Cell having increased secretion capabilities and method of use

Abstract

Provided herein is a cell having increased capabilities for secreting biologic materials comprising an exogenous constitutive promoter located upstream of the endogenous SCD1 gene. This cell allows for overexpression of SCD1 thereby increasing the secretion capabilities of the cell.

Description

[0001] CELL HAVING INCREASED SECRETION CAPABILITIES AND METHOD OF USE

[0002] Field of the Invention

[0003] The present invention is in the field of cells and cell-lines for the production of biologic materials. Such biologic materials may be used in medicaments for the treatment of mammalians, including medicaments for the treatment of humans. Provided herein is a cell having increased capabilities for secreting biologic materials comprising an exogenous constitutive promoter located upstream of the SCD1 gene. This cell allows for overexpression of SCD1 thereby increasing the secretion capabilities of the cell.

[0004] Background of the Invention

[0005] Production of biologic materials from a cell in culture has been an accepted method in the production of medicaments based on such biologic materials. In many of such methods a vector comprising the gene encoding the biologic material of interest is transfected into a host cell (host cell-line). In cell culture the cell expresses such biologic material which is being secreted from the cell. For commercial production of biologic materials, it is important that the maximum amount of biologic materials are secreted from such cells in culture. One method of increasing secretion of biologic materials from cells is to increase fluidity of the cell membrane. An important protein involved in regulating membrane fluidity is SCD1. Overexpression of SCD1 consequently can lead to increased fluidity of the cell membrane and increased secretion of biologic materials from the cell.

[0006] SCD1 gene expression was previously modulated in Lonza's CHO-K1-SV cells by inserting additional SCD1 copies into the host cell genome trough a random insertion approach. For that the mouse SCD1 gene was amplified from Mouse P19 derived cDNA using mouse sequence specific primers, cloned into a primer and stably integrated into the genome of CHO cells. This modification led to an 2,4 to 4-fold enhancement in monoclonal antibody productivity (see WO2017191165). However, such approach may not lead to a stable overexpression of SCD1 and in some instances could be non-effective.

[0007] A random integration gene editing approach for the overexpression of SCD1 gene displays some major drawbacks, especially when the modified cells are meant to be used in a pharmaceutical industry context. One of the most outstanding disadvantage of this approach is the clone-to-clone huge variability in SCD1 mRNA expression levels. The root cause of this variability can be found in the uncontrolled integration of the expression cassette into the host cell genome. Additional methods of overexpressing SCD1 are therefore needed. Provided herein is a targeted method to precisely insert a constitutive promoter upstream the endogenous SCD1 gene. Such targeted method produced a 2- to 3-fold enhancement in monoclonal antibody productivity.

[0008] Summary of the Invention

[0009] In one aspect the present invention provides a cell having increased capabilities for secreting biologic materials comprising an exogenous constitutive promoter located upstream of the SCD1 gene and which promoter regulates the expression of the SCD1 gene. The cell, in some embodiments, overexpresses SCD1.

[0010] In some embodiments, the above-described cell is a cell for the expression and production of biologic material and wherein the cell increases production / secretion of such biologic material. In a particular embodiment, the cell is a mammalian cell, more particularly, the cell is a CHO-K1 cell.

[0011] In another aspect of the present invention there is provided a method of increasing production of biologic material from a cell comprising transfecting a cell as described in the above embodiments with a vector comprising a gene encoding biologic material, growing the cells in a media and harvesting the biologic material from the cell.

[0012] The present invention is inter alia described by the following items:

[0013] [1] A cell having increased capabilities for secreting biologic materials comprising an exogenous constitutive promoter, which is located upstream of the (endogenous) SCD1 gene and which regulates the expression of the SCD1 gene.

[0014] [2] The cell of item 1, wherein the cell overexpresses SCD1.

[0015] [3] The cell of item 1 or 2, wherein the cell is a cell for the expression and production of biologic material and wherein the cell increases production / secretion of such biologic material.

[0016] [4] The cell of any of the preceding items, wherein the cell is a mammalian cell.

[0017] [5] The cell of any of the preceding items, wherein the cell is a CHO cell.

[0018] [6] The cell of any of the preceding items, wherein the cell is a CHO-K1 cell.

[0019] [7] The cell of any of the preceding items, wherein the exogenous constitutive promoter is a synthetic promoter.

[0020] [8] The cell of item 7, wherein the synthetic promoter is a chimeric (hybrid) promoter. [9] The cell of any of the preceding items, wherein the exogenous constitutive promoter comprises, or consists of, (i) optionally an enhancer, (ii) a core promoter, and (iii) an optional element (optionally comprising an Internal Ribosome Entry Site (IRES) and / or boosting transcription).

[0021]

[0010] The cell of any of the preceding items, wherein the exogenous constitutive promoter, in 5' to 3' direction, comprises, or consists of, (i) optionally an enhancer; (ii) a core promoter, and (iii) an optional element (optionally comprising an Internal Ribosome Entry Site (IRES) and / or boosting transcription).

