Novel biologic production system

Abstract

An expression system for selecting for the presence of a gene of interest, the expression system comprising: (i) a cell line in which the activity of at least one enzyme in the NAD+ synthesis pathway is at least partially inhibited, the cell line optionally further comprising a gene of interest, and the cell line optionally lacking an endogenous nucleotide sequence encoding the at least one enzyme in the NAD+ synthesis pathway; (ii) an expression vector comprising an exogenous nucleotide sequence encoding the at least one enzyme in the NAD+ synthesis pathway; and (iii) a media formulation that does not comprise nicotinamide (NAM) and / or nicotinic acid.

Description

Technical Field

[0001] This application relates to an expression system for expressing a target gene. Background Technology

[0002] Most biopharmaceuticals are produced in higher eukaryotic (usually mammalian) cell lines because this provides the “human-like” post-translational modifications required for function [1]. Cell factories are genetically modified cells optimized for the industrial production of biopharmaceuticals. Historically, several human and mouse cell lines have been used as cell factories for the production of biopharmaceuticals. The industry-standard cell factory platform for manufacturing therapeutic proteins now uses variants of Chinese hamster ovary (CHO) cells adapted to industrial bioprocessing methods [2]. Commercial CHO cell variants are natural or engineered auxotrophs for key nutrients (such as amino acids or nucleosides) essential for cell growth [3]. This can be used to provide selection systems. There are two key commercial selection systems that rely on the enzymes glutamine synthase (GS) and dihydrofolate reductase (DHFR). Both systems are enhanced by small molecular weight inhibitors of the enzymes: methionine sulfoxide imine (MSX) for GS and methotrexate (MTX) for DHFR, which allows for the isolation or amplification of cells expressing a target gene linked to a co-expressed recombinant GOI gene, resulting in higher protein expression and making these cells suitable for commercial use.

[0003] NAD + NADH, NADP + NADPH and NAD+ are essential for cell function and growth, supporting catabolism and biosynthesis through coupled dehydrogenase reactions. These reactions generate cofactors necessary to support cell division and related metabolic interconversions. + Biosynthesis is crucial for the biosynthesis of the other three nicotinamide adenine coenzymes. In mammalian cells, NAD... + Synthesis occurs via one of three pathways: the Preiss-Handler pathway; the de novo and salvage pathways (as shown in...). Figure 1 (Middle). Rescue pathways are crucial for maintaining cellular NAD. + The pool is crucial [4].

[0004] NAD + The biosynthesis of related coenzymes is accomplished via a de novo pathway from tryptophan or via a synthetic pathway using nicotinamide as a precursor. De novo pathway enzymes are mainly located in hepatocytes in mammals [4]. Nicotinamide phosphoribosylation via the synthetic pathway represents NAD in Chinese hamster ovary (CHO) cells. +The main synthetic pathway. Phosphoribosylation of nicotinic acid serves as a minor alternative precursor for synthesis using specific phosphoribosyltransferases that share a final interconversion in the de novo synthetic pathway [5, 6, 7].

[0005] Over the past 5-10 years, the importance of NAMPT in tumor cell development has received increasing attention, and high expression of NAMPT has been associated with malignant phenotypes[8].

[0006] Several NAMPT inhibitors (e.g., GMX1778 and FK866) have been developed[9] and have shown clinical promise in several tumor cell therapies[10, 11]. NAMPT plays a crucial role in the NAD+ cell pathway. + The importance of this synthesis has been evidenced by the identification of compounds that selectively activate NAMPT in cultured human lung cells, leading to NAD+ activation. + The concentration increased

[12] . These studies demonstrate that NAD+ concentrations increased. + The biosynthetic pathway is crucial for cell growth and health, and a highly manageable system has been established to enhance cellular metabolism and biosynthesis using specific and potent NAMPT inhibitors. Summary of the Invention

[0007] In a first aspect of the invention, an expression system for selecting the presence of a target gene is provided, the expression system comprising: (i) Cell lines, in which NAD + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited. The cell line optionally further includes the target gene, and the cell line optionally lacks the encoding of the NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway; (ii) an expression vector, said expression vector comprising encoding the NAD + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway; and (iii) Culture medium preparations, wherein the culture medium preparations do not include nicotinamide (NAM) and / or nicotinic acid.

[0008] In another aspect of the invention, a cell line is provided, wherein NAD+ + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited, optionally further including the target gene, and optionally lacking the encoding of said NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway.

[0009] In another aspect of the invention, an expression vector is provided, comprising encoding NAD.+ The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway, and optionally including the target gene.

[0010] In another aspect of the invention, a culture medium formulation is provided that does not include nicotinamide (NAM) and / or nicotinic acid.

[0011] In some implementations, the target gene is encoded by an expression vector and / or by a second vector.

[0012] In some embodiments, the cell line includes the expression vector.

[0013] In some implementations, NAD + The at least one enzyme in the synthetic pathway is selected from nicotinamide phosphoribosyltransferase (NAMPT), nicotinic acid phosphoribosyltransferase (NPRT) and / or quinolinate phosphoryltransferase (QPRT).

[0014] In some implementations, the NAD + At least one enzyme in the synthetic pathway is a heterologous mammalian NAMPT.

[0015] In some implementations, the NAD + The at least one enzyme in the synthetic pathway has at least 70% sequence identity with SEQ ID 1 to 8.

[0016] In some embodiments, the target gene encodes a recombinant protein, a viral vector, components of the viral vector, a viral vaccine, an intracellular protein, and / or a receptor.

[0017] In some implementations, the NAD is encoded + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway is under the control of a promoter, optionally wherein the promoter is CMV, SV40 and / or mPGK.

[0018] In some embodiments, the target gene is under the control of a promoter, optionally wherein the promoter is CMV, SV40, and / or mPGK.

[0019] In some embodiments, the cell line is a eukaryotic cell line, optionally selected from: Chinese hamster ovary KI cells; HEK cells; and / or VERO cells.

[0020] In some embodiments, the cell line is genetically engineered to lack the encoding of NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway.

[0021] In some embodiments, the culture medium formulation further includes an inhibitor, optionally wherein the inhibitor is a transferase inhibitor.

[0022] In some embodiments, the transferase inhibitor is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

[0023] In some embodiments, the culture medium preparation is a substantially serum-free and / or protein-free culture medium.

[0024] In another aspect of the invention, a method is provided for selecting cells containing a target gene using the expression system, cell line, expression vector, or culture medium formulation.

[0025] In another aspect of the invention, a method for culturing and / or amplifying the cell line using the culture medium preparation is provided.

[0026] In another aspect of the invention, use of the expression system is provided, wherein the NAD is not included. + Cells do not survive any of the at least one enzyme in the synthetic pathway. Attached Figure Description

[0027] Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, in which: Figure 1 It is shown as NAD + Synthetic pathways of synthetic routes.

[0028] Figure 2 This is a schematic diagram of an exemplary embodiment of the expression system of the present invention.

[0029] Figure 3 This is a schematic diagram representing the components of the system.

[0030] Figure 4 The study showed that the NAMPT transferase inhibitor (FK866) kills cells, and this was prevented by the addition of nicotinic acid.

