 9A and 9B show the production of an Fc fusion protein in CHO Flp-In cell pools stably overexpressing SCD 1 -V5 and SREBF 1 -V5 after transient trans fection of a nucleic acid construct encoding the fusion protein. Fig. 9 A is a western blot showing the bands

representative of the Fc fusion protein as detected by using an anti-heavy chain primary antibody and an anti-rabbit HRP conjugated secondary antibody. Fig. 9B shows the average fold change in the Fc fusion protein production in the LMM engineered cell pools compared to values generated from the control cell pool as determined by Protein A HPLC.

Figures 10A and 10B show the production of a well expressed antibody A in CHO GSKO cell pools stably overexpressing SCD1-V5, SREBF 1-V5 and SREBF410-V5 after transient transfection of a nucleic acid construct encoding antibody A heavy and light chains at 48, 72 and 96 h post transfection and in a control, Null CHOK1 SV GS-KO cell pool (a control pool of cells generated using an empty plasmid to express selection GS gene only, no LMM agents). Fig. 1 OA is a western blot showing the bands representative of antibody A as detected by using an anti-heavy chain primary antibody and an anti-rabbit HRP conjugated secondary antibody. Fig. 10B shows the average fold change in antibody production in the LMM engineered cell pools compared to values generated in the control cell pool as determined by Protein A HPLC.

Figures 11 A and 1 IB shows the relative production of a difficult to express Fc fusion protein in CHOK1SV GS-KO cell pools stably overexpressing SCD1-V5 and SREBF 1 -V5 or in a control cell pool after transient transfection of a nucleic acid construct encoding the Fc fusion protein. Fig. 1 1 A shows a western blot of the transiently produced fusion protein, as detected by using an anti-heavy chain primary antibody followed by exposure with an anti-rabbit HRP conjugated secondary antibody. Fig. 1 IB shows the average fold change in the Fc fusion protein production in the LMM engineered cell pool compared to the control cell pools as determined by Protein A HPLC.

Figures 12A and 12B show the analysis of antibody A production from supernatant harvested after 48 and 72 hours from a CHO cell line stably expressing antibody A which have

been transiently transfected with plasmid constructs containing either control (empty), SCD1-V5, SREBF1-V5 or SREBF410-V5 genes. Fig. 12A shows a western blot of the supernatants from the cells; antibody A was detected by using an anti-heavy chain primary antibody followed by exposure with an anti-rabbit HRP conjugated secondary antibody. Figure 12B shows Coomassie analysis in which the bands show the relative levels of antibody A present in the supernatant at 168 hours post transfection.

Figure 13 shows analysis of antibody A production from supernatant harvested after 48, 72, 96 and 144 hours from a CHO cell line stably expressing antibody A which had been transiently transfected with plasmid constructs containing either control (empty), SCD1-V5, SREBF 1 -V5 or SREBF410-V5 genes where protein A Octet analysis was used to determine volumetric antibody concentration (n=2).

Figure 14 shows analysis of an FC fusion protein from supernatant samples harvested after 48, 72, 96 and 144 hours from a CHO cell line stably expressing antibody A which had been transiently transfected with plasmid constructs containing either control (empty), SCD1-V5, SREBF 1-V5 or SREBF410-V5 genes where viable cell number and protein A titre

measurements were used to determine specific productivity of the FC fusion protein Error bars show standard deviation (n=3).

Figure 15A and 15B shows analysis of antibody A production from supernatant samples harvested after 48, 72, 96 and 144 hours from CHO cell pools stably integrated with control, SCD1 -V5 or SREBF 1-V5 containing vectors and subsequently stably integrated with an antibody A construct. Figure 15A shows volumetric antibody concentration whilst Figure 15B shows specific productivity of antibody A. Error bars show standard deviation (n=3).

Figure 16A and 16B shows analysis of FC fusion protein production from supernatant samples harvested after 48, 72, 96 and 144 hours from a CHO cell pools stably integrated with control, SCD1-V5, SREBF 1-V5 or SREBF410-V5 containing vectors and subsequently stably integrated with an FC fusion protein construct. Figure 16A shows volumetric FC fusion protein concentration whilst Figure 16B shows specific productivity of the FC fusion protein. Error bars show standard deviation (n=3).

Figures 17A shows western analysis of immunocytokine expression from CHO GSKO cells following transient transfection of a nucleic acid construct encoding genes appropriate for expression of the immunocytokine and either no LMM (control), SCD1 , SREBF 1 or SREB411

genes at 48 and 96 h post transfection. Supernatant samples were reduced and bands present detected using an anti heavy chain primary antibody followed by exposure to an anti-rabbit HRP conjugated secondary antibody. The lower band represents a native heavy chain antibody whilst the upper band is indicative of a heavy chain molecule fused to a cytokine. Figure 17B shows relative immunocytokine abundance of samples obtained at 96 hours post transfection.

DETAILED DESCRIPTION

As both current and next generation biologies continue to gain therapeutic utility in patients, the demand for large quantities of next generation biologic products having a high grade of quality for therapeutic use, as well as efficient means for production and efficient

development of production cell line will escalate. Furthermore, many next generation biologies are difficult to express and produce in conventional cell lines using conventional expression techniques known in the art. The current methods are not sufficient to produce these products in the large quantities and at the high grade of quality required for clinical use. As such, the present disclosure features methods and compositions for obtaining higher yields of a product, e.g., a next generation biologies, with improved quality as compared to the yield and quality obtained from current production methods. The methods and compositions described herein are also useful for engineering cells or cell lines with improved productivity, product quality, robustness, and/or culture viability, as compared to the cell expression systems currently used to produce recombinant products.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of and/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell" can mean one cell or more than one cell.

"Component of a lipid metabolism pathway", as used herein, refers to a molecule, polypeptide, or enzyme that, directly or indirectly, synthesizes a lipid, degrades a lipid, converts a lipid from one lipid species to another lipid species, or modifies a lipid. In one embodiment, the component can be an enzyme substrate, an enzyme reaction product, or an enzyme cofactor. In one embodiment, the component of a lipid metabolism pathway is a LMM. In one

embodiment, the component of a lipid metabolism pathway is provided in Table 1.

"Endogenous", as used herein, refers to any material from or naturally produced inside an organism, cell, tissue or system.

"Exogenous", as used herein, refers to any material introduced to or produced outside of an organism, cell, tissue or system. Accordingly, "exogenous nucleic acid" refers to a nucleic acid that is introduced to or produced outside of an organism, cell, tissue or system. In an embodiment, sequences of the exogenous nucleic acid are not naturally produced, or cannot be naturally found, inside the organism, cell, tissue, or system that the exogenous nucleic acid is introduced into. In embodiments, non-naturally occurring products, or products containing portions that are non-naturally occurring are exogenous materials with respect to the host cells described herein.

"Forming", as used herein, refers to introducing into the cell, synthesizing within the cell, or any other process that results in the nucleic acid encoding a LMM or an exogenous LMM being located within the cell.