[0022]

[0011] The cell of any of the preceding items, wherein the exogenous constitutive promoter (e.g. in 5' to 3' direction) comprises, or consists of, (i) optionally an enhancer selected from cytomegalovirus (CMV) enhancer (e.g. murine CMV enhancer or human CMV enhancer), cytomegalovirus (CMV) early enhancer element, SV40 enhancer, CMV intron A, an EEF1A1 intron 1 (CHEF-la intron), and human cytomegalovirus immediate early core promoter element (hCPE); (ii) a core promoter selected from elongation factor la promoter (e.g. minimal elongation factor la promoter such as minimal human elongation factor la promoter (core hEF-la promoter) or Chinese hamster elongation factor 1-alpha (CHEFla) promoter), phosphoglycerate kinase 1 promoter (e.g. phosphoglycerate kinase 1 core promoter such as human phosphoglycerate kinase 1 (hPGKl) core promoter), ACTB (P-actin) minimal promoter, RPLPO (36B4) core promoter, RPS3A minimal promoter, GAPDH (GPD) core promoter, SV40 early core promoter, CMV minimal (core / TATA), SFFV U3 core, RSV U3 core, mini-UBC, chicken p-actin (CBA), compact CBA hybrid (CBh)), and a core promoter comprising, or consisting of, the promoter region, the first exon, and the first intron of chicken beta-Actin gene; and (iii) an optional element, optionally selected from 5' untranslated region (5' UTR) of human T-cell leukemia virus (HTLV), encephalomyocarditis Virus (EMCV) IRES, poliovirus IRES, and Hepatitis C Virus (HCV) IRES, ubiquitous chromatin opening element (UCOE) such as A2UCOE (derived from the human HNRPA2B1-CBX3 housekeeping gene locus) or RPS3A-derived UCOE, Epstein-Barr virus terminal repeat fragment (EBVTR), and splice acceptor of the rabbit beta-Globin gene (e.g. 5' untranslated region (5' UTR) of human T-cell leukemia virus (HTLV)).

[0023]

[0012] The cell of any of the preceding items, wherein the exogenous constitutive promoter (e.g. in 5' to 3' direction) comprises, or consists of, an (i) enhancer selected from cytomegalovirus (CMV) enhancer (e.g. murine CMV enhancer or human CMV enhancer), SV40 enhancer, CMV intron A, an EEF1A1 intron 1 (CHEF-la intron), and human cytomegalovirus immediate early core promoter element (hCPE); (ii) a core promoter selected from elongation factor la promoter (e.g. minimal elongation factor la promoter such as minimal human elongation factor la promoter (core hEF-la promoter) or Chinese hamster elongation factor 1-alpha (CHEFla) promoter), phosphoglycerate kinase 1 promoter (e.g. phosphoglycerate kinase 1 core promoter such as human phosphoglycerate kinase 1 (hPGKl) core promoter), ACTB (P-actin) minimal promoter, RPLPO (36B4) core promoter, RPS3A minimal promoter, GAPDH (GPD) core promoter, SV40 early core promoter, CMV minimal (core / TATA), SFFV U3 core, RSV U3 core, mini-UBC, chicken p-actin (CBA), compact CBA hybrid (CBh)); and (iii) an optional element comprising an Internal Ribosome Entry Site (IRES), optionally selected from 5' untranslated region (5' UTR) of human T-cell leukemia virus (HTLV), encephalomyocarditis Virus (EMCV) IRES, poliovirus IRES, and Hepatitis C Virus (HCV) IRES (e.g. 5' untranslated region (5' UTR) of human T- cell leukemia virus (HTLV)).

[0024]

[0013] The cell of any of the preceding items, wherein the exogenous constitutive promoter comprises, or consists of, (i) a cytomegalovirus enhancer e.g. the murine cytomegalovirus enhancer (mCMV enh), (ii) a minimal elongation factor la promoter e.g. minimal human elongation factor la promoter (core hEF-la promoter, i.e. core promoter from the human elongation factor-la gene), and (iii) optionally the 5' untranslated region of the human T-cell leukemia virus (HTLV); or the CAG promoter, which comprises, or consists of, (i) cytomegalovirus (CMV) early enhancer element (C), (ii) the promoter region, the first exon, and the first intron of chicken beta-Actin gene (A), and (iii) the splice acceptor of the rabbit beta-Globin gene (G).

[0025]

[0014] The cell of any of the preceding items, wherein the exogenous constitutive promoter comprises, or consists of, preferably in 5' to 3' direction, optionally (as enhancer) a sequence as shown in SEQ ID NO: 3, or a sequence differing in less than 70, or less than 50, or less than 40, or less than 30, or less than 20, or less than 15, or in less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 3; (as core promoter) a sequence as shown in SEQ ID NO: 4, or a sequence differing in less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 4; and (as optional element) a sequence as shown in SEQ ID NO: 5, or a sequence differing in less than 50, or less than 40, or less than 30, or less than 20, or less than 15, or in less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 5.

[0026]

[0015] The cell of any of the preceding items, wherein the exogenous constitutive promoter comprises, or consists of, preferably in 5' to 3' direction, (as enhancer) a sequence as shown in SEQ ID NO: 3, or a sequence differing in less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 3; (as core promoter) a sequence as shown in SEQ ID NO: 4, or a sequence differing in less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 4; and (as optional element) a sequence as shown in SEQ ID NO: 5, or a sequence differing in less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 5.

[0016] The cell of any of the preceding items, wherein the exogenous constitutive promoter comprises, or consists of, a sequence as shown in SEQ ID NO: 6, or a sequence differing in less than 100, or less than 70, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 6.