[0031] Figure 5 The study showed that NAMPT's transferase inhibitors kill cells, and this was prevented by the addition of niacin.

[0032] Figure 6 A and Figure 6 B shows that cells (sgl-3) without endogenous NAMPT cannot survive in the absence of niacin.

[0033] Figure 7 A and Figure 7 B shows that cells without endogenous NAMPT cannot survive in the absence of niacin.

[0034] Figure 8 An exemplary implementation of the expression vector is shown.

[0035] Figure 9 Showing from Figure 10 Western blots of IgG expression in transient and stable transfections of the NAMPT vector are shown.

[0036] Figure 10 An exemplary implementation of the expression vector is shown.

[0037] Figure 11 An exemplary implementation of the mechanism of the present invention is shown.

[0038] Figure 12 The expression levels of knockout proteins transfected with the target gene under NAMPT selection are shown.

[0039] Figure 13 Another exemplary embodiment of the mechanism of the present invention is shown, which includes gene amplification using the NAMPT inhibitor FK866.

[0040] Figure 14 A through D show that the addition of the inhibitor FK866 exerts selective pressure to lead to an amplification of protein expression. Detailed Implementation

[0041] In a first aspect of the invention, an expression system for selecting the presence of a target gene is provided, the expression system comprising: (i) Cell lines, in which NAD + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited, optionally lacking the enzyme encoding NAD. + The cell line optionally further includes the target gene; an endogenous nucleotide sequence of at least one enzyme in the synthetic pathway. (ii) an expression vector, said expression vector comprising encoding the NAD + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway; and (iii) Culture medium preparations, wherein the culture medium preparations do not include nicotinamide (NAM) and / or nicotinic acid.

[0042] In another aspect of the invention, a cell line is provided, wherein NAD+ + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited, optionally lacking the encoding NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway, optionally the cell line further comprising the target gene.

[0043] In another aspect of the invention, an expression vector is provided comprising an exogenous nucleotide sequence encoding at least one enzyme in the NAD+ synthesis pathway, and optionally comprising a target gene.

[0044] In some implementations, NAD is encoded + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway can be related to the encoding NAD. + At least one enzyme in the synthetic pathway has the same endogenous nucleotide sequence. In some embodiments, the enzyme encoding NAD... + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway may be different from that encoding NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway.

[0045] In another aspect of the invention, a culture medium formulation is provided that does not include nicotinamide (NAM) and / or nicotinic acid.

[0046] An expression system is a system designed for the production of a target gene (GOI). The expression system of this invention is adapted to and capable of achieving selective expression of GOI. NAD + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited, and this leads to NAD+. + The production is reduced or completely eliminated. As used in this article, the term "inhibition" means a reduction or prevention of activity.

[0047] NAD + The activity of at least one enzyme in the synthetic pathway can be inhibited by up to about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% compared to its original activity.

[0048] NAD + The activity of at least one enzyme in the synthetic pathway can be inhibited by at least about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% compared to its pre-inhibition activity. In some embodiments, NAD... + The activity of at least one enzyme in the synthetic pathway can be substantially completely inhibited.

[0049] Inhibitors can provide information including encoding NAD +Prior to the expression vector of an exogenous nucleotide sequence of at least one enzyme in the synthetic pathway, the cell growth, survival, and / or viability of the cell line are reduced. The inhibitor can reduce cell growth, survival, and / or viability by up to about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 98%, 99%, or 100%. This can be measured using optical density (OD), VCD, or any other measurement method known in the art.

[0050] In some implementations, the inhibition method is selected from small molecules or via gene expression.

[0051] In some implementations, NAD is encoded + The inhibition of the endogenous nucleotide sequence of at least one first enzyme in the synthetic pathway is achieved through enzyme inactivation or substantially inactivation.

[0052] In implementations where the inhibition method is via gene expression, the cell line may lack the encoding of NAD. + The endogenous nucleotide sequence of at least one first enzyme in the NAD synthesis pathway. Therefore, cell lines do not substantially express NAD. + It involves at least one enzyme in the synthetic pathway and cannot substantially produce NAD. + This cell line can be referred to as an auxotrophic cell line. In some embodiments, the cell line lacks the encoding NAD. + The endogenous nucleotide sequence of an enzyme in a synthetic pathway.

[0053] In embodiments where the inhibition method is implemented via gene expression, the inhibitor can be a small interfering RNA (siRNA). In such embodiments, NAD... + The expression of at least one enzyme in the synthetic pathway is reduced by RNA interference (RNAi). The siRNA delivery system can be a non-viral or viral delivery system. Non-viral delivery systems may include polymers, optionally including lipid and / or peptide complexes. The lipid and / or peptide complexes can facilitate siRNA penetration into appropriate cellular compartments to trigger an RNA interference response.

[0054] In some implementations, the cell line is a natural variant that lacks or has substantially low expression of the NAD-encoding gene. + The endogenous nucleotide sequence of at least one first enzyme in the synthetic pathway.

[0055] In some implementations, NAD is encoded +The endogenous nucleotide sequence of at least one first enzyme in the synthetic pathway is inhibited. In some embodiments, cell lines are engineered to shut down, knock out, or otherwise remove or substantially inactivate the NAD encoding enzyme. + The endogenous nucleotide sequence of at least one first enzyme in the synthetic pathway. This improves the expression and stability of recombinant genes during scale-up (extended cell culture).

[0056] In some embodiments, the cell line is genetically engineered to lack the encoding NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway. In some embodiments, the endogenous nucleotide sequence is deleted or removed to generate the cell line. This provides the advantage of more stringent selection. In some embodiments, a knockout system is used to generate the cell line, which may include adenovirus, zinc finger, mega nuclease, TALEN, and / or other mechanisms known in the art. In some embodiments, CRISPR, such as CRISPR-Cas9 or Cas-Clover, is used to generate the cell line. This provides the advantage of rapid gene deletion.

[0057] In specific instances, CRISPR-Cas9 is used to knock out the NAMPT gene in cells. This leverages optimized delivery conditions of CRISPR components, such as single guide RNAs (sgRNAs) and the Cas9 protein, to deliver these components to specific CHO cell variants, for example, via liposome transfection or nuclear transfection, using control guide RNAs (gRNAs) that target the known genome. Three to four sgRNAs targeting NAMPT can be synthesized, including those with low off-target potential, those targeting asymmetric exons, and those targeting exons common to all isoforms. The sgRNAs can be co-delivered with Cas9 according to a predetermined protocol, and editing efficiency can be measured using sequencing and TIDE analysis. Cell sets with the highest editing levels can be further used for subsequent monoclonal derivatization. Clones can be screened genetically (to identify InDels generated in the gene and confirm clonalness) and proteomically (via Western blotting) to confirm the absence of NAMPT (high-quality commercial antibodies against NAMPT are available).

[0058] In another instance, adeno-associated virus (AAV) can be used to remove the NAD encoding. + The endogenous nucleotide sequence of at least one first enzyme in the synthetic pathway.

