RU2808756C1 - Improved transgene expression and processing - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a recombinant nucleic acid molecule for increasing the efficiency of production of a transgene expressed in a mammalian cell. The recombinant nucleic acid molecule under the invention is designed to include a 5'-3' transposon-specific terminal repeat ITR, a nucleic acid encoding a TEP transgene protein or TEP RNA, and is complemented by an epigenetic regulatory element. In addition, a recombinant expression vector, a host cell, a method of expressing the encoded TEP, a method of transfecting a cell, and a kit for performing cell transfection are described.

EFFECT: in comparison with the known analogues, the invention demonstrates advantages in terms of the magnitude and quality of expression, including high stability of transgene expression and, if required, transport of the expressed product out of the cell.

20 cl, 4 tbl, 22 dwg

Description

TECHNICAL FIELD

The present invention is directed to providing nucleic acid and protein constructs that are involved in or affect metabolic pathways mediating or influencing cellular metabolism, such as trafficking across the endoplasmic reticulum (ER) membrane and/or secretion across the cytoplasmic membrane, as well as methods for influencing on cellular metabolism. The present invention is also directed to the production and use of recombinant mammalian cells in which, for example, the movement/secretion of a wide variety of heterologous proteins (transgene expression products) is modified. These methods, nucleic acid constructs, are typically designed to improve transgene expression.

BACKGROUND OF THE ART

The production of therapeutic proteins by biotechnological methods, as well as gene and cell therapy, depend on the successful expression of transgenes introduced into the eukaryotic cell. Successful expression of a transgene typically requires integration of the transgene into the host chromosome, with expression limited by, among other things, the number of transgene copies inserted and epigenetic effects, which can cause low levels or instability of transcription and/or high clonal variability. Insufficient or reduced transport of the transgene expression product out of the cell also often limits the production of therapeutic proteins, as well as gene and cell therapy.

Publications and other materials, including patents, patent applications and accession numbers used in this application to illustrate the present invention and, in particular, to provide additional details regarding the implementation of the present invention, are incorporated herein by reference in their entirety.

Epigenetic regulators are increasingly used to enhance and stabilize transgene expression in mammalian cells to protect transgenes from negative positional effects (Bell and Felsenfeld, 1999). These epigenetic regulators include boundary or insulator elements, locus regulatory regions (LCRs), stabilizing and antirepressor (STAR) elements, universal chromatin opening elements (UCOEs), and the aforementioned matrix attachment sites (MARs). All of these epigenetic regulators have been used to produce recombinant protein in mammalian cell lines (Zahn-Zabal et al., 2001; Kim et al., 2004) and in gene therapy techniques (Agarwal et al., 1998; Allen et al., 1996; Castilla et al., 1998).

Processing and transport of the transgene expression product from the cell is often associated with various difficulties. A cell that is equipped with only the means to process and transport its native proteins can quickly become overwhelmed by the transport of certain types of transgene expression products, especially when they are produced at abnormally high levels, which are often desirable, resulting in product aggregation within the cell and/or, for example, interferes with the correct folding of a functional protein product.

Various approaches have been used to overcome the difficulties associated with transport and processing. For example, CHO cells have been engineered to have improved secretory properties by expressing SM family proteins Munc18c or Sly1, which act as regulators of membrane vesicle transport and hence exocytosis of secreted proteins (US Patent Application Publication 20090247609). X-box binding protein 1 (Xbp1), a transcription factor that regulates secretory cell differentiation and ER maintenance and expansion, or various protein disulfide isomerases (PDIs) have been used to reduce ER stress and increase protein secretion (Mohan et al. 2007). Other attempts to increase protein secretion have included expression of the chaperones ERp57, calnexin, calreticulin, and BiP1 in CHO cells (Chung et al., 2004). Expression of cold shock-induced protein, particularly cold-induced RNA-binding protein (CIRP), has been shown to increase the yield of recombinant interferon gamma. Attempts have also been made to overexpress proteins of the secretory complexes. However, for example, Lakkaraju et al. (2008) reported that expression of exogenous SRP14 in wild-type human cells (e.g., cells that were not engineered to express low levels of SRP14) did not improve the secretion efficiency of the secreted alkaline phosphatase protein.

Thus, there is a need for efficient, reliable transgene expression, for example, in recombinant protein production and for gene therapy. There is also a need to successfully transport the transgene expression product outside the cell.

Embodiments of the present invention are directed to meet this and other needs existing in the art.

BRIEF DESCRIPTION OF THE INVENTION

The present invention, in one embodiment, is directed to a recombinant nucleic acid molecule comprising:

(a) 5' and 3' transposon-specific inverted terminal repeat (ITR),

(b) at least one nucleic acid sequence encoding a transgene expression/processing protein (TEP), or a functional TEP RNA located between the 5' and 3' ITR, which is under the control of a promoter, and

(c) optionally at least one transgene, also located between the 5' and 3' ITR, which is under the control of a transgene promoter, wherein said nucleic acid molecule is optionally part of a vector.

Said recombinant nucleic acid molecule may contain at least one epigenetic regulatory element, in particular at least one MAR (matrix attachment site) element.

The MAR element may be located between the 5' and 3' ITR. A transgene, such as an antibiotic resistance gene or a gene encoding an immunoglobulin, possibly under the control of an additional promoter, may be located between the 5' and 3' ITR, for example between the 5' ITR and the MAR.

TEP protein or functional RNA TEP may be a protein or functional RNA that is directly or indirectly involved in the insertion of nucleic acid sequences into the genome, in the processing or translation of a transgene RNA product, or is involved in ER trafficking, secretion, processing, folding, ER transport. Golgi-plasma membrane, glycosylation and/or other post-translational modification of proteins, such as transgene expression products.

The TEP protein may be a protein secretion pathway protein, a recombination or DNA repair pathway protein, a protein processing protein, or a metabolic protein including chaperones such as BiP, or a combination of these proteins.

The TEP protein may be one or more of the following protein secretion pathway proteins: human SRP14 (hSRP14), hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ, and hCANX.

The TEP protein may also correspond to one or more of the following protein secretion pathway protein amino acid sequences: hSRP14 with SEQ ID NO: 13, hSec61α1 with SEQ ID NO: 15, hSec61β with SEQ ID NO: 17, hSec61γ with SEQ ID NO: : 19, hSRP54 with the sequence SEQ ID NO: 21, hSRP9 with the sequence SEQ ID NO: 23, hSRPRα with the sequence SEQ ID NO: 25, hSRPβ with the sequence SEQ ID NO: 27 and hCANX with the sequence SEQ ID NO: 29 and/or may correspond to amino acid sequences that are more than 80%, 90%, 95%, or 98% identical to the stated sequences.

The TEP protein may be one or more of the following protein processing or metabolic proteins: hUCP4, hCMPSAT, rat ST6Gal1 (rST6Gal1), hCOMSC, human T synthase, hP4HA1, hP4HB, hGILZ, hCyPB, hNRF2, hHK1, hPDI, hPIN1, hSEPW1, hCALR, hDDOST, hHSP40, hATP5A1, hSERCA2, hPDIA4, hHSC70/HSPA8, hHYOU1, hCMP-SAS, human beclin-1, hERdj3, CHO-AGE, hWip1, hRTP4, hREEP2, hDPM1 and hDRiP78.