"Heterologous", as used herein, refers to any material from one species, when introduced to an organism, cell, tissue or system from a different species. In embodiments, a heterologous material also encompasses a material that includes portions from one or multiple species or portions that are non-naturally occurring. By way of example, in an embodiment, a nucleic acid encoding a fusion protein wherein a portion of the fusion protein is human, a portion of the fusion protein is bacteria, and a portion of the fusion protein is non-naturally occurring, and the nucleic acid is introduced to a human cell, the nucleic acid is a heterologous nucleic acid.

"Lipid metabolism pathway", as used herein, refers to a process associated with the synthesis of a lipid or lipid-associated molecule, the elongation of a lipid or lipid-associated molecule, the degradation of a lipid or lipid-associated molecule, the incorporation of a lipid or lipid-associated molecule into a membrane, the state of saturation of a lipid or lipid-associated molecule (e.g., saturated or unsaturated), or conversion or modification of the chemical structure (e.g., re-esterification) of a lipid or lipid-associated molecule. In one embodiment, the lipid metabolism pathway results in lipid synthesis, lipid elongation, lipid degradation, changes in membrane composition or fluidity, formation or modulation of lipid rafts, or modification or conversion of a lipid (e.g., saturation or de-saturation of a lipid, or re-esterification of a lipid). Examples of lipid metabolism pathways include, but are not limited to: de novo lipogenesis, fatty acid re-esterification, fatty acid saturation, fatty acid de-saturation, fatty acid elongation, and phospholipid biosynthesis, and unfolded protein response.

"Lipid metabolism modulator" or "LMM", as used herein, refers to a molecule, gene product, polypeptide, or enzyme that modulates, e.g., increases or decreases, one or more of the following: the expression (e.g., transcription or translation) of a component involved in a lipid metabolism pathway; the activity (e.g., enzymatic activity) of a component, e.g., gene product, involved in a lipid metabolism pathway; the level or amount of lipids present in a cell; the level or amount of lipid rafts or rate of lipid raft formation; the fluidity, permeability, or thickness of a cell membrane, e.g., plasma membrane or an organelle membrane; the conversion of saturated lipids to unsaturated lipids or vice versa; the level or amount of saturated lipids or unsaturated lipids in a cell, e.g., monounsaturated lipids; lipid composition to achieve a favorable lipid composition that has a favorable impact on the activity of the ER; the expansion of the ER; the expansion of the Golgi; the level or amount of secretory vesicles or secretory vesicle formation; the level or rate of secretion; activation or inactivation of membrane receptors (e.g., ATR (see e.g., The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR. Zhang XH, Zhao C,

Ma ZA. J Cell Sci. 2007 Dec l ;120(Pt 23):4134-43 PMID: 18032786; ATR (ataxia telangiectasia mutated- and Rad3 -related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53. Roobol A, Roobol J, Carden MJ, Bastide A, Willis AE, Dunn WB, Goodacre R, Smales CM. Biochem J. 2011 Apr 15;435(2):499-508. doi: 10.1042/BJ20101303. PMID: 21284603) and SREPB (see e.g., Int J Biol Sci. 2016 Mar 21; 12(5):569-79. doi:

10.7150/ijbs.14027. eCollection 2016. Dysregulation ofthe Low-Density Lipoprotein Receptor Pathway Is Involved in Lipid Disorder-Mediated Organ Injury. Zhang Y, Ma KL, Ruan XZ, Liu BC); and additional receptors, see e.g., Biochim Biophys Acta. 2016 Mar 17. pii: S1388-1981(16)30071 -3. doi: 10.1016/j.bbalip.2016.03.019; and/or the unfolded protein response (UPR) . In one embodiment, the LMM comprises a polypeptide. In one embodiment, the LMM comprises a transcriptional regulator, e.g., a transcription factor. In one embodiment, the LMM comprises SREBFl or a functional fragment thereof (e.g., SREBF-410). In one embodiment, the LMM comprises an enzyme. In one embodiment, the LMM comprises SCD1 or a functional fragment thereof.

"Modification" as used herein in the expression "modification that modulates lipid metabolism" refers to an agent that is capable of effecting an increase or decrease in the expression or activity of a component, e.g., gene product, of a lipid metabolism pathway described herein. In embodiments, the modification results in increasing the expression or activity of a component of a lipid metabolism pathway, e.g., a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 99%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more increase in expression or activity of a component of a lipid metabolism pathway, e.g., as compared to the expression or activity of the component in the absence of the modification. In embodiments, the modification results in decreasing the expression or activity of a component of a lipid metabolism pathway, e.g., a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 99%%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more decrease in expression or activity of a component of a lipid metabolism pathway, e.g., as compared to the expression or activity of the component in the absence of the modification. In some embodiments where the expression or activity of a component of the lipid metabolism pathway is decreased, the component is a negative regulator of a lipid metabolism pathway. In one embodiment, the modification comprises a heterologous or exogenous nucleic acid sequence encoding a lipid metabolism modulator. In one embodiment, the modification is an exogenous lipid metabolism modulator, e.g., small molecule or polypeptide, that can be introduced to a cell, e.g., by culturing the cell in the presence of the molecule or polypeptide, to modulate the lipid metabolism of the cell.

The terms "nucleic acid", "polynucleotide", and "nucleic acid molecule", as used interchangeably herein, refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA thereof, and polymers thereof in either single-, double-, or triple-stranded form. The term "nucleic acid" includes, but is not limited to, a gene, cDNA, or an mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless specifically limited, the term encompasses molecules containing analogues or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91 -98 (1994)).

"Peptide," "polypeptide," and "protein", as used interchangeably herein, refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. In one embodiment, a protein may comprise of more than one, e.g., two, three, four, five, or more, polypeptides, in which each polypeptide is associated to another by either covalent or non-covalent bonds/interactions. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.

"Recombinant product" refers to a product that can be produced by a cell or a cell-free system. The product can be a molecule, a nucleic acid, a polypeptide, or any hybrid thereof. A recombinant product is one for at which at least one component of the product or at least one nucleotide of a sequence which controls the production or expression of the product, was formed by genetic engineering. Genetic engineering as used herein to generate a recombinant product or a construct that encodes a recombinant product encompasses recombinant DNA expression techniques known in the art (e.g., as described in Current Protocols in Molecular Biology); site-directed, scanning, or random mutagenesis; genome modification strategies employing CRISPR-based strategies; and zinc finger nuclease (ZFN)-based strategies. By way of example, in embodiments where the recombinant product is a nucleic acid, at least one nucleotide of the recombinant nucleic acid, or at least one nucleotide of a sequence that controls the production, e.g., transcription, of the recombinant nucleic acid was formed by genetic engineering. In one embodiment, the recombinant product is a recombinant polypeptide. In one embodiment, the recombinant product is a naturally occurring product. In one embodiment, the recombinant product is a non-naturally occurring product, e.g., a synthetic product. In one embodiment, a portion of the recombinant product is naturally occurring, while another portion of the recombinant product is non-naturally occurring. In another embodiment, a first portion of the recombinant product is one naturally occurring molecule, while another portion of the recombinant product is another naturally occurring molecule that is different from the first portion.