[0059] Lack or inactivation of NAD encoding +Cell lines with endogenous nucleotide sequences of at least one of the first enzymes in the synthetic pathway will be metabolically severely impaired and therefore unlikely or unable to survive in nicotinamide-deficient media, but will grow in media supplemented with nicotinic acid. Cells transfected with the vector will be selected by using a combination of nicotinamide-deficient and nicotinic acid-deficient media.

[0060] The inventors used RNA-seq studies on CHO K1 and CHO-S cells, showing no expression of mRNA encoding indole 2,3-dioxygenase, kynurenase, or quinolinate phosphoribosyltransferase. This data confirms that industrially relevant CHO cells do not express components of the de novo synthetic pathway that enables tryptophan catabolism to produce NAD precursors. In contrast, mRNAs encoding enzymes required to convert nicotinic acid (NA) or nicotinamide (NAM) to NAD were expressed, although NARPT (which processes NA) was expressed at only 12-15% of NAMPT. This demonstrates that NAMPT is a strong selection marker.

[0061] In the implementation of the inhibition method using small molecules, the inhibitor is NAD. + A small molecular weight inhibitor of at least one enzyme in the synthetic pathway.

[0062] In some embodiments, the inhibitor is a transferase inhibitor. In some embodiments, the inhibitor is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors may be selected from: (E)-daporinad (FK866); CHS-828 (GMX1778); OT-82; Padnarsertib (KPT-9274); GNE-618, STF-118804; GNE-617 hydrochloride; teglarinad chloride (GMX1777); GPP78 (CAY10618); Nampt-IN-3 (compound 35); Nampt-IN-5; CB30865 (ZM242421); LB-60-OF61, CB 300919; NAMPT inhibitor-linker 2; LB-60-OF61 hydrochloride; Nampt-IN-9 (compound 8); NAMPT / IDO1-IN-1; or combinations thereof. Transferase inhibitors can be selected based on their efficacy and / or specificity. Some transferase inhibitors are known to provide additional activity, such as that possibly associated with anticancer activity.

[0063] Preferably, the transferase inhibitor is FK866 or GMX1778. These inhibitors have the advantage that their activity profiles are known and understood. Furthermore, these inhibitors can produce up to complete inhibition of cell growth at low concentrations.

[0064] Inhibitors can isolate or amplify cells with higher gene copies, resulting in higher protein expression due to their association with co-expressed recombinant GOIs, making these cells suitable for commercial use. Therefore, inhibitors enhance cell line selection.

[0065] These inhibitors allow for enhanced selection of highly expressed clones containing multiple gene copies, and may also select for clones with additional synthetic NAD+. + Capacity of cells. This is beneficial for cell health and protein production.

[0066] In some implementations, an encoded NAD is provided. + The exogenous nucleotide sequence of at least one enzyme in the salvage pathway. This restores NAD. + A rescue approach to enable cell lines to survive. Figure 2 This exemplary implementation is shown.

[0067] Advantageously, encoding NAD + Expression of an exogenous nucleotide sequence of at least one enzyme in the salvage pathway will allow for GOI expression, therefore only NAD expression is possible. + Cells require at least one enzyme from the salvage pathway and GOI to survive. NAD + Co-expression of at least one enzyme in the salvage pathway restores the ability to synthesize key nutrients, allowing selection only of cells expressing GOI. Cells that take up the expression vector are selected using a culture medium formulation lacking key nutrients.

[0068] This expression system enjoys the advantage of generating highly selective metabolic compounds and exhibits significant advantages over known systems such as the GS system. NAD(P) + It is an essential cofactor widely involved in cellular function, various metabolic pathways, and DNA maintenance and repair. GS has a much narrower scope of involvement in cellular function and can be substituted by other amino acids (such as asparagine and glutamate), thus reducing the selectivity of GS. The claimed expression system utilizes NAD+. + The remedies are therefore more selective.

[0069] In some implementations, NAD +At least one enzyme in the synthetic pathway is selected from: nicotinamide phosphoribosyltransferase (NAMPT); nicotinic acid phosphoribosyltransferase (NPRT); quinolinate phosphoryltransferase (QPRT); indoleamine 2,3-dioxygenase 1 and / or 2 (IDO); tryptophan 2,3-dioxygenase (TDO); nicotinamide / nicotinic acid mononucleotide adenosyltransferase 1, 2 and / or 3 (NMNAT); and / or other enzymes involved in the conversion of small molecular weight metabolites and amino acids into nicotinamide precursors. In some embodiments, NAD... + At least one enzyme in the synthetic pathway is nicotinamide phosphoribosyltransferase (NAMPT), nicotinic acid phosphoribosyltransferase (NPRT), and / or quinolinate phosphoryltransferase (QPRT). These provide NAD+ for NAD+ synthesis. + It plays a key role in the synthetic pathway and thus provides the advantage of more stringent selection.

[0070] NAMPT is a target for novel anticancer drugs, and the inventors anticipate that overexpression of NAMPT will lead to an increase in nicotinamide metabolism entering the NAD pool, supporting rapid cell growth and division, and generating multiple generations of daughter cells in culture, making this enzyme an ideal metabolic selection marker. Furthermore, the inventors have shown that nicotinamide is rapidly consumed via a synthetic pathway and is associated with rapid cell growth, particularly in CHO cells.

[0071] In some implementations, NAD + At least one enzyme in the synthetic pathway is two, three, or more enzymes. This can improve the selectivity of the expression system. In one particular instance, cells can be engineered to regulate growth rates, which can be achieved by controlling NAD+. + The synthesis pathway is regulated by the activity of two, three, or more enzymes. This involves NAD. + Networks of synthesized enzymes, some of which are exemplified in Figure 1 This network can be engineered to control NAD. + synthesis.

[0072] In some implementations, NAD + At least one enzyme in the synthetic pathway is an enzyme. This provides the advantage of tight control over the system.

[0073] In some implementations, NAD +At least one enzyme in the synthetic pathway is a heterologous mammalian NAMPT. Enzymes derived from mammals can be expressed in a superior manner in mammalian cells. For example, appropriate post-translational modifications can be made, and expression is optimized for mammalian cells. In some embodiments, the mammalian NAMPT is human NAMPT, such as Gene ID: 10135, which can be found on the NCBI online National Library of Medicine.

[0074] In some implementations, NAD + At least one enzyme in the synthetic pathway has at least 70% sequence identity with any one of SEQ ID 1-8. SEQ ID 1-8 represent NAD. + Exemplary sequences of at least one enzyme in the synthetic pathway relate to humans (Homo sapiens). However, those skilled in the art may be able to identify other suitable sequences, and the nucleotide sequences encoding them. In some embodiments, NAD... + At least one enzyme in the synthetic pathway has at least 70% sequence identity with any one of SEQ ID 1 and / or 2.

[0075] In this specification, the term "identity" is used to refer to the similarity between two sequences. For the purposes of this invention, it is defined here as follows: to determine the percentage identity of two sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced into the first sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The nucleotide / amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of common identical positions (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Typically, the two sequences are of the same length. Sequence comparison is usually performed over the entire length of the two sequences being compared.