The TEP protein may also correspond to one or more of the following amino acid sequences of protein processing proteins or metabolic proteins: hUCP4 with the sequence SEQ ID NO: 31, hCMPSAT with the sequence SEQ ID NO: 33, rST6Gal1 with the sequence SEQ ID NO: 35, hCOMSC with the sequence SEQ ID NO: 35 ID NO: 37, Human T synthases with SEQ ID NO: 39, hP4HA1 with SEQ ID NO: 41, hP4HB with SEQ ID NO: 43, hGILZ with SEQ ID NO: 45, hCyPB with SEQ ID NO : 47, hNRF2 with the sequence SEQ ID NO: 49, hHK1 with the sequence SEQ ID NO: 51, hPDI with the sequence SEQ ID NO: 53, hPIN1 with the sequence SEQ ID NO: 55, hSEPW1 with the sequence SEQ ID NO: 57, hCALR with with the sequence SEQ ID NO: 59, hDDOST with the sequence SEQ ID NO: 62, hHSP40 with the sequence SEQ ID NO: 64, hATP5A1 with the sequence SEQ ID NO: 66, hSERCA2 with the sequence SEQ ID NO: 68, hPDIA4 with the sequence SEQ ID NO: 70, hHSC70 /HSPA8 with the sequence SEQ ID NO: 72, hHYOU1 with the sequence SEQ ID NO: 74, hCMP-SAS with the sequence SEQ ID NO: 76, human beclin-1 with the sequence SEQ ID NO: 78, hERdj3 with the sequence SEQ ID NO: 80, CHO-AGE with SEQ ID NO: 82, hWip1 with SEQ ID NO: 84, hRTP4 with SEQ ID NO: 86, hREEP2 with SEQ ID NO: 88, hDPM1 with SEQ ID NO: 90 and hDRiP78 having the sequence SEQ ID NO: 92 and/or may correspond to amino acid sequences more than 80%, 90%, 95% or 98% identical to the indicated sequences.

The TEP protein may be a chaperone, in particular a BiP protein, and more specifically a modified derivative of the Drosophila BiP protein (DroBiP) having 80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 60.

The MAR element can be selected from the sequences SEQ ID NO: 1 (MAR 1-68), 2 (MAR 1_6), 3 (MAR X_S29), 4 (MAR S4), 5 (chicken lysozyme MAR), or is preferably designed, in particular, the rearranged analog and/or its sequence is at least 80%, 90%, 95%, 98%, 99% or 100% identical to any of the sequences of SEQ ID NO: 1 to 5 or any of the sequences of SEQ ID NO: from 6 to 10.

The functional TEP RNA within said cell may include/consist of a nucleic acid sequence encoding a functional RNA, preferably a microRNA or short hairpin RNA (shRNA), that interferes with the expression of at least one recombination or DNA repair pathway protein, such as but not limited to : Rad51, Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Brca1, Brca2, cyclin D1, Ercc, MDC1, Bard1, ligase 1, Mre11 and/or 53BP1.

Functional TEP RNA can also interfere with the expression of genes that have at least 80%, 90%, 95%, 98%, or 100% sequence identity with Rad51 SEQ ID NO: 93, Rad51B with SEQ ID NO: 94, Rad51C with with the sequence SEQ ID NO: 95, Rad51D with the sequence SEQ ID NO: 96, Xrcc2 with the sequence SEQ ID NO: 99, Xrcc3 with the sequence SEQ ID NO: 100, Rad52 with the sequence SEQ ID NO: 97, Rad54 with the sequence SEQ ID NO: 98, Brca1 with the sequence SEQ ID NO: 101, Brca2 with the sequence SEQ ID NO: 102, cyclin D1 with the sequence SEQ ID NO: 103, Ercc1 with the sequence SEQ ID NO: 104, MDC1 with the sequence SEQ ID NO: 105, Bard1 with sequence SEQ ID NO: 106, ligase 1 with sequence SEQ ID NO: 107, Mre11 with sequence SEQ ID NO: 108 and/or 53BP1 with sequence SEQ ID NO: 109.

The length of the recombinant nucleic acid molecule may be at least 5000, 6000, 7000, 8000, 90,000 or 10,000 bp.

The 5' and 3' ITRs may be the 5' and 3' ITRs of the Sleeping Beauty transposon or preferably the PiggyBac transposon.

Following first transfection of a mammalian cell with one of said recombinant nucleic acid molecules and a second subsequent transfection with an additional recombinant nucleic acid molecule containing the transgene, insertion and/or expression of the transgene may be increased in said cell compared to a cell not subjected to said first transfection.

The TEP coding sequence or functional TEP RNA mentioned herein may be part of a vector, including an expression vector. Said vector may contain a single MAR element, two or more MAR elements, and said element(s) may be located between the 5' and 3' ITR.

For example, the specified vector may contain two MAR elements. The first MAR element may be located in the 3'-5' direction of TEP or functional TEP RNA and the second MAR element may be located in the 5'-3' direction of TEP or functional TEP RNA, wherein the first MAR element may include a MAR element 1_6 and/or an element which is at least 80%, 90%, 95%, 98%, 99% or 100% sequence identical to SEQ ID NO: 2, in particular, rearranged MARs based on MARs 1-6, and more specifically, elements that are at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identical to SEQ ID NO: 8 (MAR 1_6R2), and the second MAR element may include a MAR element 1-68 and/or an element that is at least 80%, 90%, 95%, 98%, 99% or 100% sequence identical to SEQ ID NO: 1.

The vector may also contain a single MAR element. Said MAR unit element may be located in the 5'-3' direction of TEP or functional TEP RNA, wherein said MAR unit element may be MAR 1-68, or MAR X-29 element, and/or an element whose sequence is at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1 or 3, in particular, a rearranged MAR based on MAR 1-68 or MAR X-29, in particular, an element that has at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6, 7, or 10 (MAR 1_68R, 1_68R2, or X_29R3) or 9, and may preferably be a MAR X-29 element and/or an element that is at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identical to SEQ ID NO: 3.

TEP or functional TEP RNA may be under the control of the EF1-alpha promoter and possibly followed by a poly(A) bovine growth hormone (BGH) signal.

The vector may contain promoter(s) and/or enhancer(s) or fusion products thereof, such as GAPDH, SV40p, CMV, EF1alpha cell CHO, Actb CHO and/or Hspa5 promoter(s) and/or enhancer(s) CHO, or engineered fusion products thereof, such as CGAPDH.

The promoters that are part of the vector may be GAPDH with the sequence SEQ ID NO: 111, SV40p with the sequence SEQ ID NO: 114, CMVp with the sequence SEQ ID NO: 113, EF1-alpha CHO with the sequence SEQ ID NO: 112, Actb CHO with the sequence SEQ ID NO: 115, Hspa5 CHO with the sequence SEQ ID NO: 116, and/or fusion products thereof, such as CGAPDH with the sequence SEQ ID NO: 11, or their nucleic acid sequences may be identical to these sequences by more than 80%, 90%, 95% or 98%.

The present invention is also directed to a method for expressing TEP or functional TEP RNA, comprising:

providing a recombinant mammalian cell containing a transgene, and the vector is an expression vector that expresses TEP or functional TEP RNA, wherein the TEP or functional TEP RNA expressed from said vector is capable of increasing expression of the transgene in said mammalian cell by at least 10% , at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70%.

Said vector may contain a single MAR X-29 element and/or a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 3, and wherein after more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks of culture, TEP or functional TEP RNA expressed from said vector can increase expression of the gene of interest by at least 10%, according to at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70%.

The present invention is also directed to a recombinant mammalian cell containing no more than 20, 15, 10 or 5 recombinant nucleic acid molecules, preferably integrated into the genome of said cell in single copy form.

As noted above, the TEP protein may be one or more of the following protein secretion pathway proteins: hSRP14, hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ and hCANX.