[0027]

[0017] The cell of any of the preceding items, wherein the exogenous constitutive promoter (located upstream of the endogenous SCD1 gene) is positioned at its 5' end (optionally immediately) adjacent to a left homology arm, optionally comprising, or consisting of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 70, or less than 50, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; and / or at its 3' end (optionally immediately) adjacent to a right homology arm, optionally comprising, or consisting of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 70, or less than 50, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

[0028]

[0018] The cell of any of the preceding items, wherein the exogenous constitutive promoter (located upstream of the endogenous SCD1 gene) is positioned

[0029] - at its 5' end, immediately adjacent to, or within 400, or 300, or 200, or 150, or 100, or 80, or 60, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of (the 3' end of) a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 50, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; and / or

[0030] - at its 3' end, immediately adjacent to, or within 400, or 300, or 200, or 150, or 100, or 80, or 60, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of (the 5' end of) a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 50, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

[0031]

[0019] The cell of any of the preceding items, wherein the exogenous constitutive promoter (located upstream of the endogenous SCD1 gene) is positioned at its 5' end, immediately adjacent to, or within 70, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or

[0032] 3, nucleotides of (the 3' end of) a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; and / or

[0033] - at its 3' end, immediately adjacent to, or within 70, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of (the 5' end of) a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

[0034]

[0020] The cell of any of the preceding items, wherein the cell comprises a sequence as shown in SEQ ID NO: 7, or a sequence differing in less than 200, or less than 160, or less than 140, or less than 120, or less than 100, or less than 80, or less than 70, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 7.

[0035]

[0021] The cell of any of the preceding items, wherein a ssDNA sequence is used to introduce the exogenous constitutive promoter upstream of the endogenous SCD1 gene; optionally wherein the ssDNA sequence comprises, or consists of, in 5' to 3' direction, a left homology arm, a (synthetic) constitutive promoter, and a right homology arm; wherein the ssDNA sequence optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 7, or a sequence differing in less than 200, or less than 160, or less than 140, or less than 120, or less than 100, or less than 80, or less than 70, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 7.

[0036]

[0022] The cell of any of the preceding items, wherein a ssDNA sequence is used to introduce the exogenous constitutive promoter upstream of the endogenous SCD1 gene; optionally wherein the ssDNA sequence comprises, or consists of, in 5' to 3' direction, a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 50, or less than 30, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; an exogenous constitutive promoter (sequence), which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 6, or a sequence differing in less than 100, or less than 80, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 6; and a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 50, or less than 30, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than

[0037] 2, nucleotides from SEQ. ID NO: 2.

[0038]

[0023] The cell of any of the preceding items, wherein the SCD1 gene is the endogenous Chinese hamster (Cricetulus griseus) SCD1 gene.

[0039]

[0024] The cell of any of the preceding items, wherein the SCD1 gene is the endogenous SCD1 gene of CHO (Chinese hamster ovary) cells.

[0040]

[0025] The cell of any of the preceding items, wherein the SCD1 gene is Acyl-CoA desaturase 1 of Chinese hamster (Cricetulus griseus), preferably identified by the NCBI Gene ID 100750593 (LOC100750593).

[0041]

[0026] The cell of any of the preceding items, wherein the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene identified by the NCBI Gene ID 100750593 (LOC100750593).

[0042]

[0027] The cell of any of the preceding items, wherein the SCD1 gene comprises, or consists of, a nucleotide sequence of the gene identified by the NCBI Gene ID 100750593 (LOC100750593), or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from a nucleotide sequence of the gene identified by the NCBI Gene ID 100750593 (LOC100750593).

[0043]

[0028] The cell of any of the preceding items, wherein the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1.

[0044]

[0029] The cell of any of the preceding items, wherein the SCD1 gene comprises, or consists of, the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1, or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1.

[0045]

[0030] The cell of any of the preceding items, wherein the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene with the nucleotide sequence corresponding to region 170699 to 184201 of NCBI accession number NW_003614544.1.

[0046]

[0031] The cell of any of the preceding items, wherein the SCD1 gene comprises, or consists of, the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 170699 to 184201 of NCBI accession number NW_003614544.1, or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 170699 to 184201 of NCBI accession number NW_003614544.1.

[0047]

[0032] The cell of any of the preceding items, wherein the SCD1 gene encodes a protein with an amino acid sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 96.5 %, or at least 97 %, or at least 97.5 %, or at least 98 %, or at least 98.5 %, or at least 98.8 %, or at least 99.1 %, or at least 99.4 % or at least 99.7 %, sequence identity with the protein identified by the NCBI accession number XP_027263037 (UniProt ID: A0A8C2MRM2).

[0048]

[0033] The cell of any of the preceding items, wherein the SCD1 gene encodes a protein with an amino acid sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 96.5 %, or at least 97 %, or at least 97.5 %, or at least 98 %, or at least 98.5 %, or at least 98.8 %, or at least 99.1 %, or at least 99.4 % or at least 99.7 %, sequence identity with the protein identified by the NCBI accession number XP_016834188.1.

[0049]

[0034] The cell of any of the preceding items, wherein the SCD1 gene encodes a protein identified by the NCBI accession number XP_027263037 (UniProt ID: A0A8C2MRM2), or an amino acid sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, amino acids from the protein identified by the NCBI accession number XP_027263037 (UniProt ID: A0A8C2MRM2).

[0035] The cell of any of the preceding items, wherein the SCD1 gene encodes a protein identified by the NCBI accession number XP_016834188.1, or an amino acid sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, amino acids from the protein identified by the NCBI accession number XP_016834188.1.

[0050]

[0036] The cell of any of the preceding items, wherein the exogenous constitutive promoter located upstream of the endogenous SCD1 gene is introduced by targeted gene editing, optionally selected from CRISPR (e.g. CRISPR-Cas9, CRISPR-Casl2a and Casl2b), Transcription-activator-like effector nucleases (TALENs), and Zinc-finger nucleases (ZFNs).

[0051]

[0037] A method of increasing production of biologic material from a cell comprising transfecting a cell of any of the preceding items with a vector comprising a gene encoding biologic material, growing the cells in a media and harvesting the biologic material from the cell.

[0052] Brief Description of the Drawings

[0053] Figure 1: Scheme of the constitutive promoter composition including homology arms: in the image, the full sequence of the ssDNA used [from 5' to 3': Left Homology arm (in normal letters, no background) - mCMV enhancer (with light grey background) - Core hEF-la promoter (underlined letters, no background) - HTLV (bold letters, no background) - Right homology arm (with dark grey background)].