"Recombinant polypeptide" refers to a polypeptide that can be produced by a cell described herein. A recombinant polypeptide is one for which at least one nucleotide of the sequence encoding the polypeptide, or at least one nucleotide of a sequence which controls the expression of the polypeptide, was formed by genetic engineering or manipulation (of the cell or of a precursor cell). E.g., at least one nucleotide was altered, e.g., it was introduced into the cell or it is the product of a genetically engineered rearrangement. In an embodiment, the sequence of a recombinant polypeptide does not differ from a naturally or non-naturally occurring isoform of the polypeptide or protein. In an embodiment, the amino acid sequence of the recombinant polypeptide differs from the sequence of a naturally occurring or a non-naturally isoform of the polypeptide or protein. In an embodiment, the recombinant polypeptide and the cell are from the same species. In an embodiment, the amino acid sequence of the recombinant polypeptide is

the same as or is substantially the same as, or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from, a polypeptide encoded by the endogenous genome of the cell. In an embodiment, the recombinant polypeptide and the cell are from the same species, e.g., the recombinant polypeptide is a human polypeptide and the cell is a human cell. In an embodiment, the recombinant polypeptide and the cell are from different species, e.g., the recombinant polypeptide is a human polypeptide and the cell is a non-human, e.g., a rodent, e.g., a CHO, other mammalian cell, an insect cell, a plant cell, a fungal cell, a viral cell, or a bacterial cell. In an embodiment, the recombinant polypeptide is exogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In an embodiment, the recombinant polypeptide is a human polypeptide or protein which does not differ in amino acid sequence from a naturally or non-naturally occurring isoform of the human polypeptide or protein. In an embodiment, the recombinant polypeptide differs from a naturally or non-naturally occurring isoform of the human polypeptide or protein at no more than 1 , 2, 3, 4, 5, 10, 15 or 20 amino acid residues. In an embodiment, the recombinant polypeptide differs from a naturally occurring isoform of the human polypeptide at no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% of its amino acid residues. In embodiments where a portion of the recombinant polypeptide comprises a sequence derived from a portion of a naturally or non-naturally occurring isoform of a human polypeptide, the portion of the recombinant polypeptide differs from the corresponding portion of the naturally or non-naturally occurring isoform by no more than 1, 2, 3, 4, 5, 10, 15, or 20 amino acid residues, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% of its amino acid residues.

"Homologous", "identity", or "similarity" as used herein refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten

subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

The term "next generation biologic" or "NGB" as used herein refers to a biological composition comprising a cell or a composition produced by a cell. The biological composition is selected from the group consisting of a composition with at least one natural component, a composition with at least one natural component and at least one non-natural component, a composition with at least one natural component and at least one natural structure, and a composition with at least one natural component and at least one non-natural structure, or any combinations thereof. Next generation biologies often comprise complex and/or non-natural structures. Examples of next generation biologies include, but are not limited to, fusion proteins, enzymes or recombinant enzymes, proteins or recombinant proteins, recombinant factors with extended half-lives, growth hormones with long acting therapies, multimeric glycoproteins, next generation antibodies, antibody fragments, or antibody-like proteins (ALPs), vesicles, exosomes, liposomes, viruses, and virus-like particles, mucins, nanoparticles, extracts of a cell, and a cell being used as a reagent.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

MODULATION OF LIPID METABOLISM

The present disclosure features methods and compositions for modulating lipid metabolism in a cell or a cell-free system, for example, by introducing a modification to the cell or cell-free system that results in the modulation of lipid metabolism. In embodiments, the present disclosure features the use of global regulators that impact multiple aspects of pathways or processes involved in lipid metabolism, e.g., the de novo lipogenesis, fatty acid re-esterification, fatty acid saturation or desaturation, fatty acid elongation, and phospholipid

biosynthesis pathways. By way of example, the global regulator is upstream in one or more lipid metabolism pathways or processes such that the global regulator impacts several, e.g., two or more, downstream processes or downstream components of lipid metabolism. In one embodiment, the global regulator is a transcription factor that can activate the expression of more than one, e.g., two or more, target genes involved in different lipid metabolism processes or pathways. Accordingly, without wishing to be bound by any theory, the use of a global regulator as described herein can result in a greater increase in production capacity, robustness, and survival of the cell than compared to the use of a downstream effector that modulates only a single target or other component of lipid metabolism. While not wishing to be bound by any theory, it is believed that a global or more widespread modulation of multiple lipid metabolism pathways increases the production capacity of a cell by affecting more processes involved in improving production capacity, product quality, and robustness of the cell.

Lipid metabolism pathways as described herein refer to processes that relate to the synthesis, degradation, conversion, or modification of lipids or lipid-associated molecules. Lipid molecules include, but are not limited to, fatty acids, glycerolipids, glycerophospholipids, phospholipids, saccharolipids, sphingolipids, and sterol lipids, e.g., cholesterol, and polyketides. Examples of lipid metabolism pathways include, but are not limited to: de novo lipogenesis, fatty acid re-esterification, fatty acid saturation, fatty acid de-saturation, fatty acid elongation, and phospholipid biosynthesis. In one embodiment, the methods described herein provide a cell comprising a modification that modulates lipid metabolism. The modification that modulates lipid metabolism can be an agent that increases or decreases the expression of a component involved in lipid metabolism. In one embodiment, the modification that modulates lipid metabolism comprises an exogenous nucleic acid encoding a lipid metabolism modulator (LMM). In such embodiments, the exogenous nucleic acid encoding a LMM is introduced to the cell by any of the nucleic acid delivery methods or techniques described herein, e.g., transduction or transfection

In one embodiment, the methods described herein provide a cell comprising one or more, e.g., one, two, three, four, five, six, seven, eight, nine or ten, modifications that modulate lipid metabolism. In embodiments where the cell comprises two or more modifications that modulate lipid metabolism, each modification that modulates lipid metabolism comprises an exogenous nucleic acid that encodes a LMM. In one embodiment, each of the two or more exogenous

nucleic acids that encode a LMM can be located within the same nucleic acid molecule, or are placed on two or more different nucleic acid molecules. In such embodiments where the cell comprises two or more nucleic acid sequences encoding LMMs, the LMMs are different from each other, e.g., encode a different polypeptide sequence or have a different function.

In embodiments, modulation of lipid metabolism in a cell, e.g., by introducing and expressing an exogenous nucleic acid encoding an LMM described herein, alters, e.g., increases or decreases, one or more of the following:

i) the expression (e.g., transcription and/or translation) of a component involved in a lipid metabolism pathway;

ii) the activity (e.g., enzymatic activity) of a component involved in a lipid metabolism pathway;

iii) the amount of lipids (e.g., phospholipids, or cholesterol) present in a cell;

iv) the amount of lipid rafts or rate of lipid raft formation;

v) the fluidity, permeability, and/or thickness of a cell membrane (e.g., a plasma membrane, a vesicle membrane, or an organelle membrane);

vi) the conversion of saturated lipids to unsaturated lipids or conversion of unsaturated lipids to saturated lipids;

vii) the amount of saturated lipids or unsaturated lipids, e.g., monounsaturated lipids; viii) the composition of lipids in the cell to attain a favorable composition that increases ER activity;

ix) the expansion of the ER (e.g., size of the ER, the ER membrane surface, or the amounts of the proteins and lipids that constitute and/or reside within the ER); x) the expansion of the Golgi (e.g., the number and size of the Golgi, the Golgi surface, or the number or amounts of proteins and molecules that reside within the Golgi);

xi) the amount of secretory vesicles or the formation of secretory vesicles;

xii) the amount or rate of secretion of the product;

xiii) the proliferation capacity, e.g., the proliferation rate;

xiv) culture viability or cell survival;

xv) activation of membrane receptors;

xvi) the unfolded protein response (UPR);

xvii) the yield or rate of production of the product;

xviii) the product quality (e.g., aggregation, glycosylation heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling); and /or

xix) cell growth/proliferation or cell specific growth rate.