[0076] Technicians will be aware that several different computer programs can be used to determine the identity between two sequences. For example, mathematical algorithms can be used to compare sequences and determine the percentage identity between two sequences. In a preferred embodiment, the sequence alignment software Clone Manager 9 (Sci-Ed software - www.scied.com) is used to determine the percentage identity between two nucleic acid sequences using global DNA alignment (parameters: two strands, scoring matrix: linear (mismatch 2, open gap 4, extended gap 1)).

[0077] Alternatively, the Needleman and Wunsch (1970) algorithm can be used with a Blosum 62 or PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage identity between two amino acid or nucleic acid sequences. This algorithm has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ). Another method for assessing the percentage identity between two amino acid or nucleic acid sequences is to use the BLAST sequence comparison tool available at the National Center for Biotechnology Information (NCBI) website (www.blast.ncbi.nlm.nih.gov), such as BLASTn for nucleotide sequences or BLASTp for amino acid sequences using the default parameters.

[0078] Expression systems, cell lines, expression vectors, and culture medium formulations offer the advantage of providing more stringent selection in terms of cell health. They enable rapid and specific isolation of desired cells and, through their metabolically selective properties, enhance cell growth, leading to the selection of a greater number of recombinant clones, higher biomass in cultures, and, in particular, improved overall cellular performance as a platform for biopharmaceutical production. Based on NAD... + The selection of at least one enzyme in the synthetic pathway implies that the system is also compatible with a wide range of mammalian cell types, including CHO cells.

[0079] In some embodiments, the target gene is encoded by an expression vector and / or a second vector. In some embodiments, the expression vector includes both NAD and other encoding vectors. + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway also includes GOI. This provides the advantage of selecting cells that have been successfully transformed with expression vectors and will therefore be able to express GOI.

[0080] In some embodiments, the cell line includes both an expression vector and a second vector. For example, the cell line can be co-transformed with the expression vector and the second vector. In this embodiment, cells successfully transformed with the expression vector will survive and will also include the second vector. This provides the advantage that the cell line can be provided in a kit that includes the cell line, culture medium preparation, and expression vector. Users can co-transform cells with the expression vector and the second vector including GOI, thereby using the expression system to select from cells successfully co-transformed with both the expression vector and the second vector.

[0081] In some embodiments of the invention, the vector can be inserted into a cell line using any suitable means. Means for inserting the vector into cells can be selected from the group consisting of: plasmid transformation (including transfection), viral transduction, or non-viral transfection. Examples of non-viral transfection include the use of materials such as polymers, lipids, peptides, inorganic materials, and / or hybrid materials. Viral transduction can use viral vectors such as adeno-associated virus (AAV), herpes simplex virus (HSV), adenovirus, retrovirus, lentivirus, or baculovirus. The vector can be a lentiviral vector or an AAV vector, although in some embodiments, the vector can be one that allows NAD expression. + At least one second enzyme in the synthetic pathway and any suitable carrier for GOI.

[0082] Transfection can be transient or stable. Transient transfection refers to DNA remaining in the cell nucleus rather than being integrated into the genome. Transient transfection offers the advantage of being able to test whether the target gene is expressed within the expression system. Stable transfection refers to DNA being integrated into the genome. Stable transfection offers the advantage of cell lines being able to be stored and revived as needed. Furthermore, due to genetic variation, some cells may exhibit improved expression, and these can be selected and reused for industrial applications such as manufacturing.

[0083] The target gene (GOI) refers to the desired nucleotide sequence. GOIs can encode recombinant proteins, viral vectors, components of viral vectors, viral vaccines, intracellular proteins and / or receptors, and / or receptors.

[0084] Recombinant proteins can be secreted proteins, such as proteases and glycosidases. Recombinant proteins can also be cell surface proteins, such as MHC proteins; cell lineage markers, such as IGR and TCR. Recombinant proteins can also be serum proteins.

[0085] Recombinant proteins can be therapeutic proteins or proteins used for therapeutic purposes. Recombinant proteins can be single proteins, at least one protein, or protein-based complexes. Recombinant proteins can be antibodies. Recombinant proteins can be selected from: antibodies; antibodies containing non-natural amino acids, optionally used to prepare antibody-drug conjugates; multispecific antibodies; engineered domain proteins, such as engineered domain antibodies (such as bispecific, trispecific, and multispecific forms, and / or fusion combinations including protein-Fc structures); and / or monoclonal antibodies (mAbs). In some embodiments, the antibody is trastuzumab, also known as Herceptin®. In some embodiments, the recombinant protein can be selected from fluorescent secreted proteins: sEGFP; sRFP; smCherry; Intracellular proteins can be those involved in redirecting and regulating cellular function.

[0086] The viral vector may be selected from: adeno-associated virus (AAV); herpes simplex virus (HSV); adenovirus; retrovirus; lentivirus; and / or baculovirus.

[0087] The expression vector and / or selection vector can be deoxyribonucleic acid (DNA). In specific instances, the expression vector is determined according to... Figure 8 .

[0088] In some implementations, the target gene and / or gene encoding NAD + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway is under the control of a promoter, which may optionally be a polymerase type 2 promoter.

[0089] In some embodiments, the promoter is selected from the group consisting of: human cytomegalovirus immediate early promoter (hCMV), mouse cytomegalovirus immediate early promoter (mCMV), human elongation factor 1α promoter (hEF1α), mouse elongation factor 1α promoter (mEF1α), chicken β-actin heterozygous promoter (CAG), mouse phosphoglycerate kinase promoter (mPGK) or human ubiquitin C promoter (UBC), liver-specific promoters (including thyroxine transporter (TTR) promoter and α-1-antitrypsin promoter (hAAT)), ubiquitous promoters (including CAG, CMV, CBh, and SV40), CBh promoter, thyroxine transporter (TTR) promoter, or α-1-antitrypsin promoter (hAAT). In some embodiments, the promoter is a neuron-specific enolase (NSE) promoter. In some embodiments, the promoter is a neuron-specific enolase (NSE) promoter selected from the group consisting of: synapticin I (SYN) promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, tubulin α I promoter, neuron-specific enolase promoter, or platelet-derived growth factor β chain promoter. In some embodiments, the promoter is a synthetic promoter. In some embodiments, the promoter is a modified promoter selected from any of those listed herein.

[0090] In some implementations, the target gene is under the control of the CMV, SV40, and / or mPGK promoters. In some implementations, the gene encoding NAD... + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway is controlled by the mPGK promoter. Therefore, expression vectors and / or second vectors may include at least one promoter. The target gene and NAD+ are contained within them. +In embodiments where at least one enzyme in the synthetic pathway is encoded in an expression vector, the expression vector may include two promoters. SV40, CMV, and mPGK offer advantages suitable for expression control.

[0091] Promoters can be selected to improve expression. Promoters can be selected to improve tissue- and / or cell-specific expression. For example, SV40 is a weak promoter. As used herein, the term weak promoter indicates relatively less or infrequent RNA polymerase binding, resulting in lower transcription and lower levels of expressed protein. When combined with selection markers, weaker promoters enhance selection strength.

[0092] In some implementations, the expression vector and / or the second vector includes at least one inverted terminal repeat (ITR) sequence. This mediates intermolecular recombination of genes.