[0054] Figure 2: (A) Junction PCR analysis of modified clones derived from the SCD1#1 pool. (B) SCD1 5' Junction PCR (upper panel) and SCD1 3' Junction PCR (lower panel) results. Expected amplicons (±10% of the predicted size) were observed in all clones and the positive control plasmid. No amplification was detected in the reference clone.

[0055] Figure 3: RT-ddPCR analysis on modified and reference clones. RNA from all the pools was extracted and analysed with a duplex ddPCR reaction to quantify SCD1 mRNA (target) and GUSB mRNA (reference transcript). SCD1 / GUSB ratios of all the modified clones (dark grey) are shown in comparison to the reference clone (light grey).

[0056] Figure 4: (A) PA-HPLC titer analysis on modified and reference clones cell culture medias. Cell culture media harvested after four days of culture was subjected to PA-HPLC. Titers (mg / L) of all the modified clones are shown in comparison to the reference clone. Data are shown as mean ± Standard Deviation. (B) Specific productivity (Qp) of modified and reference clones. Titer values of each clone were divided by Integral Viable Cell density (IVCD). Qp values of all the modified clones are shown in comparison to the reference clone. Data are shown as mean ± Standard Deviation. Detailed Description of the Invention

[0057] In a first aspect, the present invention provides a cell having increased capabilities for secreting biologic materials comprising an exogenous constitutive promoter located upstream of the (endogenous) SCD1 gene and which promoter regulates the expression of the SCD1 gene. In some embodiments the cell overexpresses SCD1.

[0058] In some embodiments the present invention is as defined in the following: in a targeted method for inserting an exogenous constitutive promoter for SCD1, the constitutive promoter was inserted upstream the endogenous SCD1 gene of CHO cells capable of expressing biologic materials of intertest. The introduced modification not only produced a prominent enhancement in the SCD1 mRNA expression but also produced a 2 to 3-fold increase in the production of biologic materials (for example monoclonal antibody productivity).

[0059] In some embodiments of the present invention, the cell overexpresses SCD1. In some embodiments of the present invention, the cell is a cell for the expression and production of biologic material and wherein the cell increases production / secretion of such biologic material. In some embodiments of the present invention, the cell is a mammalian cell. In some embodiments of the present invention, the cell is a CHO cell. In specific embodiments of the present invention, the cell is a CHO-K1 cell.

[0060] In some embodiments of the present invention, the exogenous constitutive promoter is a synthetic promoter. In some embodiments of the present invention, the synthetic promoter is a chimeric promoter. In some embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, (i) optionally an enhancer, (ii) a core promoter, and (iii) an optional element (e.g. an element having a sequence comprising an Internal Ribosome Entry Site (IRES) and / or boosting transcription). In further embodiments of the present invention, the exogenous constitutive promoter, in 5' to 3' direction, comprises, or consists of, (i) optionally an enhancer, (ii) a core promoter, and (iii) an optional element optionally comprising an Internal Ribosome Entry Site (IRES) (e.g. an element having a sequence comprising an IRES and / or boosting transcription).

[0061] In specific embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, (i) optionally an enhancer selected from cytomegalovirus (CMV) enhancer (e.g. murine CMV enhancer or human CMV enhancer, e.g. one or more individual elements such as the cytomegalovirus (CMV) early enhancer element or the complete enhancer), cytomegalovirus (CMV) early enhancer element, SV40 enhancer, CMV intron A, an EEF1A1 intron 1 (CHEF-la intron), and human cytomegalovirus immediate early core promoter element (hCPE); (ii) a core promoter selected from elongation factor la promoter (e.g. minimal elongation factor la promoter such as minimal human elongation factor la promoter (core hEF-la promoter) or Chinese hamster elongation factor 1-alpha (CHEFla) promoter), phosphoglycerate kinase 1 promoter (e.g. phosphoglycerate kinase 1 core promoter such as human phosphoglycerate kinase 1 (hPGKl) core promoter), ACTB (P-actin) minimal promoter, RPLPO (36B4) core promoter, RPS3A minimal promoter, GAPDH (GPD) core promoter, SV40 early core promoter, CMV minimal (core / TATA), SFFV U3 core, RSV U3 core, mini-UBC, chicken p-actin (CBA), compact CBA hybrid (CBh)), and a core promoter comprising, or consisting of, the promoter region, the first exon, and the first intron of chicken beta-Actin gene; and (iii) an optional element (e.g. a sequence comprising an IRES and / or boosting transcription), optionally selected from 5' untranslated region (5' UTR) of human T-cell leukemia virus (HTLV), encephalomyocarditis virus (EMCV) IRES, poliovirus IRES, hepatitis C virus (HCV) IRES, ubiquitous chromatin opening element (UCOE) such as A2UCOE (derived from the human HNRPA2B1-CBX3 housekeeping gene locus) or RPS3A-derived UCOE, Epstein-Barr virus terminal repeat fragment (EBVTR), and splice acceptor of the rabbit beta-Globin gene (e.g. 5' UTR of human T-cell leukemia virus (HTLV) or a variant thereof).