In an embodiment, modulation of lipid metabolism results in an increase in any of the properties listed above, e.g., a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, increase in any of the properties listed above as compared to a cell without modulation of lipid metabolism. In an embodiment, modulation of lipid metabolism results in a decrease in any of the properties listed above, e.g., a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, decrease in any of the properties listed above as compared to a cell without modulation of lipid metabolism.

In an embodiment, a modification that modulates lipid metabolism increases or decreases the expression or activity of a component involved in one or more lipid metabolism pathways.

In embodiments where the modification that modulates lipid metabolism results in an increase in the expression, e.g., transcription or translation, or an increase in the activity of a component of a lipid metabolism pathway, the component is a positive regulator of the lipid metabolism pathway. In embodiments where the modification that modulates lipid metabolism results in a decrease in the expression, e.g., transcription or translation, or a decrease in the activity of a component of a lipid metabolism pathway, the component is a negative regulator of the lipid metabolism pathway. Assays for quantifying the expression, e.g., transcription and/or translation, of a gene of the lipid metabolism pathway, are known in the art, and include quantifying the amount of mRNA encoding the gene; or quantifying the amount of the gene product, or polypeptide; PCR-based assays, e.g., quantitative real-time PCR; Northern blot; or microarray. Assays for quantifying the activity of a component of the lipid metabolism pathway, e.g., an enzyme of the lipid metabolism pathway, will be specific to the particular component of the lipid metabolism pathway.

In embodiments where the modulation of the lipid metabolism of a cell results in an increase in the level or amount of lipids in the cell, the total level or total amount of lipids in the cell can be increased. In another embodiment, the level or amount of one or more species of lipids, e.g., a phospholipid or cholesterol, in the cell can be increased. An increase in the level or amount of lipids in the cell (e.g., total or a select lipid species) comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or a one-fold, two-fold, three-fold, four- fold, or five-fold, 10-fold, 20-fold, 50-fold, or 100-fold, increase in the level or amount of lipids in the cell after modulation of lipid metabolism, e.g., live cells, as compared to cells that do not comprise a modification that modulates lipid metabolism. Assays for quantifying the level or amount of lipids in a cell are known in the art, and include enzymatic assays and oxidation assays and measurement by mass spectrometry of lipid components in a particular compartment (e.g., organelle) or from the total cell.

In one embodiment, a modification that modulates lipid metabolism results in increased cell survival. For example, cell survival can be measured by determining or quantifying cell apoptosis, e.g., the number or amount of cells that have been killed or died. An increase in cell survival comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or a one-fold, two-fold, three-fold, fourfold, or five-fold, 10-fold, 20-fold, 50-fold, or 100-fold„ increase in the number of cells after modulation of lipid metabolism, e.g., live cells, as compared to cells that do not comprise a modification that modulates lipid metabolism. Alternatively, an increase in cell survival comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more decrease in the number of apoptotic cells after modulation of lipid metabolism, e.g., as compared to cells without modulation of lipid metabolism. Methods for detecting cell survival or apoptosis are known in the art, e.g., Annexin V assays, and are described herein in the Examples.

In one embodiment, a modification that modulates lipid metabolism results in increased culture viability. For example, culture viability can be measured by determining or quantifying the number or amount of live cells, e.g., live cells in a culture or population of cells, or cells that have a characteristic related to being viable, e.g., proliferation markers, intact DNA, or do not display apoptotic markers. An increase in culture viability comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or a one-fold, two-fold, three-fold, four-fold, or five-fold, 10-fold, 20-fold, 50-fold, or 100-fold, or more increase in the number of cells, e.g., live cells, after modulation of lipid metabolism, e.g., as compared to cells without modulation of lipid metabolism. Methods for determining culture viability are known in the art, and are described herein in Example 3. Other methods for assessing culture viability include, but are not limited to, trypan blue exclusion methods followed by counting using a hemocytometer or Vi-CELL (Beckman-Coulter). Other methods for determining viable biomass include methods using radiofrequency impedance or capacitance (e.g., Carvell and Dowd, 2006, Cytotechnology, 50:35-48), or using Raman spectroscopy (e.g., Moretto et al., 2011, American Pharmaceutical Review, Vol. 14).

In one embodiment, a modification that modulates lipid metabolism results in increased cell proliferation. For example, the ability of a cell to proliferate can be measured by

quantifying or counting the number of cells, cell doublings, or growth rate of the cells.

Alternatively, proliferating cells can be identified by analysis of the genomic content of the cells (e.g., replicating DNA), e.g., by flow cytometry analysis, or presence of proliferation markers, e.g., Ki67, phosphorylated cyclin-CDK complexes involved in cell cycle. An increase in the ability to proliferate comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or one-fold, twofold, three-fold, four- fold, five-fold, 10-fold, 20-fold, 50-fold, or 100-fold, or more increase in the number of cells, or number of cells expressing a proliferation marker, after modulation of lipid metabolism. Alternatively, an increase in the ability to proliferate comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or one-fold, two-fold, three-fold, four-fold, five-fold, 10-fold, 20-fold, 50-fold, or 100-fold, or more increase in the doubling or growth rate of the cells after modulation of lipid metabolism. Cell counting can be performed using a cell counting machine, or by use of a hemacytometer.

In one embodiment, a modification that modulates lipid metabolism results in an increase in production capacity, e.g., the amount, quantity, or yield of product produced, or the rate of production. An increase in the amount, quantity, or yield of the product produced comprises 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more increase in the amount, quantity, or yield of the product produced after modulation of lipid metabolism, e.g., as compared to the amount, quantity, or yield of the product produced by a cell without modulation of the lipid metabolism. An increase in the rate of production comprises 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or byl-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more increase in the amount, quantity, or yield of the product produced after modulation of lipid metabolism, after modulation of lipid metabolism, e.g., as compared to the rate of production of a cell without modulation of the lipid metabolism. In one embodiment, the rate of production is determined by determining the amount, quantity, or yield of the product produced in a specific unit of time.