[0093] In some embodiments, the expression vector and / or the second vector includes a polyadenine region. The polyadenine (polyA) region encodes a polyadenine tail, a long chain of adenine nucleotides, which increases the stability of the mRNA.

[0094] In some embodiments, the expression vector and / or the second vector includes a transcriptional regulatory element. A transcriptional regulatory element is a nucleotide sequence of a gene that participates in the regulation of gene transcription. Advantageously, this can provide epigenetic control, such as preventing or limiting epigenetic repression during cell culture. An example of this is the ubiquitous chromatin opening element (UCOE).

[0095] In some embodiments, the expression vector and / or the second vector may include other selection markers, such as puromycin, glutamine synthase, or DHFR. In some embodiments, the selection marker is located downstream of the promoter. This is advantageously particularly suitable for bispecific and other more complex molecules or targets. Furthermore, these may be particularly advantageous in systems having more than one vector.

[0096] In some embodiments, the expression vector and / or the second vector includes an inducible element. This inducible element provides the advantage of allowing cells to reach high density before gene construct expression is initiated.

[0097] In some implementations, the target gene and / or gene encoding NAD + Exogenous nucleotide sequences of at least one enzyme in the synthetic pathway can be codon-optimized. This is the process of changing codons from those rare in the host to those preferred by the host. The target gene and / or encoding NAD... +Codons in the exogenous nucleotide sequence of at least one enzyme in the synthetic pathway can be selected to closely mimic codon usage in a selected cell line, such that the frequency of codon usage in the gene is substantially the same as that in genes encoding the same or similar proteins in the cell line. This enhances heterologous expression levels.

[0098] In some embodiments, the cell line is a eukaryotic cell line. In other embodiments, the cell line is mammalian. This provides the advantage of the cell line providing appropriate post-translational modifications for the expressed GOI.

[0099] In some implementations, the cell line is plant, insect, yeast, or prokaryotic.

[0100] In some embodiments, the cell line is selected from: Chinese hamster ovary (CHO) cells; HEK cells; and / or VERO cells. In some embodiments, the CHO cells are CHO K1 cells, CHO DG44 cells, CHO DXB11 cells, or CHO-S cells. In some embodiments, the cell line is a tumor cell; and / or CAR-T cells.

[0101] CHO cells exhibit inefficient metabolism similar to tumor cells, such as generating energy from glucose via anaerobic rather than aerobic pathways, utilizing amino acids as energy and as a biosynthetic backbone to support rapid biosynthesis, leading to the production of toxic metabolic byproducts. Therefore, metabolism in tumor cells can reasonably be expected to be similar in CHO cells. CHO cells have the benefit of serving as a good model for tumor cells, and the use of expression systems in these cells is particularly advantageous.

[0102] In some embodiments, the cell line may be provided in suspension, adherent, or continuous perfusion modes. The cell line may be provided, for example, in a culture medium formulation.

[0103] In some implementations, the cell lines are optimized for selection by feeding with inhibitors, optionally transferase inhibitors.

[0104] In some embodiments, the cell line is provided together with the inhibitor, for example, in a culture medium formulation. The inhibitor is optionally an inhibitor as described herein. The inhibitor can inhibit NAD. + The activity of at least one enzyme in the NAD synthesis pathway. Inhibitors can inhibit endogenous and / or exogenous NAD. + The activity of at least one enzyme in the synthetic pathway. In some embodiments, the inhibitor is compared with exogenous or endogenous NAD, respectively. + At least one enzyme in the synthetic pathway more significantly inhibits endogenous or exogenous NAD. + The activity of at least one enzyme in the synthetic pathway. This activity can be inhibited as described herein.

[0105] In such embodiments, cell lines may include those encoding NAD. + The endogenous nucleotide sequence of at least one enzyme in the NAD synthesis pathway. Inhibitors can inhibit endogenous NAD. + At least one enzyme in the synthetic pathway reduces the growth and / or survival of the cells. This means including exogenous NAD. + Cells express higher levels of NAD+ at least one enzyme in the synthesis pathway. + It synthesizes at least one enzyme in the pathway, thereby restoring that pathway. These cells also express GOI, so inhibitors provide selection for these cells.

[0106] Culture medium preparations refer to the culture medium in which cells grow and survive. In some embodiments, the culture medium does not include nicotinamide (NAM) and / or nicotinic acid.

[0107] In some embodiments, the culture medium formulation does not include small molecular weight metabolites and / or amino acids that may serve as potential precursors to nicotinamide. This provides the advantage of more controlled selection. In some embodiments, the culture medium formulation is substantially serum-free, protein-free, and / or tryptophan-free. In some embodiments, the culture medium formulation is Ham's F12. + 8 mM glutamine medium. In some embodiments, the medium formulation is supplemented with 10% (v / v) fetal bovine serum. In some embodiments, the medium is metabolite-deficient. In some embodiments, the medium is a commercially available protein-free CHO medium, such as CDCHO, CD FortiCHO, or Excell.

[0108] In some embodiments, the culture medium formulation includes amino acids that support protein synthesis. For example, the culture medium formulation may include tryptophan.

[0109] Culture medium formulations can be selected to suit the growth and selection of cell lines. In some embodiments, the culture medium formulation is optimized, and fed-batch methods have been developed to further maximize selection and increase recombinant gene expression. In some embodiments, the culture medium formulation includes serum. This provides the advantage of viral vectors, such as lentiviruses and AAVs as described herein, operating in the presence of serum in the culture medium.

[0110] The known industry-standard CD-CHO medium contains 0.1–1.0 mg nicotinamide / mL. Other commercially available media with publicly available compositions, such as DMEM, contain 1–4 mg nicotinamide / mL. The earliest medium formulation developed by Ham in 1965 used nicotinamide, thus nicotinamide-containing medium formulations became the standard for all other universal media. Ham likely preferred nicotinamide over nicotinic acid because it supports cell growth or has greater stability in cell culture. The inventors have performed RNA-seq experiments using several different CHO cell variants, showing that the raw transcript count for NAMPT was ten times higher than that encoding NAPRT, further supporting this cellular advantage of using nicotinamide to support NAD synthesis.

[0111] In some embodiments, the culture medium formulation is a basal culture medium. In such embodiments, the culture medium formulation further includes a transferase inhibitor.

[0112] In some embodiments, the culture medium formulation further includes an inhibitor as described herein. The inhibitor may be a transferase inhibitor as described herein.

[0113] In another aspect of the invention, a method is provided for selecting cells containing a target gene using the expression system, cell line, expression vector, or culture medium formulation.

[0114] In another aspect of the invention, a method is provided for culturing and / or amplifying the cell line using the aforementioned culture medium preparation. As discussed herein, NAD... + Expression of at least one enzyme in the synthetic pathway restores the cell’s ability to synthesize key nutrients, allowing only those cells expressing GOI to be cultured and / or expanded.

[0115] In another aspect of the invention, use of the expression system is provided, wherein NAD is not included. + Cells do not survive if at least one enzyme in the synthetic pathway is involved.