[0062] In further specific embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, (i) optionally an enhancer selected from cytomegalovirus (CMV) enhancer (e.g. murine CMV enhancer or human CMV enhancer, e.g. individual elements such as the cytomegalovirus (CMV) early enhancer element or the complete enhancer), SV40 enhancer, CMV intron A, an EEF1A1 intron 1 (CHEF-la intron), and human cytomegalovirus immediate early core promoter element (hCPE); (ii) a core promoter selected from elongation factor la promoter (e.g. minimal elongation factor la promoter such as minimal human elongation factor la promoter (core hEF-la promoter) or Chinese hamster elongation factor 1-alpha (CHEFla) promoter), phosphoglycerate kinase 1 promoter (e.g. phosphoglycerate kinase 1 core promoter such as human phosphoglycerate kinase 1 (hPGKl) core promoter), p-actin (ACTB) minimal promoter, RPLPO (36B4) core promoter, RPS3A minimal promoter, GAPDH (GPD) core promoter, SV40 early core promoter, CMV minimal (core / TATA), SFFV U3 core, RSV U3 core, mini-UBC, chicken p-actin (CBA), and compact CBA hybrid (CBh)); and (iii) an optional element optionally comprising an Internal Ribosome Entry Site (IRES), optionally selected from 5' untranslated region (5' UTR) of human T-cell leukemia virus (HTLV), encephalomyocarditis Virus (EMCV) IRES, poliovirus IRES, and hepatitis C virus (HCV) IRES (e.g. 5' UTR of human T-cell leukemia virus (HTLV)).

[0063] In some embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, the CAG promoter, which is composed of the following (regulatory) elements: (i) cytomegalovirus (CMV) early enhancer element (C); (ii) the promoter region, the first exon, and the first intron of chicken beta-Actin gene (A); and (iii) the splice acceptor of the rabbit beta-Globin gene (G). In particular embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, (i) a cytomegalovirus enhancer e.g. the murine cytomegalovirus enhancer (mCMV enh); (ii) a minimal elongation factor la promoter e.g. minimal human elongation factor la promoter (core hEF-la promoter, i.e. core promoter from the human elongation factor-la gene); and (iii) optionally the 5' untranslated region of the human T-cell leukemia virus (HTLV).

[0064] In some embodiments the "murine cytomegalovirus enhancer" comprises, or consists of, a sequence as shown in SEQ ID NO: 3, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 3.

[0065] In some embodiments the "minimal human elongation factor la promoter" comprises, or consists of, a sequence as shown in SEQ ID NO: 4, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 4.

[0066] In some embodiments the "5' untranslated region of the human T-cell leukemia virus (HTLV)" comprises, or consists of, a sequence as shown in SEQ ID NO: 5, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 5.

[0067] In specific embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, preferably in 5' to 3' direction, (i) optionally as enhancer a sequence as shown in SEQ ID NO: 3, or a sequence differing in less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 3; (ii) as core promoter a sequence as shown in SEQ ID NO: 4, or a sequence differing in less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 4; and (iii) as optional element a sequence as shown in SEQ ID NO: 5, or a sequence differing in less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 5.

[0068] In some embodiments of the present invention, the exogenous constitutive promoter comprises, or consists of, a sequence as shown in SEQ ID NO: 6, or a sequence differing in less than 90, or less than 70, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 6. In some embodiments of the present invention, the exogenous constitutive promoter is positioned at its 5' end (optionally immediately) adjacent to (the 3' end of) a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; and / or at its 3' end (optionally immediately) adjacent to (the 5' end of) a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

[0069] In further embodiments of the present invention, the exogenous constitutive promoter is positioned at its 5' end, immediately adjacent to, or within 300, or 200, or 150, or 100, or 80, or 60, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of (the 3' end of) a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; and / or at its 3' end, immediately adjacent to, or within 300, or 200, or 150, or 100, or 80, or 60, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of, (the 5' end of) a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

[0070] In some embodiments of the present invention, the exogenous constitutive promoter is introduced upstream of the endogenous SCD1 gene using a ssDNA sequence for example using targeted gene editing (e.g. CRISPR, TALEN, ZFN, etc.); optionally wherein the ssDNA sequence comprises, or consists of, in 5' to 3' direction, a left homology arm, a (synthetic) constitutive promoter, and a right homology arm; wherein ssDNA sequence optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 7, or a sequence differing in less than 100, or less than 80, or less than 70, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 7.

[0071] In some embodiments of the present invention, the exogenous constitutive promoter located upstream of the endogenous SCD1 gene is introduced by targeted gene editing, optionally selected from CRISPR (e.g. CRISPR-Cas9, CRISPR-Casl2a and Casl2b), Transcription-activator-like effector nucleases (TALENs), and Zinc-finger nucleases (ZFNs). In some embodiments of the present invention, the exogenous constitutive promoter located upstream of the endogenous SCD1 gene is introduced by CRISPR (e.g. CRISPR-Cas9, CRISPR-Casl2a and Casl2b), optionally using a crRNA sequence comprising, or consisting of a sequence as defined in SEQ ID NO: 24, or a sequence differing in less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 24 and a ssDNA as defined in any of the embodiments herein.

[0072] In further embodiments of the present invention, the exogenous constitutive promoter is introduced upstream of the endogenous SCD1 gene using a ssDNA sequence; optionally wherein the ssDNA sequence comprises, or consists of, in 5' to 3' direction, a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 30, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than

[0073] 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; an (exogenous) constitutive promoter (sequence), which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 6, or a sequence differing in less than 80, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than

[0074] 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 6; and a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 30, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

[0075] In some embodiments of the present invention, the SCD1 gene is the endogenous Chinese hamster (Cricetulus griseus) SCD1 gene. In some embodiments of the present invention, the SCD1 gene is the endogenous SD1 gene of CHO (Chinese hamster ovary) cells.

[0076] In some embodiments of the present invention, the SCD1 gene is Acyl-CoA desaturase 1 of Chinese hamster (Cricetulus griseus), preferably identified by the NCBI Gene ID 100750593 (LQC100750593).