In one embodiment, a modification that modulates lipid metabolism results in an increase in the quality of the product, e.g., aggregation, glycosylation status or heterogeneity,

fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling. An increase quality of the product comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more of: an increase in the amount or quantity of non-aggregated product, an increase in the ratio of non-aggregated product to aggregated product, or decrease in the amount or quantity of aggregated product, after modulation of lipid metabolism e.g., as compared to that observed in a cell without modulation of the lipid metabolism. An increase quality of the product comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more of: an increase in the amount or quantity of properly folded or assembled product, an increase in the ratio of properly folded or assembled product to misfolded, unfolded, partially assembled, or non-assembled product, or decrease in the amount or quantity of misfolded, unfolded, partially assembled, or non-assembled product, after modulation of lipid metabolism e.g., as compared to that observed in a cell without modulation of the lipid metabolism. An increase quality of the product comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more of: an increase in the amount or quantity of non-fragmented or full-length product, or a decrease in the amount or quantity of fragmented product after modulation of lipid metabolism, e.g., as compared to that observed in a cell without modulation of the lipid metabolism. An increase quality of the product comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,

90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more of: an increase in the amount or quantity of functional product, or a decrease in the amount or quantity of non-functional or dysfunctional product after modulation of lipid metabolism, e.g., as compared to that observed in a cell without modulation of the lipid metabolism. An increase quality of the product comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold, 100-fold or more of: an increase or decrease in the glycan heterogeneity after modulation of lipid metabolism, e.g., as compared to that observed in a cell without modulation of the lipid metabolism. An increase quality of the product comprises a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more, or by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more of: an increase in the amount or quantity of functional product, or a decrease in the amount or quantity of non-functional or dysfunctional product after modulation of lipid metabolism, e.g., as compared to that observed in a cell without modulation of the lipid metabolism.

LIPID METABOLISM MODULATORS

As described herein, modulation of the lipid metabolism can be achieved by expressing or introducing a LMM, or by altering the regulation of a LMM. In one embodiment, an LMM is overexpressed in a cell, e.g., by introducing an exogenous nucleic acid encoding a LMM or by increasing expression by introducing promoter elements or other regulatory transcriptional elements. In another embodiment, the expression or activity of an LMM is inhibited or decreased, e.g., by introducing an inhibitor of the LMM or an exogenous inhibitory nucleic acid, e.g., an R A interfering agent. Examples of inhibitory nucleic acids include short interfering R As (siR As) and short hairpin R As (shRNAs) that target the LMM, e.g, the mR A encoding the LMM. In one embodiment, the activity or expression of an LMM is increased or decreased by altering the post-translational modifications or other endogenous regulatory mechanisms that regulate LMM activity or expression. Regulation by post-translational modifications include, but are not limited to, phosphorylation, sumoylation, ubiquitination, acetylation, methylation, or glycosylation can increase or decrease LMM expression or activity.

By way of example, regulation of post-translational modifications can be achieved through modulation of the enzyme or molecule that modifies the LMM, or modification of the LMM such that the post-translational modification cannot occur or occurs more frequently or constitutively. Regulation of the LMM can also include modulating endogenous regulatory mechanisms that can increase or decrease LMM expression or activity, e.g., increase or decrease one or more of: miRNA regulation, protein cleavage, expression of specific isoforms, alternative splicing, and degradation.

In one embodiment, the LMM modulates, e.g., increases or decreases, the expression, e.g., transcription, or activity of a component of the lipid metabolism pathway. In another embodiment, the LMM modulates, e.g., increases or decreases, the synthesis, degradation, elongation, or structural conformation (e.g., saturation or desaturation, or esterification) of a lipid or lipid-associated molecule. Exemplary LMMs and/or components of the lipid metabolism pathway are listed, but not limited, to those listed in Table 1.

Table 1. Lipid Metabolism Pathways and Components/Gene Products Thereof

KDSR (3-ketosphinganine reductase)

LCS (polypeptide N-acetylgalactosaminyltransferase)

PAP (phosphatidic acid phosphatase)

PEMT (phosphatidylethanolamine N-methyltransferase)

PGP (phosphatidylglycerophosphatase)

PGS (CDP-diacylglycerol-glycerol-3 -phosphate 3- phosphatidyltransferase)

PIS (CDP-diacylglycerol-inositol 3-phosphatidyltransferase)

PSD (phosphatidylserine decarboxylase)

PSS1 (phosphatidylserine synthase 1)

PSS2 (phosphatidylserine synthase 2)

SGMS (ceramide choline phosphotransferase)

SNAT (sphingosine N-acyltransferase)

SPK (sphinganine kinase)

SPP (sphingosine- 1 -phosphate phosphatase)

SPT (serine Co-palmitoyltransferase)

Fatty Acid Desaturation SCD1 (stearoyl CoA desaturase-1)

SCD2 (stearoyl CoA desaturase-2)

SCD3 (stearoyl CoA desaturase-3)

SCD4 (stearoyl CoA desaturase-4)

SCD5 (Steoryl CoA desaturase-5)

PED (plasmanylethanolamine desaturase)

Regulation of SREBF1 and other SIP (site- 1 protease)

pathways S2P (site-2 protease)

SCAP (SREBF cleavage-activating protein)

INSIG1 (insulin induced gene 1)

INSIG2 (insulin induced gene 2)

HMG CoA reductase (2-hydroxy-3-methylgulatryl-CoA reductase)

PPAR receptors, e.g., PPARa, PPARy

In one embodiment, the LMM comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity or homology with a component, e.g., gene product, involved in a lipid metabolism pathway, e.g., provided in Table 1 ; or differs by 1 , 2, or 3 or more amino acid residues but no more than 50, 40, 30, 20, 15, or 10 amino acid residues from the amino acid sequence of a component, e.g., gene product, involved in the lipid metabolism pathway, e.g., provided in Table 1.

In one embodiment, the LMM comprises a functional fragment of a component involved in the lipid metabolism pathway, e.g., provided in Table 1. A functional fragment of an LMM as

described herein may comprise one or more functional domains of the LMM. By way of example, a functional fragment of a LMM that is a transcription factor comprises a DNA binding domain and a transactivation domain. By way of example, a functional fragment of a LMM that is an enzyme comprises a domain with enzymatic activity. A functional fragment of an LMM as described herein retains functional activity, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%), 80%), or 90%> of the functional activity, of the full-length LMM. Functional fragments of an LMM can be experimentally determined by one skilled in the art, or can be predicted using algorithms based on sequence homology of functional domains. Exemplary LMMs are further described below.

In any of the embodiments of the methods described herein, the LMM is a transcriptional regulator. In one embodiment, the LMM is a transcription factor or transcriptional activator, that binds to the DNA or associates in a complex that binds to DNA, and recruits or associates in a complex that recruits RNA polymerase for transcription of one or more gene products involved in lipid metabolism. In one embodiment, the LMM binds to a sterol binding element and/or E-box promoter sequences. In one embodiment, the LMM comprises sterol regulatory element binding factor 1 (SREBF1) or sterol regulatory element binding factor 2 (SREBF2) or a functional fragment or isoform thereof.

In an embodiment, the LMM comprises a global transcriptional activator or transcription factor. In one embodiment, the LMM is capable of modulating the transcription of two or more, e.g., two, three, four, five, six, or more, components of a lipid metabolism pathway, e.g., as provided in Table 1. In another embodiment, the LMM is capable of modulating the transcription of one or more, e.g., one, two, three, four, or five, or more, components of two or more lipid metabolism pathways, e.g., components and pathways as provided in Table 1.