[0116] Those skilled in the art will understand that all aspects of the invention, whether relating to, for example, compositions, their uses, or methods of processing, are equally applicable to all other aspects of the invention. In particular, aspects concerning compositions may be described in more detail, for example, than other aspects of the invention (e.g., uses of the compositions). However, those skilled in the art will understand that where more detailed information has been given with respect to a particular aspect of the invention, that information generally applies equally to other aspects of the invention.

[0117] Example Material: The CHO-K1 cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC) (catalog number 85051005).

[0118] Cell culture medium, cells were cultured in Ham's F12 supplemented with 10% (v / v) fetal bovine serum (New Zealand certified). + Initial growth was performed in 8 mM glutamine (Sigma Merck). Subsequent cell cultures were conducted in CD CHO medium (Gibco). A proprietary modified serum-free medium (free of nicotinamide, nicotinic acid, and tryptophan) was synthesized.

[0119] The plasmids were synthesized using VectorBuilder. The first series of plasmids was designed for EGFP expression, utilizing the mouse NAMPT gene (codon-optimized for CHO cells using SnapGene software) as a selectable marker under the control of the SV40 promoter, with either EGFP or IgG as a recombinant protein downstream of the cytomegalovirus (CMV) promoter. The second plasmid was designed for NAMPT protein expression, using the same gene sequence, under the control of the CMV promoter, with puromycin as a selectable marker downstream of the SV40 promoter.

[0120] The reagents used for CRISPR editing (including the targeted design guide RNA) were procured by the University of Manchester Genome Editing Facility.

[0121] Method summary: Develop a serum-free, suspension CHO-K1 master cell bank.

[0122] Cells were adapted for suspension and serum-free growth by sequentially altering the relative mixing of serum-containing (Hams F12) and serum-free (CD-CHO) media, supplemented with 0.1% (w / v) anti-caking agent, and cultured under shaking conditions. Over a 4-month period, the percentage of serum-containing medium was gradually reduced until the serum-free cell line was harvested. At this stage, the master cell bank was preserved.

[0123] Transfection procedure For each transfection reaction, approximately 4 x 10⁴ samples were harvested by centrifugation at 100g for 5 minutes. 7Cells were collected and washed with cold PBS. The cells were centrifuged again, and the pellet was resuspended in 950 μL PBS. Three electroporation cuvettes were prepared, two containing 20 μg plasmid and one containing sterile water as a control. 950 μL of cell-PBS suspension was added to each cuvette, and the mixture was incubated on ice for 5 minutes. Transfection was performed via electroporation using the Bio-Rad Gene Pulser Xcell system. Two consecutive pulses (300 V, 950 μF, and 20 ms) were applied to each DNA / cell mixture. The cells were then placed on ice for 5 minutes and transferred to CDCHO medium, with or without added nicotinic acid (50 nM). Transient transfection studies were conducted over a 7-day period. For stable transfection, passages were performed every 3–4 days, diluting the cell count and replacing the medium with the appropriate CD-CHO medium type.

[0124] Analysis program Cell cultures are evaluated using a variety of standards and in-house methods, including cell density and viability, metabolomics profiling (enzymatic, GC-MS, LC-MS), karyotype analysis, flow cytometry, RNA isolation, characterization and evaluation (PCR, qRT-PCR), and protein isolation and evaluation (Western blotting, ELISA).

[0125] result: The applicant has demonstrated that NAMPT CRISPR gene knockout cells do not grow in the absence of niacin. Figure 4 and Figure 5 This study demonstrated that the NAMPT transferase inhibitor (FFK866) was lethal to CHO-K1 cells. The lethal effect of the NAMPT transferase inhibitor was overcome by the presence of nicotinic acid. The basal medium contained nicotinamide. This illustrates the effect of NAMPT (and NAD) on cell death. + ) is of core importance to cell health.

[0126] Figure 6 and Figure 7 The study showed that NAMPT gene knockout in a cell line generated cells that could not survive in the absence of niacin. CRISPR NAMPT knockout is lethal to the cells. This was prevented with niacin.

[0127] Figure 6 and Figure 7 Two different knockout gRNA sequences were used. Figure 6 During the study, CRISPR sg1-3 cell lines were maintained in CD CHO medium supplemented with nicotinic acid (50 μM). At time zero (t=0), the medium was switched to either a medium with or without nicotinic acid. Control cells were maintained in basal medium (containing nicotinamide) throughout the study. FK866 was added at a concentration of 50 nM.

[0128] exist Figure 7 During the study, CRISPR sg2 cell lines were maintained in CD CHO medium supplemented with nicotinic acid (50 μM). At time zero (t=0), the medium was switched to either a medium with or without nicotinic acid. Control cells were maintained in basal medium (containing nicotinamide) throughout the study. FK866 was added at a concentration of 50 nM.

[0129] Figure 8 The example shown is the generation of a proprietary expression vector using GFP expression and Herceptin bioanalyst molecules.

[0130] Figure 9 IgG expression from the NAMPT vector in transient and stable transfections is shown. This stable and transient expression supports strong expression of the Herceptin bioanalyst in the host CHO-K1 cell line and both gene knockout cell lines. * indicates that for transient transfection, the culture medium sample was collected on day 7 of batch growth. ** indicates that for stable transfection, cells were cultured in 10 mL of medium in T75 flasks for 5 days, followed by replenishment of an additional 10 mL of fresh medium. Culture medium samples for stable transfection were obtained 2 days after medium replenishment. 30 μL of undiluted culture medium sample was loaded into each well.

[0131] Figure 10 It shows the transfection to Figure 9 A diagram of carriers within a cell.

[0132] Figure 11 An exemplary embodiment of the mechanism of the present invention is shown. In this embodiment, [the following is an example of the implementation of the mechanism]: Figure 10 The vector was transfected into cells to generate a recombinant NAMPT KO cell line.

[0133] Figure 12 The expression levels of knockout proteins transfected with the target gene under NAMPT selection are shown, using... Figure 11 The mechanism is illustrated. In this embodiment, batch cultures are grown in CDCHO medium. Fed batch cultures are supplemented with commercial feed during culture to replenish nutrients consumed by the cells.

[0134] Figure 13 Another exemplary embodiment of the mechanism of the present invention is shown. In this embodiment, [the following is an example of the implementation of the invention:] Figure 10 The vector was transfected into cells to generate a recombinant NAMPT KO cell line. The inhibitor FK866 was added to exert selection pressure on the cells.

[0135] Figure 14A through D illustrate how the addition of the inhibitor FK866 exerts selective pressure, leading to amplification of protein expression. In these experiments, untreated cells and cells treated with the 5 nM FK866 inhibitor were compared. The results show that the amount of recombinant protein produced after FK866 inhibition was greater than that produced by untreated cells. For example, the highest volumetric titer obtained after treating cells with 5 mM FK866 was 4000 mg / L, compared to a maximum of 2000 mg / L in untreated cells. The use of an inhibitor to amplify selection and thus increase expression highlights the potency of this expression system. Advantageously, enzyme inhibition leads to increased gene expression, demonstrating the essentiality of the enzyme.

[0136] Therefore, the applicant has demonstrated that NAMPT is a selection biomarker that drives high protein expression with reduced screening effort. Volumetric titers up to 4 g / L and productivity up to 50 pcd can be achieved using this biomarker.