[0077] In some embodiments of the present invention, the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene identified by the NCBI Gene ID 100750593 (LQC100750593).

[0078] In some embodiments of the present invention, the SCD1 gene comprises, or consists of, a nucleotide sequence of the gene identified by the NCBI Gene ID 100750593 (LQC100750593), or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from a nucleotide sequence of the gene identified by the NCBI Gene ID 100750593 (LOC100750593).

[0079] In some embodiments of the present invention, the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1.

[0080] In some embodiments of the present invention, the SCD1 gene comprises, or consists of, the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1, or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1.

[0081] In some embodiments of the present invention, the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene with the nucleotide sequence corresponding to region 170699 to 184201 of NCBI accession number NW_003614544.1.

[0082] In some embodiments of the present invention, the SCD1 gene comprises, or consists of, the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 170699 to 184201 of NCBI accession number NW_003614544.1, or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 170699 to 184201 of NCBI accession number NW_003614544.1.

[0083] In some embodiments of the present invention, the SCD1 gene encodes a protein with an amino acid sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 96.5 %, or at least 97 %, or at least 97.5 %, or at least 98 %, or at least 98.5 %, or at least 98.8 %, or at least 99.1 %, or at least 99.4 % or at least 99.7 %, sequence identity with the protein identified by the NCBI accession number XP_027263037 (UniProt ID: A0A8C2MRM2).

[0084] In some embodiments of the present invention, the SCD1 gene encodes a protein with an amino acid sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 96.5 %, or at least 97 %, or at least 97.5 %, or at least 98 %, or at least 98.5 %, or at least 98.8 %, or at least 99.1 %, or at least 99.4 % or at least 99.7 %, sequence identity with the protein identified by the NCBI accession number XP_016834188.1.

[0085] In some embodiments of the present invention, the SCD1 gene encodes a protein identified by the NCBI accession number XP_027263037 (UniProt ID: A0A8C2MRM2), or an amino acid sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, amino acids from the protein identified by the NCBI accession number XP_027263037 (UniProt ID: A0A8C2MRM2).

[0086] In some embodiments of the present invention, the SCD1 gene encodes a protein identified by the NCBI accession number XP_016834188.1, or an amino acid sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, amino acids from the protein identified by the NCBI accession number XP_016834188.1.

[0087] In a second aspect, the present invention provides a method of increasing production of biologic material from a cell comprising transfecting a cell of any of the embodiments of the first aspect of the present invention described herein above with a vector comprising a gene encoding biologic material, growing the cells in a media and harvesting the biologic material from the cell.

[0088] Examples

[0089] The single strand DNA (ssDNA) transgene containing the promoter sequence and the homology arms were synthetized. The ssDNA was transfected alongside CRISPR / HiFi-Cas9 ribonucleoprotein into the CHO cells. After a genomic characterization of the transfected cell pool, monoclonal populations were produced. Clonal populations were screened at the genomic and transcriptomic level. The productivity of correctly modified clones was determined by Pa-HPLC (Protein A High-performance liquid chromatography).

[0090] It was demonstrated that the modified cells display a higher monoclonal antibody productivity as compared to the parental clone. The amount of fold increase was 2-3 times over the productivity compared to non-modified cells.

[0091] The NCBI annotation of the Chinese hamster (Cricetulus griseus) genome does not explicitly define the sequence of a SCD1 gene. A gene annotated in the Chinese hamster (Cricetulus griseus) genome as Acyl-CoA Desaturase 1 was identified as a putative SCD1 orthologue. The identified gene was selected as the target for overexpression in subsequent experimental procedures in Chinese hamster ovary (CHO) cells.

[0092] To achieve overexpression of SCD1, a targeted genome editing strategy was employed to precisely insert a synthetic promoter upstream of the endogenous SCD1 gene. The promoter consisted of the murine cytomegalovirus enhancer, the minimal human elongation factor la promoter, and the 5' untranslated region (UTR) of the human T-cell leukemia virus. A synthetic promoter was selected in preference to classical viral promoters, as synthetic promoters are typically engineered to drive higher levels of gene expression and exhibit reduced susceptibility to epigenetic silencing mechanisms. The complete promoter sequence is depicted in Figure 1. The DNA donor was synthetized by and purchased from Azenta (GENEWIZ).

[0093] A crRNA was designed to target the 220 bp region immediately upstream of its transcription start site (TSS). The selected crRNA sequence, with its relative features, is shown in Table 1.

[0094] Table 1. Features of selected crRNA. Following crRNA selection, a ssDNA HDR donor template was constructed. The donor was optimized to include only the constitutive promoter flanked by homology arms. The promoter consisted of the murine cytomegalovirus enhancer, the minimal human elongation factor la promoter, and the 5' untranslated region (UTR) of the human T-cell leukemia virus. Homology arms were designed starting exactly from the double strand break point. Homology arms' length was set at 300 nucleotides as suggested by IDT guidelines. The complete ssDNA donor sequence is depicted in Figure 1. The ssDNA donor was synthetized by and purchased from Azenta (GENEWIZ).

[0095] Table 2. Sequences

[0096] In case of discrepancies between the information in Table 2 above and the information in the enclosed sequence listing file, the information in Table 2 above shall prevail.

[0097] Experiments were conducted using a Merck proprietary bi-specific monoclonal antibody-expressing CHO-K1 clone. 1 x 106viable cells were transfected using the AMAXA 4-D Nucleofector system (Lonza) using the SF Cell Line 4D-Nucleofecto X Kit S. The electroporation protocol used was specific for CHO- K1 cells (Amaxa 4-D Nucleofector electroporation protocol code: DT-133).