The TEP protein may also correspond to one or more of the following protein secretion pathway protein amino acid sequences: hSRP14 with SEQ ID NO: 13, hSec61α1 with SEQ ID NO: 15, hSec61β with SEQ ID NO: 17, hSec61γ with SEQ ID NO: : 19, hSRP54 with the sequence SEQ ID NO: 21, hSRP9 with the sequence SEQ ID NO: 23, hSRPRα with the sequence SEQ ID NO: 25, hSRPβ with the sequence SEQ ID NO: 27 and hCANX with the sequence SEQ ID NO: 29, and/ or may correspond to amino acid sequences that are more than 80%, 90%, 95%, or 98% identical to the stated sequences.

The TEP protein may be one or more of the following protein processing or metabolic proteins: hUCP4, hCMPSAT, rST6Gal1, hCOMSC, human T synthase, hP4HA1, hP4HB, hGILZ, hCyPB, hNRF2, hHK1, hPDI, hPIN1, hSEPW1, hCALR, hDDOST, hHSP40, hATP5A1, hSERCA2, hPDIA4, hHSC70/HSPA8, hHYOU1, hCMP-SAS, human beclin-1, hERdj3, CHO-AGE, hWip1, hRTP4, hREEP2, hDPM1 and hDRiP78.

The TEP protein may also correspond to one or more of the following amino acid sequences of protein processing proteins or metabolic proteins: hUCP4 with the sequence SEQ ID NO: 31, hCMPSAT with the sequence SEQ ID NO: 33, rST6Gal1 with the sequence SEQ ID NO: 35, hCOMSC with the sequence SEQ ID NO: 35 ID NO: 37, Human T synthases with SEQ ID NO: 39, hP4HA1 with SEQ ID NO: 41, hP4HB with SEQ ID NO: 43, hGILZ with SEQ ID NO: 45, hCyPB with SEQ ID NO : 47, hNRF2 with the sequence SEQ ID NO: 49, hHK1 with the sequence SEQ ID NO: 51, hPDI with the sequence SEQ ID NO: 53, hPIN1 with the sequence SEQ ID NO: 55, hSEPWI with the sequence SEQ ID NO: 57, hCALR with with the sequence SEQ ID NO: 59, hDDOST with the sequence SEQ ID NO: 62, hHSP40 with the sequence SEQ ID NO: 64, IATP5A1 with the sequence SEQ ID NO: 66, hSERCA2 with the sequence SEQ ID NO: 68, hPDIA4 with the sequence SEQ ID NO: 70, hHSC70/HSPA8 with the sequence SEQ ID NO: 72, hHYOU1 with the sequence SEQ ID NO: 74, hCMP-SAS with the sequence SEQ ID NO: 76, human beclin-1 with the sequence SEQ ID NO: 78, hERdj3 with the sequence SEQ ID NO: 80, CHO-AGE with SEQ ID NO: 82, hWip1 with SEQ ID NO: 84, hRTP4 with SEQ ID NO: 86, hREEP2 with SEQ ID NO: 88, hDPM1 with SEQ ID NO: 90 and hDRiP78 having the sequence SEQ ID NO: 92, and/or may correspond to amino acid sequences more than 80%, 90%, 95% or 98% identical to the indicated sequences.

The TEP protein may be a chaperone, particularly a BiP protein, and more specifically an engineered derivative of the Drosophila BiP protein (DroBiP) that is 80%, 90%, 95%, or 100% sequence identical to SEQ ID NO: 60.

The functional TEP RNA within a recombinant mammalian cell may include/consist of a nucleic acid sequence(s) encoding a functional RNA, preferably a microRNA or shRNA, that interferes with the expression of at least one recombination protein, preferably a homologous recombination gene such as but not limited to the following: Rad51, Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Brca1, Brca2, cyclin D1, Ercc1, MDC1, Bard1, ligase 1, Mre11 and/or 53BP1. The nucleic acid sequence may be at least 80%, 90%, 95%, 98% or 100% identical to Rad51 SEQ ID NO: 93, Rad51B to SEQ ID NO: 94, Rad51C to SEQ ID NO: 95 , Rad51D with the sequence SEQ ID NO: 96, Xrcc2 with the sequence SEQ ID NO: 99, Xrcc3 with the sequence SEQ ID NO: 100, Rad52 with the sequence SEQ ID NO: 97, Rad54 with the sequence SEQ ID NO: 98, Brca1 with the sequence SEQ ID NO: 101, Brca2 with SEQ ID NO: 102, Cyclin D1 with SEQ ID NO: 103, Ercc1 with SEQ ID NO: 104, MDC1 with SEQ ID NO: 105, Bard1 with SEQ ID NO: 106 , ligase 1 with the sequence SEQ ID NO: 107, Mre11 with the sequence SEQ ID NO: 108 and/or 53BP1 with the sequence SEQ ID NO: 109.

The recombinant mammalian cell may be a primary stem cell, a hamster cell, such as a Chinese hamster ovary (CHO) cell, or a human cell, such as a HEK293 cell.

The present invention is also directed to a recombinant mammalian cell containing:

a.) at least one functional TEP RNA and/or at least one recombinant nucleic acid sequence encoding a TEP protein or encoding a functional TEP RNA,

And

b.) a recombinant nucleic acid molecule containing:

(i) at least one transgene of interest, and

(ii) possibly a MAR element.

As noted above, the TEP protein may be one or more of the following protein secretion pathway proteins: hSRP14, hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ and hCANX.

The TEP protein may also correspond to one or more of the following protein secretion pathway protein amino acid sequences: hSRP14 with SEQ ID NO: 13, hSec61α1 with SEQ ID NO: 15, hSec61β with SEQ ID NO: 17, hSec61γ with SEQ ID NO: : 19, hSRP54 with the sequence SEQ ID NO: 21, hSRP9 with the sequence SEQ ID NO: 23, hSRPRα with the sequence SEQ ID NO: 25, hSRPβ with the sequence SEQ ID NO: 27, and hCANX with the sequence SEQ ID NO: 29 and/ or may correspond to amino acid sequences that are more than 80%, 90%, 95%, or 98% identical to the stated sequences.

The TEP protein may be one or more of the following protein processing or metabolic proteins: hUCP4, hCMPSAT, rST6Gal1, hCOMSC, human T synthase, hP4HA1, hP4HB, hGILZ, hCyPB, hNRF2, hHK1, hPDI, hPIN1, hSEPW1, hCALR, hDDOST, hHSP40, hATP5A1, hSERCA2, hPDIA4, hHSC70/HSPA8, hHYOU1, hCMP-SAS, human Beclin-1, hERdj3, CHO-AGE, hWip1, hRTP4, hREEP2, hDPM1 and hDRiP78.