[0137] The applicant also demonstrated that NAMPT is a more potent selective marker compared to other prior biomarkers, such as glutamine synthase (GS), which can be bypassed by other amino acids. Glutamine's effects on cellular functions, such as energy production and biosynthesis, can be mitigated by other amino acids. Aspartate and asparagine can also be used to substitute for energy production, bypassing glutamine. Cells grown in glutamine-free medium under the GS expression system showed a significant increase in the metabolism of a group of amino acids, highlighting that cells bypassed GS. In contrast, in the absence of NAMPT / nicotinic acid and / or nicotinamide, there was no alternative pathway in CHO, resulting in immediate cell death.

[0138] References [1] Tripathi, NK&Srivastava, A (2019) Front BioengBiotechnolhttps: / / doi.org / 10.3389 / fbioe.2019.00420 [2] Wurm, MJ&Wurm, FM (2021) Biotechnol J 16: 2100165 [3] Zhang, Q et al (2020) Biotechnol Bioeng 117: 2401 [4] Hayes, MP et al (1997) Biochem J 326: 351 [5] Vaziri, H et al (2001) Cell 107: 149 [6] Rajman, L et al (2018) Cell Metabolism 27:529 [7] Hara N et al (2007) Metabolism&Bioenergetics 282: 24574. [8] Shackleford, RE et al (2013) Genes Cancer 4: 447 [9] Sampath, D et al (2015) Pharmacol Ther 151: 16

[10] Tan, B et al (2015) J Biol Chem 290: 15812

[11] Khan, HY et al (2022) Cancers 14: 160

[12] Gardell, SJ et al (2019) Nat Commun 10: 3241 Sequence SEQ ID 1: NAMPT >sp|P43490|NAMPT_Human Nicotinamide phosphoribosyltransferase OS=Homo sapiens OX=9606 GN=NAMPT PE=1 SV=1 MNPAAEAEFNILLATDSYKVTHYKQYPPNTSKVYSYFECREKKTENSKLRKVKYEETVFY GLQYILNKYLKGKVVTKEKIQEAKDVYKEHFQDDVFNEKGWNYILEKYDGHLPIEIKAVP EGFVIPRGNVLFTVENTDPECYWLTNWIETILVQSWYPITVATNSREQKKILAKYLLETS GNLDGLEYKLHDFGYRGVSSQETAGIGASAHLVNFKGTDTVAGLALIKKYYGTKDPVPGY SVPAAEHSTITAWGKDHEKDAFEHIVTQFSSVPVSVVSDSYDIYNACEKIWGEDLRHLIV SRSTQAPLIIRPDSGNPLDTVLKVLEILGKKFPVTENSKGYKLLPPYLRVIQGDGVDINT LQEIVEGMKQKMWSIENIAFGSGGGLLQKLTRDLLNCSFKCSYVVTNGLGINVFKDPVAD PNKRSKKGRLSLHRTPAGNFVTLEEGKGDLEEYGQDLLHTVFKNGKVTKSYSFDEIRKNA QLNIELEAAHH SEQ ID 2: NAPRT >sp|Q6XQN6|PNCB_Nicotinate phosphoribosyltransferase OS=Homo sapiens OX=9606 GN=NAPRT PE=1 SV=2 MAAEQDPEARAAARPLLTDLYQATMALGYWRAGRARDAAEFELFFRRCPFGGAFALAAGL RDCVRFLRAFRLRDADVQFLASVLPPDTDPAFFEHLRALDCSEVTVRALPEGSLAFPGVP LLQVSGPLLVVQLLETPLLCLVSYASLVATNAARLRLIAGPEKRLLEMGLRRAQGPDGGL TASTYSYLGGFDSSSNVLAGQLRGVPVAGTLAHSFVTSFSGSEVPPDPMLAPAAGEGPGV DLAAKAQVWLEQVCAHLGLGVQEPHPGERAAFVAYALAFPRAFQGLLDTYSVWRSGLPNF LAVALALGELGYRAVGVRLDSGDLLQQAQEIRKVFRAAAAQFQVPWLESVLIVVSNNIDE EALARLAQEGSEVNVIGIGTSVVTCPQQPSLGGVYKLVAVGGQPRMKLTEDPEKQTLPGS KAAFRLLGSDGSPLMDMLQLAEEPVPQAGQELRVWPPGAQEPCTVRPAQVEPLLRLCLQQ GQLCEPLPSLAESRALAQLSLSRLSPEHRRLRSPAQYQVVLSERLQALVNSLCAGQSP SEQ ID 3: QPRT >sp|Q15274|NADC_Nicotinate-nucleotide pyrophosphorylase [carboxylating] OS=Homo sapiens OX=9606 GN=QPRT PE=1 SV=3 MDAEGLALLLPPVTLAALVDSWLREDCPGLNYAALVSGAGPSQAALWAKSPGVLAGQPFF DAIFTQLNCQVSWFLPEGSKLVPVARVAEVRGPAHCLLLGERVALNTLARCSGIASAAAA AVEAARGAGWTGHVAGTRKTTPGFRLVEKYGLLVGGAASHRYDLGGLVMVKDNHVVAAGG VEKAVRAARQAADFTLKVEVECSSLQEAVQAAEAGADLVLLDNFKPEELHPTATVLKAQF PSVAVEASGGITLDNLPQFCGPHIDVISMGMLTQAAPALDFSLKLFAKEVAPVPKIH SEQ ID 4: IDO >sp|Q6ZQW0|I23O2_Human indoleamine 2,3-dioxygenase 2 OS=Homo sapiens OX=9606 GN=IDO2 PE=1 SV=5 MEPHRPNVKTAVPLSLESYHISEEYGFLLPDSLKELPDHYRPWMEIANKLPQLIDAHQLQ AHVDKMPLLSCQFLKGHREQRLAHLVLSFLTMGYVWQEGEAQPAEVLPRNLALPFVEVSR NLGLPPILVHSDLVLTNWTKKDPDGFLEIGNLETIISFPGGESLHGFILVTALVEKEAVP GIKALVQATNAILQPNQEALLQALQRLRLSIQDITKTLGQMHDYVDPDIFYAGIRIFLSG WKDNPAMPAGLMYEGVSQEPLKYSGGSAAQSTVLHAFDEFLGIRHSKESGDFLYRMRDYM PPSHKAFIEDIHSAPSLRDYILSSGQDHLLTAYNQCVQALAELRSYHITMVTKYLITAAA KAKHGKPNHLPGPPQALKDRGTGGTAVMSFLKSVRDKTLESILHPRG SEQ ID 5: TDO >sp|P48775|T23O_Human tryptophan 2,3-dioxygenase OS=Homo sapiens OX=9606 GN=TDO2 PE=1 SV=1 MSGCPFLGNNFGYTFKKLPVEGSEEDKSQTGVNRASKGGLIYGNYLHLEKVLNAQELQSE TKGNKIHDEHLFIITHQAYELWFKQILWELDSVREIFQNGHVRDERNMLKVVSRMHRVSV ILKLLVQQFSILETMTALDFNDFREYLSPASGFQSLQFRLLENKIGVLQNMRVPYNRRHY RDNFKGEENELLLKSEQEKTLLELVEAWLERTPGLEPHGFNFWGKLEKNITRGLEEEFIR IQAKEESEEKEEQVAEFQKQKEVLLSLFDEKRHEHLLSKGERRLSYRALQGALMIYFYRE EPRFQVPFQLLTSLMDIDSLMTKWRYNHVCMVHRMLGSKAGTGGSSGYHYLRSTVSDRYK VFVDLFNLSTYLIPRHWIPKMNPTIHKFLYTAEYCDSSYFSSDESD SEQ ID 6: NMNA1 >sp|Q9HAN9|NMNA1_Human nicotinamide / nicotinate mononucleotide adenylyltransferase 1 OS=Homo sapiens OX=9606 GN=NMNAT1 PE=1 SV=1 MENSEKTEVVLLACGSFNPITNMHLRLFELAKDYMNGTGRYTVVKGIISPVGDAYKKKGL IPAYHRVIMAELATKNSKWVEVDTWESLQKEWKETLKVLRHHQEKLEASDCDHQQNSPTL ERPGRKRKWTETQDSSQKKSLEPKTKAVPKVKLLCGADLLESFAVPNLWKSEDITQIVAN YGLICVTRAGNDAQKFIYESDVLWKHRSNIHVVNEWIANDISSTKIRRALRRGQSIRYLV PDLVQEYIEKHNLYSSESEDRNAGVILAPLQRNTAEAKT SEQ ID 7: NMNA2 >sp|Q9BZQ4|NMNA2_Human Nicotinamide / Nicotinic Acid Mononucleotide Adenosyltransferase 2 OS=Homo sapiens OX=9606GN=NMNAT2 PE=1 SV=1 MTETTKTHVILLACGSFNPITKGHIQMFERARDYLHKTGRFIVIGGIVSPVHDSYGKQGL VSSRHRLIMCQLAVQNSDWIRVDPWECYQDTWQTTCSVLEHHRDLMKRVTGCILSNVNTP SMTPVIGQPQNETPQPIYQNSNVATKPTAAKILGKVGESLSRICCVRPPVERFTFVDENA NLGTVMRYEEIELRILLLCGSDLLESFCIPGLWNEADMEVIVGDFGIVVVPRDAADTDRI MNHSSILRKYKNNIMVVKDDINHPMSVVSSTKSRLALQHGDGHVVDYLSQPVIDYILKSQ LYINASG SEQ ID 8: NMNA3 >sp|Q96T66|NMNA3_Human Nicotinamide / Nicotinic Acid Mononucleotide Adenosyltransferase 3 OS=Homo sapiens OX=9606 GN=NMNAT3 PE=1 SV=2 MKSRIPVVLLACGSFNPITNMHLRMFEVARDHLHQTGMYQVIQGIISPVNDTYGKKDLAA SHHRVAMARLALQTSDWIRVDPWESEQAQWMETVKVLRHHHSKLLRSPPQMEGPDHGKAL FSTPAAVPELKLLCGADVLKTFQTPNLWKDAHIQEIVEKFGLVCVGRVGHDPKGYIAESP ILRMHQHNIHLAKEPVQNEISATYIRRALGQGQSVKYLIPDAVITYIKDHGLYTKGSTWK GKSTQSTEGKTS。