[0098] Transfection reagents were present in the final electroporation mix as follows: ~l,98pM High Fidelity ( Hi Fi) Cas9 ribonucleoprotein (RNP) complex (Alt-R® CRISPR-Cas9 crRNA, Alt-R® CRISPR-Cas9 tracrRNA, Alt-R® S.p. HiFi Cas9 Nuclease V3 from IDT), 2pg of ssDNA donor, lpM IDT HDR enhancer (Cat# 10007921, IDT), and 2pM IDT electroporation enhancer (Cat# 1076301, IDT). Following transfection experiment, the transfected cell pool was generated (SCD1#1 pool).

[0099] Monoclonal populations were subsequently isolated from the SCD1#1 pool by seeding cells at a 1 cell / well density in 96-well plates using the Paia single-cell dispenser (Bio-Techne) and incubated under static conditions for the single-cell cloning phase. A total of 603 monoclonal populations were generated, and following initial outgrowth, were transferred in 24-well plates for the screening phase.

[0100] As a first line of screening, the gene copy number of the constitutive promoter was determined for each monoclonal population. This evaluation was conducted using the QX ONE Droplet Digital PCR system (Bio-Rad). The ddPCR reaction mix was prepared using the ddPCR Supermix for Probes (no dUTP) following manufacturer instruction (Cat# 1863024, Bio-Rad). Each set of primers was added at the final concentration of 900 nM, while TaqMan Probes were added at 250 nM as final concentration. The analysis was performed by measuring the concentration of the constitutive promoter in relation to the reference assay designed on beta2-microglobulin (B2M) gene. Starting from concentration data the GCN was automatically computed by the instrument. Information about the ddPCR assays is listed in Table 3.

[0101] Table 3. 5'-3' sequence of primer and probes for ddPCR experiments.

[0102] Clones displaying a GCN close to one or two were selected and marked respectively as heterozygous and homozygous for the insertion of the constitutive promoter. The list of selected clones with their respective GCN values are listed in Table 4.

[0103] Table 4. GCN values and genotype of selected clones.

[0104] To determine if the insertions detected by ddPCR were indeed targeted insertions of the constitutive promoter upstream the endogenous SCD1 gene, all selected clones were subjected to 5' and 3' Junction PCRs. As a positive control, a synthetic plasmid containing the exact sequence that should be amplified by both 5' and 3' Junction PCRs was used. As negative control, to ensure specificity, DNA from untransfected CHO-K1 cells was used. Information about the Junction PCR primer sets is listed in Table 5. Table 5. Primer sets used for Junction PCRs.

[0105] All eight clones showed an appropriate PCR band pattern, thus suggesting that the integration, or at least one of the two integrations in case of a homozygous genotype, is placed in the expected genomic locus (Figure 2). These results testify the generation of 8 clones which have integrated the constitutive promoter upstream the endogenous SCD1 gene.

[0106] A further characterization of modified clones was put in place to verify whether the presence of the constitutive promoter upstream the endogenous SCD1 gene was really promoting its overexpression. To do so, a SCD1 mRNA expression analysis was performed using the QX ONE Droplet Digital PCR system. The ddPCR reaction mix was prepared using the One-Step RT-ddPCR Advanced Kit for Probes following manufacturer instruction (Cat# 1864022, Bio-Rad). Primers were used at a final concentration of 900 nM, and TaqMan probes at 250 nM. All the analyses were performed by measuring the concentration of the target assay on the SCD1 mRNA in relation to the reference assay designed on Glucuronidase beta (GUSB) gene. Detailed ddPCR assays information is provided in Table 6.

[0107] Table 6. 5 '-3' sequence of primer and probes for RT-ddPCR mRNA expression analysis.

[0108] All the clones tested showed a certain degree of SCD1 mRNA overexpression. In Figure 3, mRNA expression levels of modified and reference clones are plotted. On average, modified clones displayed a 20-fold increase in SCD1 mRNA expression as compared to the reference clone.

[0109] To determine if SCD1 overexpression have indeed an effect in ameliorating the productivity in the tested cellular model, a batch experiment was performed to compare the titer and the specific productivity of engineered clones with the reference clone's ones. Cells were cultured for four days and Protein A High Performance Liquid Chromatography (Pa-HPLC) was exploited to determine the molecule titer level in the tested samples. An average 40% increase in titer levels was detected (Figure 4A).

[0110] Specific productivity was subsequently computed to take also into account the number of cells which have effectively produced those titer levels during the four days of culture. Qpfor each sample was calculated by dividing its titer value by the corresponding Integral Viable Cell Density (IVCD). IVCD was calculated across three timepoints of culture (Day 0, Day 2, and Day4) using the equation below, where x is the viable cell number recorded at a timepoint, x-i is the viable cell number recorded at the previous timepoint, d is the day in which the timepoint was taken and d-i is the day of the previous timepoint. IVCD-i was the IVCD recorded at the previous timepoint:

[0111] Modified clones showed an average 2-fold increase in specific productivity as compared to the reference clone; with clone #8 being the top performer and reaching a 2,5-fold increase (Figure 4B).

Claims

Claims1. A cell having increased capabilities for secreting biologic materials comprising an exogenous constitutive promoter which is located upstream of the endogenous SCD1 gene and regulates the expression of the SCD1 gene.

2. The cell of claim 1, wherein the cell overexpresses SCD1.

3. The cell of claim 1 or 1, wherein the cell is a cell for the expression and production of biologic material and wherein the cell increases production / secretion of such biologic material.