Sterol regulatory element binding factor 1 (SREBF1) is a global transcriptional activator which upregulates the transcription of genes involved in lipogenesis, fatty acid re-esterification, fatty acid desaturation and elongation, and phospholipid biosynthesis by binding to sterol regulatory element (SRE) and E-box promoter sequences (Hagen, Rodriguez-Cuenca et al. 2010) present in the promoter regions of target genes. Transcription of the SREBF1 gene itself is endogenously regulated by the presence of the sterol regulatory element (SRE) amongst other transcriptional regulating elements in the promoter region of the gene. On top of this, a multitude of posttranslational regulating mechanisms including phosphorylation, ubiquitination, sumoylation, acetylation, fatty acid-mediated modifications and proteolytic processing make for a tightly controlled but adaptable homeostatic system fixed around SREBF1.

Full-length SREBF1 is synthesized and localizes primarily to the endoplasmic reticulum (ER). Membrane integral SREBFl forms a complex with SREBF cleavage-activating protein (SCAP) which can facilitate migration of SREBFl to the Golgi. However, when high sterol levels (particularly cholesterol) are present, a conformational change in SCAP is induced which aids binding to the membrane integral protein insig (insulin induced gene), thus inhibiting migration of this complex. In the absence of sterols, insig does not bind to SCAP, therefore allowing COPII mediated vesicle formation, and subsequent migration of the

SREBF:SCAP complex to the Golgi. Sequential proteolytic cleavage occurs in the Golgi mediated by site-1 protease (SIP) and site -2 protease (S2P) proteins liberating the N-terminal basic helix loop helix leucine zipper (bHLHlz) of SREBFl which is immediately present in the cytoplasm, but migrates to the nucleus. Lysine residues present on the cleaved SREBFl are ubiquitinated and degraded by the 26S proteasome but this ubiquitination can be inhibited through acetylation of the lysine residues which allows migration to the nucleus. Finally, nuclear SREBFl can bind to sterol regulatory element (SRE) sequences upstream of a number of genes responsible for de novo lipogenesis (fatty acid synthase (FAS) and acetyl coA carboxylase (ACQ), fatty acid re-esterfication (diacylglycerol acyltransferase (DGAT), glycerol-3 -phosphate (GPAT) and lipoprotein lipase (LPL)), phospholipid biosynthesis

(CTP:phosphocholine cytidylyltransferase (CCT)), fatty acid desaturation (stearoyl-coA desaturase 1 (SCD1)). Nuclear SREBFl is also capable of activating transcription of the full length SREBFl gene itself, but this is also dependent on activation of the liver X receptor (LXR) promoter sequence also located upstream of the gene (Brown, Goldstein 1997-BROWN, M.S. and GOLDSTEIN, J.L., 1997. The SREBP Pathway: Regulation of Cholesterol Metabolism by Proteolysis of a Membrane-Bound Transcription Factor. Cell, 89(3), pp. 331-340) (Hagen, Rodriguez-Cuenca- HAGEN, R.M., RODRIGUEZ-CUENCA, S. and VIDAL-PUIG, A., 2010. An allostatic control of membrane lipid composition by SREBPl . FEBS letters, 584(12), pp. 2689-2698).

What is claimed is:

1. A method for producing a product, e.g., a polypeptide, e.g., a recombinant polypeptide, by a cell, comprising:

i) providing a cell comprising a modification that modulates lipid metabolism, and ii) culturing the cell, e.g., under conditions suitable for modulation of lipid metabolism by the modification,

thereby producing the product, e.g., polypeptide, e.g., recombinant polypeptide.

2. The method of claim 1, wherein the cell comprises an exogenous nucleic acid that encodes the polypeptide, e.g., a polypeptide selected from Table 2 or 3.

3. The method of either of claims 1 or 2, wherein the modification provides increased production or improved quality of the polypeptide as compared to a cell not having the modification.

4. The method of any of claims 1-3, comprising forming, in the cell, an exogenous nucleic acid encoding a lipid metabolism modulator (LMM) or an exogenous LMM, and optionally wherein forming comprises introducing an exogenous nucleic acid encoding a lipid metabolism modulator, e.g., which increases the expression of an endogenous nucleic acid encoding a LMM.

5. The method of any of claims 1-4, comprising a second modification that modulates lipid metabolism and wherein the second modification comprises a second exogenous nucleic acid encoding a second LMM, e.g., a LMM different from the LMM of the first modification.

6. The method of claim 5, wherein:

i) the second exogenous nucleic acid and the first exogenous nucleic acid are disposed on the same nucleic acid molecule, or

ii) the second exogenous nucleic acid and the first exogenous nucleic acid are disposed on different nucleic acid molecules.

7. The method of either of claims 5 or 6, wherein the second modification provides increased production or improved quality of the product, as compared to a cell not having the second modification.

8. The method of any of claims 5-7, comprising forming, in the cell, a second exogenous nucleic acid encoding a second LMM or a second exogenous LMM, and optionally wherein forming comprises introducing the second exogenous nucleic acid encoding a second LMM, e.g., which increases the expression of an endogenous nucleic acid encoding a LMM.

9. The method of any of claims 1-8, wherein modulating lipid metabolism comprises, e.g., results in, altering one or more of the following (e.g., as compared with a cell not having a modification):

i) the expression (e.g., transcription and/or translation) of a component involved in a lipid metabolism pathway;

ii) the activity (e.g., enzymatic activity) of a component involved in a lipid metabolism pathway;

iii) the amount of lipids (e.g., phospholipids, or cholesterol) present in a cell;

iv) the amount of lipid rafts or rate of lipid raft formation;

v) the fluidity, permeability, and/or thickness of a cell membrane (e.g., a plasma membrane, a vesicle membrane, or an organelle membrane);

vi) the conversion of saturated lipids to unsaturated lipids or conversion of unsaturated lipids to saturated lipids;

vii) the amount of saturated lipids or unsaturated lipids, e.g., monounsaturated lipids; viii) the composition of lipids in the cell to attain a favorable composition that increases ER activity;

ix) the expansion of the ER (e.g., size of the ER, the ER membrane surface, or the amounts of the proteins and lipids that constitute and/or reside within the ER); x) the expansion of the Golgi (e.g., the number and size of the Golgi, the Golgi surface, or the number or amounts of proteins and molecules that reside within the Golgi);

xi) the amount of secretory vesicles or the formation of secretory vesicles;

xii) the amount or rate of secretion of the product;

xiii) the proliferation capacity, e.g., the proliferation rate;

xiv) culture viability or cell survival;

xv) activation of membrane receptors;

xvi) the unfolded protein response (UPR);

xvii) the yield or rate of production of the product;

xviii) the product quality (e.g., aggregation, glycosylation heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling); and /or

cell growth proliferation or cell specific growth rate.

10. The method of any of claims 1 -9, wherein:

i) production of the product, e.g., recombinant polypeptide, is increased by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, e.g., as compared to the level or quantity of product produced by a cell without modulation of the lipid metabolism, or

ii) the quality of the product, e.g., recombinant polypeptide, is increased, e.g., by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, e.g., as compared to the quality of product produced by a cell without modulation of the lipid metabolism.