The TEP protein may also correspond to one or more of the following amino acid sequences of protein processing proteins or metabolic proteins: hUCP4 with the sequence SEQ ID NO: 31, hCMPSAT with the sequence SEQ ID NO: 33, rST6Gal1 with the sequence SEQ ID NO: 35, hCOMSC with the sequence SEQ ID NO: 35 ID NO: 37, Human T synthases with SEQ ID NO: 39, hP4HA1 with SEQ ID NO: 41, hP4HB with SEQ ID NO: 43, hGILZ with SEQ ID NO: 45, hCyPB with SEQ ID NO : 47, hNRF2 with the sequence SEQ ID NO: 49, hHK1 with the sequence SEQ ID NO: 51, hPDI with the sequence SEQ ID NO: 53, hPIN1 with the sequence SEQ ID NO: 55, hSEPW1 with the sequence SEQ ID NO: 57, hCALR with with the sequence SEQ ID NO: 59, hDDOST with the sequence SEQ ID NO: 62, hHSP40 with the sequence SEQ ID NO: 64, IATP5A1 with the sequence SEQ ID NO: 66, hSERCA2 with the sequence SEQ ID NO: 68, hPDIA4 with the sequence SEQ ID NO: 70, hHSC70/HSPA8 with the sequence SEQ ID NO: 72, hHYOU1 with the sequence SEQ ID NO: 74, hCMP-SAS with the sequence SEQ ID NO: 76, human beclin-1 with the sequence SEQ ID NO: 78, hERdj3 with the sequence SEQ ID NO: 80, CHO-AGE with SEQ ID NO: 82, hWip1 with SEQ ID NO: 84, hRTP4 with SEQ ID NO: 86, hREEP2 with SEQ ID NO: 88, hDPM1 with SEQ ID NO: 90 and hDRiP78 having the sequence SEQ ID NO: 92 and/or may correspond to amino acid sequences more than 80%, 90%, 95% or 98% identical to the indicated sequences.

The TEP protein may be a chaperone, in particular a BiP protein, and more specifically, a synthetic derivative of the Drosophila BiP protein (DroBiP), the sequence of which is 80%, 90%, 95% or 100% identical to the sequence of SEQ ID NO: 60.

The functional RNA may be a transiently transfected siRNA or shRNA that is transcribed from said at least one isolated nucleic acid sequence, wherein said siRNA or said processed shRNA is an antisense RNA of 20, 21, 22, 23, 24 or 25 base pairs in length , which is fully complementary to 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the mRNA of at least one target gene that is part of the recombinant pathway NGJK (non-homologous end joining), GR (homologous recombination), OMJK (microhomology-mediated end joining) or is a DNA repair protein such as MDC1 (mediator of DNA damage checkpoint 1).

Said at least one target gene, which may be part of:

- DNA repair and NGSC, represents

53BP1 (p53 tumor suppressor binding protein 1),

- GR, represents

Rad51 (DNA repair protein RAD51), Rad51B (homologue 2 of DNA repair protein RAD51), Rad51C (homologue 3 of DNA repair protein RAD51),

Rad51D (homologue 4 of DNA repair protein RAD51), Rad52 (DNA repair protein RAD52), Rad54 (DNA repair and recombination protein RAD54),

Xrcc2 (repair after X-ray irradiation, correcting (complementing) impaired repair in Chinese hamster cells 2),

Xrcc3 (repair after X-ray irradiation, complementing impaired repair in Chinese hamster cells 3),

Brca1 (breast cancer 1, early onset),

Brca2 (breast cancer 2, early onset),

Bard1 (BRCA1-associated RING domain 1),

- OMSK represents

Ercc1 (excision repair cross-complementing defective repair in rodents, complementation group 1),

Mre11 (meiotic recombination 11)

ligase 1 (DNA ligase 1),

and/or

is a DNA repair protein MDC1.

The target genes may be nucleic acids that have at least 80%, 90%, 95%, 98%, or 100% sequence identity with Rad51 of SEQ ID NO: 93, Rad51B with SEQ ID NO: 94, Rad51C with sequence SEQ ID NO: 95, Rad51D with SEQ ID NO: 96, Xrcc2 with SEQ ID NO: 99, Xrcc3 with SEQ ID NO: 100, Rad52 with SEQ ID NO: 97, Rad54 with SEQ ID NO: 98 , Brca1 with the sequence SEQ ID NO: 101, Brca2 with the sequence SEQ ID NO: 102, cyclin D1 with the sequence SEQ ID NO: 103, Ercd with the sequence SEQ ID NO: 104, MDC1 with the sequence SEQ ID NO: 105, Bard1 with the sequence SEQ ID NO: 106, ligase 1 with the sequence SEQ ID NO: 107, Mre11 with the sequence SEQ ID NO: 108 and/or 53BP1 with the sequence SEQ ID NO: 109.

Said at least one transgene can express a therapeutic protein, such as an immunoglobulin, a hormone, such as erythropoietin, or a growth factor, and optionally the insertion and/or expression of the transgene is increased in the recombinant mammalian cell compared to a cell not containing the recombinant nucleic acid molecule(s).

A recombinant mammalian cell may contain at least two functional TEP RNAs, wherein one or both TEP RNAs are transiently transfected siRNAs, or expressed from the specified isolated nucleic acid sequence(s) encoding the functional TEP RNA.

The recombinant mammalian cell may contain a MAR element.

The present invention is also directed to a method for transfecting mammalian cells, in particular hamster cells, comprising:

transfection, possibly upon first transfection, of said mammalian cells

(i) at least one of said recombinant nucleic acid molecules according to any one of claims. 1-13 and/or

(ii) at least one isolated functional TEP RNA and at least one transgene, which is optionally part of a recombinant nucleic acid molecule, which is optionally transfected in a second, subsequent transfection, possibly together with an isolated nucleic acid or mRNA expressing a transposase that recognizes the indicated 5'- and 3'-ITRs.

A recombinant mammalian cell can be transfected with more than one nucleic acid molecule, including at least two, at least three, or at least four of the recombinant nucleic acid molecules encoding one, two, or three of the following proteins: hSRP14, hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ and hCANX.

It can also be transfected with hSRP14 with SEQ ID NO: 13, hSec61α1 with SEQ ID NO: 15, hSec61β with SEQ ID NO: 17, hSec61γ with SEQ ID NO: 19, hSRP54 with SEQ ID NO: 21, hSRP9 with the sequence SEQ ID NO: 23, hSRPRα with the sequence SEQ ID NO: 25, hSRPβ with the sequence SEQ ID NO: 27, and hCANX with the sequence SEQ ID NO: 29, and/or amino acid sequences more than 80% identical, 90 %, 95% or 98% of the specified sequences.

Any of the recombinant nucleic acid molecules can be part of a vector, and co-transfection with said vectors can be carried out.

Cotransfection with vectors encoding multiple TEP proteins, preferably cotransfection with recombinant nucleic acid molecules encoding the SRP14, SRP9 and SRP54 proteins, can increase the insertion and/or expression of the transgene in the cell compared to a cell not so cotransfected. Cotransfection with vectors containing nucleic acid sequences that are at least 80%, 90%, 95%, 98% or 100% identical to the sequences of SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 20 is also included in scope of the present invention.

A plurality of said mammalian cells that stably express said TEP protein or functional TEP RNA may be obtained to produce recombinant mammalian cells, and wherein said number of recombinant mammalian cells may be independent of the presence of said MAR element. These mammalian cells can be transfected two and possibly three times.

Preferably, at least 30%, 40% or 45% of said mammalian cells may become recombinant mammalian cells and express said transgene.

A mammalian cell can be transfected with said at least one isolated functional TEP RNA and a vector containing said at least one transgene and optionally 3'-ITR and 5'-ITR flanking said at least one transgene, optionally together with an isolated nucleic acid or An mRNA expressing a transposase that recognizes the indicated 5' and 3' ITRs.

The transgene may be a therapeutic protein such as an immunoglobulin, hormone, cytokine or growth factor.

The recombinant nucleic acid molecule may optionally contain a selection marker, and wherein said at least one transgene is expressed

(a) without selection for the specified marker, or

(b) with selection for the specified marker, for example, by adding a selection agent to the culture medium, and

(c) in the absence of a transposase, or

(d) in the presence of transposase.