4. The cell of any of the preceding claims wherein the cell is a mammalian cell.

5. The cell of any of the preceding claims, wherein the cell is a CHO cell, optionally a CHO-K1 cell.

6. The cell of any of the preceding claims, wherein the exogenous constitutive promoter is a synthetic promoter.

7. The cell of any of the preceding claims, wherein the exogenous constitutive promoter comprises, or consists of, (i) optionally an enhancer, (ii) a core promoter, and (iii) an optional element optionally comprising an Internal Ribosome Entry Site (IRES).

8. The cell of any of the preceding items, wherein the exogenous constitutive promoter comprises, or consists of, (i) optionally an enhancer selected from cytomegalovirus (CMV) enhancer, cytomegalovirus (CMV) early enhancer element, SV40 enhancer, CMV intron A, an EEF1A1 intron 1 (CHEF-la intron), and human cytomegalovirus immediate early core promoter element (hCPE); (ii) a core promoter selected from elongation factor la promoter (e.g. minimal human elongation factor la promoter (core hEF-la promoter) or Chinese hamster elongation factor 1-alpha (CHEFla) promoter), phosphoglycerate kinase 1 core promoter such as human phosphoglycerate kinase 1 (hPGKl) core promoter), p-actin (ACTB) minimal promoter, RPLPO (36B4) core promoter, RPS3A minimal promoter, GAPDH (GPD) core promoter, SV40 early core promoter, CMV minimal (core / TATA), SFFV U3 core, RSV U3 core, mini-UBC, chicken p-actin (CBA), compact CBA hybrid (CBh)), and a core promoter comprising, or consisting of, the promoter region, the first exon, and the first intron of chicken beta-Actin gene; and (iii) an optional element selected from 5' untranslated region (5' UTR) of human T-cell leukemia virus (HTLV), encephalomyocarditis virus (EMCV) IRES, poliovirus IRES, hepatitis C virus (HCV) IRES,ubiquitous chromatin opening element (UCOE) such as A2UCOE or RPS3A-derived UCOE, Epstein-Barr virus terminal repeat fragment (EBVTR), and splice acceptor of the rabbit betaGlobin gene.

9. The cell of any of the preceding claims, wherein the exogenous constitutive promoter comprises, or consists of,(i) a cytomegalovirus enhancer, optionally which is the murine cytomegalovirus enhancer (mCMV enh), (ii) a minimal elongation factor la promoter, optionally which is the minimal human elongation factor la promoter, (iii) and optionally the 5' untranslated region of the human T-cell leukemia virus (HTLV); or the CAG promoter, which comprises, or consists of, (i) of cytomegalovirus (CMV) early enhancer element (C), (ii) the promoter region, the first exon, and the first intron of chicken beta-Actin gene (A), and (iii) the splice acceptor of the rabbit beta-Globin gene (G).

10. The cell of any of the preceding claims, wherein the exogenous constitutive promoter comprises, or consists of, a sequence as shown in SEQ ID NO: 6, or a sequence differing in less than 70, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 6.

11. The cell of any of the preceding claims, wherein the exogenous constitutive promoter is positioned at its 5' end, immediately adjacent to, or within 200, or 150, or 100, or 80, or 60, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of a left homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 1, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 1; and / or at its 3' end, immediately adjacent to, or within 200, or 150, or 100, or 80, or 60, or 50, or 40, or 30, or 20, or 15, or 10, or 5, or 3, nucleotides of a right homology arm, which optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 2, or a sequence differing in less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 2.

112. The cell of any of the preceding claims, wherein the exogenous constitutive promoter is introduced upstream of the endogenous SCD1 gene using a ssDNA sequence; optionally wherein the ssDNA sequence comprises, or consists of, in 5' to 3' direction, a left homology arm, a constitutive promoter, and a right homology arm; wherein the ssDNA sequence optionally comprises, or consists of, a sequence as shown in SEQ ID NO: 7, or a sequence differing in less than 100, or less than 80, or less than 70, or less than 60, or less than 50, or less than 40, or less than 30, or less than 25, or less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from SEQ ID NO: 7.

13. The cell of any of the preceding claims, wherein the SCD1 gene is the endogenous Chinese hamster (Cricetulus griseus) SCD1 gene, optionally the acyl-CoA desaturase 1 of Chinese hamster (Cricetulus griseus); preferably identified by the NCBI Gene ID 100750593 (LQC100750593), or comprises, or consists of, a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from a nucleotide sequence of the gene identified by the NCBI Gene ID 100750593 (LQC100750593).

14. The cell of any of the preceding claims, wherein the SCD1 gene comprises, or consists of, a nucleotide sequence having at least 90 %, or at least 92 %, or at least 94 %, or at least 95 %, or at least 96 %, or at least 97 %, or at least 98 %, or at least 98.5 %, or at least 99 %, or at least 99.2 %, or at least 99.4 %, or at least 99.5 %, or at least 99.6 % or at least 99.7 %, or at least 99.8 %, or at least 99.9 %, or at least 99.92 %, or at least 99.94 %, or at least 99.96 %, or at least 99.98 %, or at least 99.99 %, sequence identity with the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1; and / or wherein the SCD1 gene comprises, or consists of, the nucleotide sequence of the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1, or a nucleotide sequence differing in less than 200, or less than 150, or less than 100, or less than 70, or less than 50, or less than 40, or less than 30, or less than 25, less than 20, or less than 15, or less than 10, or less than 8, or less than 6, or less than 5, or less than 4, or less than 3, or less than 2, nucleotides from the nucleotide sequenceof the gene with the nucleotide sequence corresponding to region 17174783 to 17188455 of NCBI accession number NC_048596.1.

15. A method of increasing production of biologic material from a cell comprising transfecting a cell of any of the preceding claims with a vector comprising a gene encoding biologic material, growing the cells in a media and harvesting the biologic material from the cell.

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