11. The method of any of claims 1 -10, wherein:

i) the modification results in modulating, e.g., increasing, the expression or activity of a lipid metabolism gene product, e.g., a lipid metabolism gene product selected from Table 1 , ii) the expression or activity of a transcription regulator, e.g., a transcription factor, that modulates the expression of a lipid metabolism gene product, e.g., a lipid metabolism gene product selected from Table 1 , is upregulated by the modification, or

iii) both (i) and (ii).

12. The method of any of claims 1 -1 1 , wherein the lipid metabolism modulator comprises:

i) a transcription regulator, e.g., a transcription factor, that mediates, e.g., upregulates, the expression of a lipid metabolism gene product, e.g., a lipid metabolism gene product selected from Table 1 ; SREBFl; or SREBF2, or a functional fragment or analog of any of the same, or ii) SCDl or a functional fragment thereof.

13. The method of any of claims 1 -12, wherein the modification results in modulating, e.g., increasing, one or more of de novo lipogenesis, fatty acid re-esterification, fatty acid saturation or desaturation, fatty acid elongation, and phospholipid biosynthesis.

14. The method of any of claims 1 -13, wherein the LMM comprises an enzyme that modulates, e.g., increases, the conversion of saturated to unsaturated, e.g., monounsaturated, lipids, and optionally wherein the enzyme comprises SCDl , SCD2, SCD3, SCD4, or SCDl, or a functional fragment or analog thereof.

15. The method of claim 11 , wherein the modification comprises:

i) a lipid metabolism gene product, e.g., a lipid metabolism gene product selected from

Table 1 , or a functional fragment or analog thereof, or

ii) a cis or trans regulatory element that increases the expression of a nucleic acid that encodes a lipid metabolism gene product, e.g., a lipid metabolism gene product selected from Table 1.

16. The method of claim 11 , wherein the LMM comprises one or more gene products, or functional fragments or analogs thereof, from:

i) the lipid biosynthesis (lipogenesis) pathway, e.g., a gene product selected from Table 1 , ii) the fatty acid re-esterification pathway, e.g., a gene product selected from Table 1, iii) the phospholipid biosynthesis pathway, e.g., a gene product selected from Table 1, or iv) the fatty acid saturation or desaturation pathway, e.g., a gene product selected from Table 1.

17. The method of claim 11 , wherein the lipid metabolism modulator comprises:

i) at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with the amino acid sequence of SREBFl; e.g., SEQ ID NOs:l or 34, or a functional fragment thereof, e.g., SEQ ID NOs: 26, 27, or 36; or differs by 1 , 2, or 3 or more amino acid residues but no more than 50, 40, 30, 20, 15, or 10 amino acid residues from the amino acid sequence of SREBFl, e.g., SEQ ID NOs: 1 or 34, or a functional fragment thereof, or a functional fragment thereof, e.g., SEQ ID NOs: 26, 27, or 36, or

ii) at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with the amino acid sequence of SCD1; e.g., SEQ ID NO:3, or a functional fragment thereof; or differs by 1 , 2, or 3 or more amino acid residues but no more than 50, 40, 30, 20, 15, or 10 amino acid residues from the amino acid sequence of SCD1, e.g., SEQ ID NO: 3, or a functional fragment thereof.

18. The method of claim 11 , wherein the nucleic acid encoding the lipid metabolism modulator comprises at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with any of the nucleic acid sequences selected from SEQ ID NOs: 2, 4, or 32.

19. The method of any of claims 1 1-18, wherein the nucleic acid encoding the lipid metabolism modulator comprises a plasmid or a vector, and optionally wherein the nucleic acid encoding the lipid metabolism modulator is introduced into the cell by transfection or transduction.

20. The method of any of claims 1 1-19, wherein the nucleic acid encoding the lipid metabolism modulator:

i) is integrated into the chromosomal genome of the cell, and optionally wherein the LMM is stably expressed, or

ii) is not integrated into the chromosomal genome of the cell, and optionally wherein the LMM is transiently expressed.

21. The method of any of claims 1 -20, further comprising introducing to the cell an exogenous nucleic acid encoding the product, e.g., polypeptide, e.g., recombinant polypeptide, wherein:

i) the exogenous nucleic acid encoding the recombinant polypeptide is introduced after step i) or step ii), or

ii) the exogenous nucleic acid encoding the recombinant polypeptide is introduced prior to step i) or step ii).

22. The method of claim 21 , wherein the recombinant polypeptide is:

i) a therapeutic polypeptide,

ii) an antibody molecule, e.g., an antibody or an antibody fragment thereof, a monoclonal antibody, or a bispecific molecule, or

iii) selected from Table 2 or 3.

23. The method of either of claims 21 or 22, wherein the exogenous nucleic acid encoding the recombinant polypeptide is:

i) integrated into the chromosomal genome of the cell, or

ii) not integrated into the chromosomal genome of the cell.

24. The method of any of claims 1 -23, wherein the cell is:

i) a eukaryotic cell, e.g., an animal cell, a mammalian cell, a human cell, a rodent cell, a CHO cell, a yeast cell, an insect cell, or a plant cell, or

ii) a prokaryotic cell.

25. The method of any of claims 1 -24, wherein the cell is a selected from HeLa,

HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK, VERO, SP2/0, NSO, YB2/0, EB66, C127, L cell, COS, e.g., COS1 and COS7, QCl -3, , CHO-K1 , CHOKISV, Potelligent CHOKISV (FUT8-KO), CHO GS-KO, Exceed (CHOKISV GS-KO), CHO-S, CHO DG44, CHO DXB11 , CHOZN, or a CHO-derived cell.

26. The method of any of claims 1 -25, wherein the method further comprises introducing a modification to the cell that improves ER processing capacity (ER expansion) or secretion, optionally wherein improving ER processing capacity (ER expansion) comprises introducing a nucleic acid encoding PD 1 , BiP, ERO, or XBP 1 , and optionally wherein improving secretion comprises modulating SNARE machinery, e.g., introducing a nucleic acid encoding a SNARE component.

27. The method of any of claims 1 -26, further comprising one or more of the following: i) obtaining from the cell, or a descendent of the cell, the polypeptide,

ii) obtaining from medium conditioned by the cell, or a descendent of the cell, the polypeptide,

iii) separating the polypeptide from at least one cellular or medium component, or iv) analyzing the polypeptide, e.g., for activity or for the presence of a structural moiety.

28. A method of engineering a cell having increased production capacity and/or improved quality of production comprising introducing to the cell an exogenous nucleic acid encoding a lipid metabolism modulator, thereby engineering a cell having increased production capacity and/or improved quality of production.

29. The method of claim 28, wherein the exogenous nucleic acid encoding a lipid metabolism modulator is introduced to the cell by transfection, transduction, e.g., viral transduction, electroporation, nucleofection, or lipofection, and optionally wherein the exogenous nucleic acid encoding a lipid metabolism modulator is integrated into the

chromosomal genome of the cell.