Functional TEP RNA may be encoded by recombinant nucleic acid sequences encoding shRNA or microRNA, or may include/consist of a miRNA that interferes with the expression of at least one homologous recombination gene, such as, but not limited to: Rad51, Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Brca1, Brca2, cyclin D1, Ercc1, MDC1, Bard1, ligase 1, Mre11 and/or 53BP1.

Functional TEP RNA can also interfere with the expression of genes that have at least 80%, 90%, 95%, 98%, or 100% sequence identity with Rad51 SEQ ID NO: 93, Rad51B with SEQ ID NO: 94, Rad51C with with the sequence SEQ ID NO: 95, Rad51D with the sequence SEQ ID NO: 96, Xrcc2 with the sequence SEQ ID NO: 99, Xrcc3 with the sequence SEQ ID NO: 100, Rad52 with the sequence SEQ ID NO: 97, Rad54 with the sequence SEQ ID NO: 98, Brca1 with the sequence SEQ ID NO: 101, Brca2 with the sequence SEQ ID NO: 102, cyclin D1 with the sequence SEQ ID NO: 103, Ercc1 with the sequence SEQ ID NO: 104, MDC1 with the sequence SEQ ID NO: 105, Bard1 with sequence SEQ ID NO: 106, ligase 1 with sequence SEQ ID NO: 107, Mre11 with sequence SEQ ID NO: 108 and/or 53BP1 with sequence SEQ ID NO: 109.

Incorporation and/or expression of the transgene can be increased in such a cell compared to a cell not transfected with said isolated nucleic acid molecules and/or at least one of said isolated functional TEP RNAs.

The present invention is also directed to a kit containing in one container at least one vector containing any of the specified recombinant nucleic acid molecules according to paragraphs. 1-13, and in a second optional container containing a vector encoding a suitable transposase, and in an additional container containing instructions for use of said vector or vectors.

The above set is provided wherein more than one vector is provided in one or more containers and wherein the TEP proteins are at least two of the following proteins: chaperone, SRP14, SRP9, SRP54, SR or translocon.

The above set is proposed, in which the functional TEP RNA(s) within the specified vector(s) includes/consists of nucleic acid sequences encoding microRNAs, miRNAs or shRNAs that interfere with the expression of at least one homologous recombination gene such as, but not limited to: Rad51, Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Brca1, Brca2, cyclin D1, Ercc1, MDC1, Bard1, ligase 1, Mre11 and/or 53BP1, and preferably in in an additional container, said kit contains one or more siRNAs that interfere with the expression of at least one other homologous recombination gene, such as, but not limited to: Rad51, Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Brca1, Brca2, cyclin D1, Ercc, MDC1, Bard1, ligase 1, Mre11 and/or 53BP1.

The homologous recombination gene may correspond to nucleic acids that are at least 80%, 90%, 95%, 98%, or 100% sequence identical to Rad51 with the sequence SEQ ID NO: 93, Rad51B with the sequence SEQ ID NO: 94, Rad51C with the sequence SEQ ID NO: 95, Rad51D with SEQ ID NO: 96, Xrcc2 with SEQ ID NO: 99, Xrcc3 with SEQ ID NO: 100, Rad52 with SEQ ID NO: 97, Rad54 with SEQ ID NO: 98 , Brca1 with the sequence SEQ ID NO: 101, Brca2 with the sequence SEQ ID NO: 102, cyclin D1 with the sequence SEQ ID NO: 103, Ercc1 with the sequence SEQ ID NO: 104, MDC1 with the sequence SEQ ID NO: 105, Bard1 with the sequence SEQ ID NO: 106, ligase 1 with the sequence SEQ ID NO: 107, Mre11 with the sequence SEQ ID NO: 108 and/or 53BP1 with the sequence SEQ ID NO: 109.

The present invention is also directed to the use of recombinant nucleic acids described in this application and/or recombinant mammalian cells described in this application, preferably to enhance the integration and/or expression of a transgene.

The present invention is also directed to an expression vector comprising:

(a) a transgene that is flanked in the 3'-5' direction by a promoter and in the 5'-3' direction by a polyadenylation signal,

(b) a single MAR element in the 5'-3' direction of the polyadenylation signal, or

(c) a first MAR element in the 3'-5' direction from the transgene of interest and a second MAR element in the 5'-3' direction from the insertion site of the specified transgene.

The single or first and second MAR elements can be selected from MAR elements 1_68, 1_6, 1_6R2, 1_68R, 1_68R2, X_29R3 or X_29 or elements whose sequences are at least 80%, 90%, 95%, 98%, 99% or 100 % identical to SEQ ID NO: 1, 2, 3, 6, 7, 8, 9 or 10.

The single or first and second MAR element(s) may be selected from rearranged MAR elements 1_6R2, 1_68R, 1_68R2 or X_29R3 or elements whose sequences are at least 80%, 90%, 95%, 98%, 99% or 100% identical sequences of SEQ ID NO: 6, 7, 8, or 10, wherein optionally said MAR element(s) increases expression of the transgene of interest by at least 10%, at least 20%, at least 30%, at least 40 %, at least 50%, at least 60%, or at least 70% compared to their non-rebuilt counterparts.

The promoter may be the EF1-alpha promoter and the polyadenylation signal is the BGH poly(A) signal.

Said vector may contain promoter(s) and/or enhancer(s) or fusion products thereof, such as GAPDH, CGAPD, CSV40p, CMVp, Ef1alpha CHO, Actb CHO or HspaS CHO.

The promoters may be GAPDH having SEQ ID NO: 111, CGAPDH having SEQ ID NO: 11, SV40p having SEQ ID NO: 114, CMVp having SEQ ID NO: 113, EF1-alpha CHO having SEQ ID NO: :112, Actb CHO with the sequence SEQ ID NO: 115 and/or HspaS CHO with the sequence SEQ ID NO: 116 and nucleic acid sequences that are more than 80%, 90%, 95% or 98% identical to these sequences.

The promoter may be a GAPDH promoter and include a CMV enhancer.

The first and/or second MAR, enhancer, promoter, transgene of interest, and polyadenylation signal may be located between the 5' and 3' ITR.

In some embodiment, said expression vector may comprise:

(a) a MAR unit element in the 5'-3' direction from the polyadenylation signal, wherein said MAR unit element is preferably a MAR 1-68 or MARX-29 element and/or an element which is at least 80% sequenced, 90 %, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1 or 3, in particular, a rearranged MAR based on MAR 1-68 or MAR X-29, in particular, an element whose sequence is at least is 80%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 6, 7 or 10 (MAR elements 1_68R, 1_68R2 or X_29R3) or SEQ ID NO: 9, and is preferably an element MAR X-29, and/or its sequence is at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 3, or

(b) a first MAR element in the 3'-5' direction from the transgene of interest and a second MAR element in the 5'-3' direction from said transgene of interest, wherein said first MAR element preferably includes element 1_6, and/or sequence thereof at least at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 2, in particular, identical to rearranged MAR elements based on MARs 1-6, in particular, elements whose sequence is at least at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8 (MAR 1_6R2), and a second MAR element, which preferably includes MAR element 1-68, and/or sequence thereof is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1.

The expression vector may contain a single MAR element, and the single MAR element may be located in the 5'-3' direction from the polyadenylation site and is MAR 1-68 or MAR X-29, and/or at least 80% of the sequence thereof , 90%, 95%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1 or 3, particularly identical to rearranged MAR elements based on MAR 1-68 or MAR X-29, particularly to elements, sequence which is at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequences of SEQ ID NO: 6, 7 or 10 (MAR elements 1_68R, 1_68R2 or X_29R3) or 9, and may preferably be a MAR X-29 derived element and/or its sequence is at least 80%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 3.