Claims

1. An expression system for selecting the presence of a target gene, the expression system comprising: (i) Cell lines, in which NAD + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited. The cell line optionally further includes the target gene, and the cell line optionally lacks the encoding of the NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway; (ii) an expression vector, said expression vector comprising encoding the NAD + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway; and (iii) Culture medium preparations, wherein the culture medium preparations do not include nicotinamide (NAM) and / or nicotinic acid.

2. A cell line, wherein, NAD + The activity of at least one enzyme in the synthetic pathway is at least partially inhibited, optionally further including the target gene, and optionally lacking the encoding of said NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway.

3. An expression vector comprising encoding NAD + The exogenous nucleotide sequence of at least one enzyme in the synthetic pathway, and optionally including the target gene.

4. A culture medium preparation that does not include nicotinamide (NAM) and / or nicotinic acid.

5. The expression system, cell line, or expression vector according to any one of claims 1 to 3, wherein, The target gene is encoded by the expression vector and / or by a second vector.

6. The expression system or cell line according to any one of claims 1 to 3 or 5, wherein, The cell line includes the expression vector.

7. The expression system, cell line, or expression vector according to any one of claims 1 to 3 or 5 to 6, wherein, The NAD + The at least one enzyme in the synthetic pathway is selected from nicotinamide phosphoribosyltransferase (NAMPT), nicotinic acid phosphoribosyltransferase (NPRT) and / or quinolinate phosphoryltransferase (QPRT).

8. The expression system, cell line, or expression vector according to claim 7, wherein, The NAD + At least one enzyme in the synthetic pathway is a heterologous mammalian NAMPT.

9. The expression system, cell line, or expression vector according to claim 8, wherein, The NAD + The at least one enzyme in the synthetic pathway has at least 70% sequence identity with SEQ ID 1 to 8.

10. The expression system, cell line, or expression vector according to any one of claims 1 to 3 or 5 to 9, wherein, The target gene encodes a recombinant protein, a viral vector, components of the viral vector, a viral vaccine, an intracellular protein, and / or a receptor.

11. The expression system, cell line, or expression vector according to any one of claims 1 to 3 or 5 to 10, wherein, The exogenous nucleotide sequence encoding at least one enzyme in the NAD+ synthesis pathway is under the control of a promoter, optionally wherein the promoter is CMV, SV40 and / or mPGK.

12. The expression system, cell line, or expression vector according to any one of claims 1 to 3 or 5 to 11, wherein, The target gene is under the control of a promoter, optionally wherein the promoter is CMV, SV40 and / or mPGK.

13. The expression system or cell line according to any one of claims 1 to 2 or 5 to 12, wherein, The cell line is a eukaryotic cell line, optionally selected from: Chinese hamster ovary KI cells; HEK cells; and / or VERO cells.

14. The expression system or cell line according to any one of claims 1 to 2 or 5 to 13, wherein, The cell line was genetically engineered to lack the encoding of NAD. + The endogenous nucleotide sequence of at least one enzyme in the synthetic pathway.

15. The expression system or culture medium formulation according to any one of claims 1 to 14, wherein, The culture medium formulation further includes inhibitors, optionally wherein... The inhibitor is a transferase inhibitor.

16. The expression system or culture medium formulation according to claim 15, wherein, The transferase inhibitor is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

17. The expression system or culture medium formulation according to any one of claims 1 to 16, wherein, The culture medium preparation is a substantially serum-free and / or protein-free culture medium.

18. A method for selecting cells comprising a target gene using an expression system, cell line, expression vector, or culture medium formulation according to any one of claims 1 to 17.

19. A method for culturing and / or amplifying cell lines according to any one of claims 1, 2, 5 to 14 using a culture medium preparation according to any one of claims 4 and 15 to 17.

20. Use of the expression system according to any one of claims 1 to 17, wherein, Excluding NAD + Cells cannot survive if at least one enzyme in the synthetic pathway is present.

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