30. The method of either of claims 28 or 29, further comprising introducing to the cell an exogenous nucleic acid encoding a recombinant polypeptide, wherein the exogenous nucleic acid encoding a recombinant polypeptide is introduced:

i) prior to introducing the exogenous nucleic acid encoding the LMM, or

ii) after introducing the exogenous nucleic acid encoding the LMM.

31. The method of any of claims 28-30, wherein:

i) the production capacity, e.g., the amount of recombinant polypeptide produced, is increased by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, e.g., as compared to the production capacity of a cell without modulation of the lipid metabolism,

ii) the quality of production, e.g., the quality of the recombinant polypeptide, is increased by, e.g., 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%,

80%, 85%, 90%, 85%, or 100%, or more, , e.g., as compared to the quality of production of a cell without modulation of the lipid metabolism, or

iii) both (i) and (ii).

32. The method of any of claims 28-31 , wherein the LMM is selected from a group consisting of SREBF1 , SREBF2, SCD1, SCD2, SCD3, SCD4, SCD5, and wherein the cell produces a product, e.g., a polypeptide, e.g., recombinant polypeptide as provided in Table 2 or 3, or a functional fragment thereof.

33. A cell produced by any of claims 28-32.

34. A cell comprising an exogenous nucleic acid encoding a lipid metabolism modulator (LMM), optionally wherein the exogenous nucleic acid encoding a lipid metabolism modulator is integrated into the chromosomal genome of the cell.

35. The cell of claim 34, wherein the cell is a eukaryotic cell, e.g. a CHO cell or a human cell.

36. The cell of either of claims 34 or 35, wherein the cell is selected from the group consisting of CHO-Kl , CHOKISV, Potelligent CHOKI SV (FUT8-KO), CHO GS-KO, Exceed (CHOKI SV GS-KO), CHO-S, CHO DG44, CHO DXB11, CHOZN, or a CHO-derived cell.

37. The cell of claim 34, wherein the LMM modulates the expression of a product, e.g., a Next generation biologic (NGB) described herein, e.g., a bispecific antibody, a fusion protein, or a glycosylated protein.

38. The engineered cell of claim 37, wherein the LMM is selected from a group consisting of SREBFl, SREBF2, SCDl , SCD2, SCD3, SCD4, and SCD5, or a functional fragment thereof.

39. The engineered cell of claim 37, wherein the LMM alters one or more characteristics of the cell selected from the group consisting of:

i) the expression (e.g., transcription and/or translation) of a component involved in a lipid metabolism pathway;

ii) the activity (e.g., enzymatic activity) of a component involved in a lipid metabolism pathway;

iii) the amount of lipids (e.g., phospholipids, or cholesterol) present in a cell;

iv) the amount of lipid rafts or rate of lipid raft formation;

v) the fluidity, permeability, and/or thickness of a cell membrane (e.g., a plasma membrane, a vesicle membrane, or an organelle membrane);

vi) the conversion of saturated lipids to unsaturated lipids or conversion of unsaturated lipids to saturated lipids;

vii) the amount of saturated lipids or unsaturated lipids, e.g., monounsaturated lipids; viii) the composition of lipids in the cell to attain a favorable composition that increases ER activity;

ix) the expansion of the ER (e.g., size of the ER, the ER membrane surface, or the amounts of the proteins and lipids that constitute and/or reside within the ER); x) the expansion of the Golgi (e.g., the number and size of the Golgi, the Golgi surface, or the number or amounts of proteins and molecules that reside within the Golgi);

xi) the amount of secretory vesicles or the formation of secretory vesicles;

xii) the amount or rate of secretion of the product;

xiii) the proliferation capacity, e.g., the proliferation rate;

xiv) culture viability or cell survival;

xv) activation of membrane receptors;

xvi) the unfolded protein response (UPR);

xvii) the yield or rate of production of the product;

xviii) the product quality (e.g., aggregation, glycosylation heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling); and /or

xix) cell growth/proliferation or cell specific growth rate.

40. The engineered cell of any of claims 37-39, wherein the cell is selected from a group consisting of:

i) a eukaryotic cell, e.g., a CHO cell selected from the group consisting of CHO-K1 , CHOKISV, Potelligent CHOKISV (FUT8-KO), CHO GS-KO, Exceed (CHOKI SV GS-KO), CHO-S, CHO DG44, CHO DXB1 1, CHOZN, or a CHO-derived cell,

ii) a prokaryotic cell,

iii) an insect cell,

iv) a plant cell,

v) a yeast cell, or

vi) an algae cell.

41. A CHO cell engineered to express a lipid metabolism modulator (LMM), wherein the LMM modulates one or more characteristics of the CHO cell, wherein the engineered CHO cell is selected based on modulation of one or more characteristics selected from the group consisting of:

i) the expression (e.g., transcription and/or translation) of a component involved in a lipid metabolism pathway;

ii) the activity (e.g., enzymatic activity) of a component involved in a lipid metabolism pathway;

iii) the amount of lipids (e.g., phospholipids, or cholesterol) present in a cell;

iv) the amount of lipid rafts or rate of lipid raft formation;

v) the fluidity, permeability, and/or thickness of a cell membrane (e.g., a plasma membrane, a vesicle membrane, or an organelle membrane);

vi) the conversion of saturated lipids to unsaturated lipids or conversion of unsaturated lipids to saturated lipids;

vii) the amount of saturated lipids or unsaturated lipids, e.g., monounsaturated lipids; viii) the composition of lipids in the cell to attain a favorable composition that increases ER activity;

ix) the expansion of the ER (e.g., size of the ER, the ER membrane surface, or the amounts of the proteins and lipids that constitute and/or reside within the ER); x) the expansion of the Golgi (e.g., the number and size of the Golgi, the Golgi surface, or the number or amounts of proteins and molecules that reside within the Golgi);

xi) the amount of secretory vesicles or the formation of secretory vesicles;

xii) the amount or rate of secretion of the product;

xiii) the proliferation capacity, e.g., the proliferation rate;

xiv) culture viability or cell survival;

xv) activation of membrane receptors;

xvi) the unfolded protein response (UPR);

xvii) the yield or rate of production, e.g., expression level, of the product, e.g., a Next generation biologic (NGB), e.g., a bispecific antibody; a fusion protein; or a glycosylated protein;

xviii) the product quality (e.g., aggregation, glycosylation heterogeneity, fragmentation, proper folding or assembly, post-translational modification, or disulfide bond scrambling); and /or

xix) cell growth proliferation or cell specific growth rate.

42. The engineered CHO cell of claim 41 , wherein the LMM is selected from a group consisting of SREBFl, SREBF2, SCDl , SCD2, SCD3, SCD4, and SCD5, or a functional fragment thereof.

43. The engineered CHO cell of claim 41 , wherein the product, e.g., NGB, is selected from a group consisting of a bispecific antibody molecule, a fusion protein, and a glycosylated protein.

44. The engineered CHO cell of claim 41 , wherein the CHO cell is selected from the group consisting of CHO-Kl , CHOKISV, Potelligent CHOKI SV (FUT8-KO), CHO GS-KO, Exceed (CHOKISV GS-KO), CHO-S, CHO DG44, CHO DXBl 1, CHOZN, or a CHO-derived cell.