The first MAR element may be in the 3'-5' direction from the transgene of interest and the second MAR element may be in the 5'-3' direction from the said transgene of interest, wherein the first MAR element may include the MAR 1_6 element, and/or its sequence is at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 2, and said second MAR element may include MAR element 1_68, and/or its sequence may be at least 80%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 1.

The present invention is also directed to a method for expressing a transgene, comprising:

providing a recombinant mammalian cell containing one of the above vectors containing said transgene and expressing said transgene, wherein said MAR element(s) can increase expression of said transgene preferably by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70%. Said vector may contain a single MAR X_29 element and/or a nucleic acid the sequence of which is at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 3, and more than than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks of culture, said MAR element can increase expression of the transgene of interest by at least 10%, at least 20%, at least 30% , at least 40%, at least 50%, at least 60% or at least 70%.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Construction of a vector carrying a transposon.

To test whether the addition of a MAR element to the PB transposon (PiggyBac) could influence transposition efficiency and transgene expression, and to evaluate whether the location of the MAR in the construct had any influence on these effects, a series of donor transposon constructs containing a green fluorescent protein (GFP) gene and a puromycin resistance gene (Puro) into which MAR 1_68 or a control neutral DNA spacer sequence was inserted at various positions in the plasmid. The original transposon-carrying plasmid, Puro-GFP, without insertion, was used as a transposition control to distinguish the contribution of increased transposon size from the effect of the addition of a MAR or spacer sequence.

Fig. 2. Vectors carrying transposons: efficiency of transposition.

The transposition efficiency of the various transposon-carrying constructs was measured by assessing (A) the percentage of cells expressing GFP after transfection and three weeks of culture without antibiotic selection and (B) by counting puromycin-resistant colonies.

Fig. 3. Vectors carrying transposons: level of expression.

Analysis of the level of expression provided by various transposon-carrying vectors transfected with (+PB) or without (-PB) a transposase expression plasmid by measuring GFP fluorescence levels in CHO cells after 3 weeks of culture without selection (A) or with selection (B ) by puromycin resistance after transfection, taking into account the fluorescence of only GFP-positive cells.

Fig. 4. Influence of MAR and transposase on transgene integration into the genome.

The number of inserted copies of the GFP transgene was determined using quantitative PCR, and values were normalized to the cellular B2M gene using genomic DNA isolated from unselected CHO cells (A) or puromycin-resistant cells (B) obtained as described in captions for figures 1-3. Values represent means ± SEM (n=3). *P<0.05.

Fig. 5. Expression of the transgene relative to the transgene.

Assessment of the internal expression potential of vectors, regardless of their ability to integrate into the genome, without selection (A) and with selection (B) with puromycin.

Fig. 6. The influence of the expression of secretory proteins from vectors based on transposons and plasmid vectors on the expression of a recombinant protein (transgene). Transposon-based vectors or conventional plasmid vectors have been constructed to express the secretory proteins SRP9, SRP14, SRP54, the alpha and beta subunits (SR) of the SRP receptor, or the translocon. Transposon-based vectors were cotransfected with the PiggyBac transposase expression vector (right panel), whereas non-transposon-based plasmid vectors were transfected alone (left panel) into a cell clone expressing the infliximab antibody as described in this text. After three weeks of culture with selection (left panel) or without selection (right panel), levels of secreted infliximab antibody in the culture supernatants were analyzed. It can be seen that the specific productivity, which is the relative expression in a cell containing the transgene expression/processing protein (TEP) coding sequence or functional TEP RNA, increased with the use of the transposon-carrying vector from 0.25-1.5 to 1-2.5, respectively, compared to the original cell without TEP.

Fig. 7. Expression of recombinant protein in suspensions of CHO-M cells exposed to electromagnetic radiation.

(A) CHO-M cells that were electroporated once or twice with a MAR X-29-carrying vector expressing GFP and a transposon in the presence (+PB) or absence (-PB) of piggyBac transposase. The percentage of cells stably expressing GFP after 3 weeks of culture in the absence of selection is shown.

(B) Average GFP fluorescence value in GFP-positive cells.

(C) cDNAs encoding the antibody light and heavy chains of bevacizumab (Beva), adalimumab (Adal), and rituximab (Ritu) were inserted in place of GFP into transposon-carrying plasmids containing MAR X29. CHO-M cells were electroporated three times at 12-day intervals with light and heavy chain transposon-carrying constructs along with an expression vector carrying the piggyBac transposase. Shown are the levels of immunoglobulin secreted into culture supernatants by a population of polyclonal cells grown without selection (open bars). Alternatively, unselected polyclonal cell populations were sorted by panning cells exposed to immunoglobulins using magnetic beads. Levels of immunoglobulins secreted by unsorted populations are shown (filled bars).

(D) Transfected cell populations were screened for immunoglobulin-expressing colonies using a colony picker, and two clones expressing each of the three immunoglobulins were grown in fed-batch cultures in centrifuge tube bioreactors. Levels of secreted immunoglobulins are shown and were determined as for panel (C).

Fig.8. Heterologous expression of SRP14 improves trastuzumab secretion and restores infliximab secretion.

HP and LP CHO-K1 clones expressing the immunoglobulins trastuzumab (A) or infliximab (B) at the highest levels obtained were stably retransfected with the SRP14 expression vector, and monoclonal populations were isolated. Cell growth and productivity under batch culture conditions were assessed for the resulting subclones, designated A through E. Cell concentration (cells/mL) and IgG titer (μg/mL) were plotted for each sampling day over the 7 days of culture. (C) Distribution of specific productivity of trastuzumab (HP) and infliximab (LP) subclones after transfection with the SRP14 expression vector (lanes S) compared with that of the original HP and LP (−) clones. (D) Relative SRP14 mRNA levels for 5 individual SRP14-LP subclones A-E and the original control clone LP were determined and plotted against specific IgG productivity for the 4 resulting cultures. Mean values and standard deviations of mRNA levels and specific productivity were expressed as folds higher than the corresponding values for the control clone LP.

Fig. 9. Heterologous expression of SRP14 mediates high yield of difficult to express immunoglobulin during production.

Transfected with the vector carrying SRP14, the HP B subclone expressing trastuzumab (A) and the LP E subclone expressing infliximab (B) analyzed in FIG. 8 were cultured in 125 mL vented shake flasks in fed-batch cultures with a working volume of 25 mL, and the viable cell concentration and IgG titer were determined over an 11-day period.

Fig. 10. Expression of SRP14 disrupts light chain aggregation in CHO cell lineages.

(A) Collected supernatants and pellets of Triton X100 (Tx100) permeabilized cells by centrifugation were analyzed by SDS-PAGE as depicted in panels indicating Tx-100-soluble and Tx-100-insoluble aggregates, respectively, for subclone E of SRP14 -LP, derived from LP, and subclone B of SRP14-HP, derived from HP (lanes S), or for CHO subclones expressing the control GFP protein (lanes G). Arrows indicate underprocessed free light chain (LC) and aggregated (aggregated) LC. (B) A displacement assay was performed on the various LC, HC, and IgG aggregation intermediates obtained in the SRP14-LP clone E and the LP control clone E, the results of which are shown in this figure.

Fig. 11. Effect of co-expression of SRP, SR and translocon subunits on immunoglobulin secretion.

(A) LP E clone expressing infliximab was retransfected with different combinations of transposon-based expression vectors carrying SRP, SR, and translocon. The specific productivity of the resulting cell populations was then assessed during batch culture and reported as % of picograms per cell per day (pcd) values for LP control cells. The range diagrams show the average, upper and lower quartiles of normalized specific productivity determined on day 3 for independent cultivation series. (B) A subclone of an E cell expressing SRP14 and producing infliximab was retransfected with different combinations of transposon-based expression vectors carrying the SR and the translocon. The specific productivity of cell populations is presented similarly to panel A.

Fig. 12. Model for restoration of infliximab secretion by SRP14-expressing clones

Model of folding and secretion of IgG by low-producing clones before (A) and after (B) overexpression of SRP/translocon subunits. The results obtained indicate that newly synthesized LC, produced by clones with low production capacity, is insufficiently processed and has incorrect folding. Insufficient processing of the infliximab LC signal peptide may lead to saturation of the ER cotranslational translocation machinery (panel A, number 1). Its aggregation in the ER within IgG-incapable aggregated forms of LC (panel A, number 2) causes ER stress and causes the formation of an autophagosome-like structure (panel A, number 3). Overexpression of SRP14 and other SRP/translocon protein components completely restored infliximab IgG processing and secretion (panel B). The elongation arresting activity of SRP14 probably delays the movement of LC into the ER during translation of its mRNA (panel B, number 1). This, in turn, will promote proper processing of LC and its proper interaction with folding chaperones in the ER (panel B, number 2). Maintaining the newly synthesized LC in a state that allows IgG assembly thus restores the high secretion yield of fully assembled antibodies (panel B, number 3).

Fig. 13. Effect of suppression of GR and NGSC by siRNA molecules on expression.

Fold differences in the percentage of GFP-positive cells (compared to cells transfected with the control GFP plasmid, represented in this figure as 1.0), representing the frequencies of recombination events in untreated cells (mock), cells treated with negative control siRNA (miOtr), siRNA against NGSC factors (siRNA against Ku70, Ku80 and the catalytic subunit of DNA-dependent protein kinase (miKu70+80+DNA-PKx)) and siRNA against GR (MnRad51). The GFP lanes show the positive control of GFP-expressing cells. The lanes labeled “uncleaved GR” and “uncleaved NGSC” show negative control cells, i.e. cells transfected with circular reporter plasmids GR and NGSC. Lanes labeled “GR I-Scel” and “NGSC I-Scel” represent cells transfected with Seel-cleaved reporter plasmids that restore GFP expression after DNA cleavage repair by homologous recombination or nonhomologous end joining, respectively. This figure shows the efficiency of inhibition of GR or NGSC by siRNA molecules, as evidenced by the percentage of GFP-positive cells, which was normalized to the percentage of dsRed-positive cells and expressed as the fold change relative to the percentage of control GFP-expressing cells, which was set to 1. Mean value for 3 experiments, whiskers show the standard error of the mean. Statistical significance was determined using an unpaired Student's t test; significance level p<0.05 (*) and p<0.01 (**).

Fig. 14. Influence of MAR elements on the suppression of NGSC by miRNA molecules.

The fold increase in GFP expression and incorporation in CHO cells treated with siRNA molecules directed against NGSC factors and re-transfected with GFP or MAR-GFP plasmids is shown. Average GFP fluorescence, copy number, and fluorescence per copy of GFP are shown as the fold excess of the result obtained for untreated cells (labeled “mock”) transfected with the GFP plasmid. A) Flow cytometry results, B) genomic GFP copy number analysis by qPCR, C) average fluorescence of each integrated GFP gene (calculated for each experiment as the ratio of expression to copy number). Average of 3 or more experiments; statistical significance was determined using an unpaired Student's t test. Asterisks indicate significant differences between the siRNA-treated sample and the corresponding untreated control; significance levels: p<0.05 (*), p<0.01 (**); whiskers indicate standard error of the mean.

Fig. 15. Influence of MAR elements on the suppression of GR by miRNA molecules.

The fold increase in GFP expression and incorporation in CHO cells treated with siRNA molecules directed against GR factors and re-transfected with GFP or MAR-GFP plasmids is shown. Average GFP fluorescence, copy number, and fluorescence per copy of GFP are shown as the fold excess of the result obtained for untreated cells (labeled “mock”) transfected with the GFP plasmid. A) Flow cytometry results, B) genomic GFP copy number analysis by qPCR, C) average fluorescence of each integrated GFP gene (calculated for each experiment as the ratio of expression to copy number). Average of 3 or more experiments; statistical significance was determined using an unpaired Student's t test. Asterisks indicate significant differences between the siRNA-treated sample and the corresponding untreated control; significance levels: p<0.05 (*), p<0.01 (**); whiskers indicate standard error of the mean.

Fig. 16. Influence of MAR elements on the suppression of OSCs by siRNA molecules.

The expression and integration of GFP in CHO cells treated with siRNA molecules directed against OMC factors (and some GR factors) and re-transfected with GFP (A) or MAR-GFP (B) plasmids is shown. Average GFP fluorescence, copy number, and fluorescence per copy of GFP are shown as the fold excess of the result obtained for untreated cells (labeled “mock”) transfected with the GFP plasmid. The figures show the results of flow cytometry. Shown is the average for the number of experiments listed below the graph. Cells transfected with miMDC1 expressed GFP even without MAR at a level 11.8 times higher than that of cells not transfected with miMDC. Particularly good results could also be achieved for some plasmids that did not contain MAR, namely miBard1 and miLigl.

Fig. 17. Effect of siRNA-mediated suppression of GR protein.

The figure shows that higher expression of GFP and immunoglobulin can be achieved by CHO-M cells stably expressing shRNA directed against Rad51. CHO-M cells were transfected with a PiggyBac transposon-derived expression vector expressing shRNA directed against Rad51, and the resulting polyclonal cell population, and the cell clones derived from it, were re-transfected with a plasmid expressing GFP along with the original CHO-M cells . GFP fluorescence of the original CHO-M, the population of cells expressing Rad51-shRNA, and the resulting clones was assessed 10 days after selection based on the stable expression of the GFP gene and the puromycin resistance gene. The fluorescence profiles of the two most fluorescent clones are shown next to those for the combined population of cells and parent cells (A), as well as the percentage of cells in the M1, M2 and M3 sectors 10 days after selection based on puromycin resistance (B), which is depicted in the panel A as horizontal bars labeled 1, 2, and 3. The proportion of high-expressing M3 cells was monitored for an additional 68 days of culture without selection to show that it was possible to obtain more stable, higher-level expression in clones of cells expressing shRNA compared to parental CHO-M cells (C). Alternatively, expression plasmids encoding the light and heavy chains of the infliximab antibody were transfected into representative clones, and the specific productivity of secreted immunoglobulin was assessed after selection for an additional three weeks of antibiotic-free culture.

Fig. 18. The effect of various recombinant human MAR elements located in the 3'-5' direction on the percentile of cells with high and very high production (% M3/M2), assessed using fluorescence-excited cell sorting (FACS) analysis. GFP fluorescence for two MAR constructs.

(A) The indicated MAR elements were rearranged derivatives of MAR X-29 (X_29R2 (SEQ ID NO: 9), X_29R3 (SEQ ID NO: 10)), MAR 1-42 (1_42R2Bis, 1_42R3), MAR 1-6 (1_6R2 (SEQ ID NO: 8), 1_6R3) or MAR 1-68 (1_68R2 (SEQ ID NO: 7)), as evidenced by the names of these structures. (B) Representative FACS profiles obtained for the best 3'-5' MAR elements (MAR 1_68R (SEQ ID NO: 6)).

Fig. 19. Stability of expression for two MAR vectors.

Polyclonal populations constructed from vectors containing MAR derivatives 1_68R2, 1_6R2 and X_29R3 were examined for 5 weeks of culture without selection, and GFP fluorescence was assessed weekly during this period. The percentile of the M3 subpopulation was assessed: MAR element 1_6R2, located in the 3'-5' direction, and unrearranged MAR 1-68, located in the 5'-3' direction, represented the best combination tested for a vector with two MAR elements. Subpopulations M1 and M2 are also shown.

Fig. 20. Expression vectors containing a single genetic element.

MAR 1_68 and X_29 were studied and used in combination with the LmnB2 replicator. MAR elements were positioned in the 5'-3' direction of the transgene expression cassette and assessed in a transgene transfection assay over two months. A polyclonal population of stably transfected cells was subjected to antibiotic resistance selection for two weeks and examined for GFP fluorescence by fluorescence-excited cell sorting (FACS) analysis for seven weeks. The proportion of high-producing M3 cells is shown in graph (A), while graph (B) shows typical FACS profiles.

Fig. 21. Expression vectors containing a single genetic element X-29.

Analysis of the stability of the X_29 vector: showed that the expression vector containing a single X_29 in the 5'-3' direction from the expression cassette was stable and made it possible to obtain a very high percentile of the M2 and M3 subpopulations even after 14 weeks of cultivation (27 passages).

Fig. 22. Comparative analysis of stably transfected CHO populations after 24 weeks of antibiotic selection.

A vector with a single MAR X_29 located 5'-3' of the expression cassette (Puro_CGAPD_GFP_gastrin_X29) increases the appearance of cells expressing high levels of GFP, as well as the stability of expression over time, compared to a vector with two MAR elements: with 1_6R2 as MAR located in the 3'-5' direction, and 1_68 as a MAR located in the 5'-3' direction (Puro_1_6R2_CGAPD_GFP_gastrin_1_68).

DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS FOR IMPLEMENTING THE PRESENT INVENTION

The term transgene, as used herein, is an isolated deoxyribonucleotide (DNA) sequence encoding a given mature protein (also referred to herein as protein-coding DNA), a protein precursor, or functional RNA that does not encode a protein (non-coding RNA). The transgene is isolated and inserted into a cell to produce the transgene product. Some preferred transgenes of the present invention are transgenes encoding immunoglobulins (Ig), Fc fusion proteins and other proteins, in particular proteins with therapeutic activity ("biotherapeutic agents"). For example, some immunoglobulins, such as infliximab (Remicade) or other secreted proteins, such as coagulation factor VIII, are quite difficult to express due to cellular difficulties, mostly uncharacterized. With the help of recombinant nucleic acid molecules, vectors and methods according to the present invention, these difficulties can be detected and/or solved. This typically improves the quantity of therapeutic proteins that can be produced and/or their quality, such as their processing and homogeneity of post-translational modifications such as glycosylation.

As used herein, the term transgene shall not, in the context of protein-coding DNA, include nontranscribed flanking regions such as RNA transcription initiation signals, polyadenylation sites, promoters, or enhancers. Other preferred transgenes include DNA sequences encoding functional RNAs. Thus, the term transgene in the context of the present invention refers to a DNA sequence that is introduced into a cell, such as a eukaryotic host cell, by transfection (which also includes, in the context of the present invention, transduction, i.e., introduction through viral vectors) , and which encodes the product of interest, also referred to herein as a “transgene expression product,” eg, “heterologous proteins.” The transgene may be operably linked to a sequence that encodes a signal peptide, which in turn mediates and/or promotes movement and/or secretion across the endoplasmic reticulum and/or cytoplasmic membrane and is removed before or during secretion.

Small interfering RNAs (siRNAs) are double-stranded RNA molecules, typically 20-25 base pairs in length, that play a role in RNA interference (RNAi) by interfering with the expression of certain genes with complementary nucleotide sequences. siRNA can be directly introduced into cells or can be expressed in a cell via a vector. The isolated TEP siRNA referred to herein is a 20-25 base pair siRNA that is typically introduced directly into a cell, i.e., without expression by a nucleic acid that has been introduced into the cell.

Small/short hairpin RNA (shRNA) is an RNA sequence that forms a tight hairpin bend that can be used to silence the expression of a target gene through RNAi. Expression of shRNAs in cells is usually accomplished by delivery of plasmids or viral vectors such as retroviral vectors. To produce shRNA molecules, the siRNA sequence is usually modified to introduce a short loop between two siRNA strands. The nucleic acid encoding the shRNA is then delivered via a vector into the cell, and it is transcribed as a short hairpin RNA (shRNA), which is possibly processed by the Dicer in the usual manner into functional siRNA.

Mi/shRNA is capable of reducing the expression of a target gene in a sequence-dependent manner. The shRNA can hybridize to a region of the mRNA transcript encoding the target gene product, thereby inhibiting the expression of the target gene through RNA interference. Bifunctional shRNAs have more than one target, such as the coding region as well as some untranslated regions of the mRNA. Incorporation into the cell genome promotes long-term or constitutive silencing of the gene, which can spread to daughter cells.

A microRNA (miRNA) is a small RNA molecule, e.g., 20 to 24 nucleotides in length, particularly 22 nucleotides, that is involved in the transcriptional and posttranscriptional regulation of gene expression by pairing with complementary sequences within the mRNA. Gene silencing can occur either by inhibiting the transcription of the transgene, by degrading the mRNA, or by preventing the translation of the mRNA. MicroRNAs can be expressed by delivery of plasmids or viral vectors such as retroviral vectors. Alternatively, RNA molecules that inhibit or mimic miRNAs can be synthesized and directly transfected into cells.

A “transgene expression/processing protein (TEP) or functional TEP RNA coding sequence” allows the TEP protein to be expressed or overexpressed after its transfer into a cell, whereas a sequence coding for non-coding functional RNA molecules inhibits the expression of cellular proteins, respectively. TEP proteins may be identical or similar to cellular proteins, or they may be proteins from a different cell or species. Cellular proteins whose expression is, for example, inhibited by functional RNA molecules are constitutive proteins of the cell into which the functional RNA molecules have been introduced. The TEP protein can also complement the expression of another cellular protein and, as a result, preferentially enhances the expression of the transgene. These proteins may be involved in recombination; in the processes of mRNA translation; in ER trafficking, secretion, processing or folding of polypeptides, ER-Golgi-plasma membrane transport, glycosylation and/or other post-translational modification. Functional RNA molecules include, for example, siRNA, shRNA, microRNA, lasso-spliced RNA, small sense RNA (svRNA), antisense RNA (aRNA), ribozyme RNA, and other RNAs, particularly those that can repress the expression of a target gene. . In a particularly preferred embodiment, these proteins are involved in the "protein secretion pathway" or "recombination pathway", but also include some protein processing proteins or metabolic proteins described below.

Functional TEP RNAs can not only be expressed from the nucleic acid sequence described above, but they can be directly introduced into a cell. This is particularly true for isolated TEP siRNAs.

The term "isolated nucleic acid molecule" in the context of the present invention is equivalent to the term "recombinant nucleic acid molecule", i.e. a nucleic acid molecule that does not exist in a given form in nature, but which has been constructed from parts that exist in nature.

A nucleic acid sequence, such as DNA or RNA, is complementary to another DNA or RNA if the nucleotides, such as two strands of single-stranded DNA or two strands of single-stranded RNA, can form stable hydrogen bonds, such as the hydrogen bond between guanine (G) and cytosine ( WITH). In a cell, complementary base pairing allows cells, for example, to copy information from one generation to the next. In RNA interference (RNAi), complementary base pairing allows the silencing or complete knockout of some target genes. Essentially, a siRNA, shRNA, or microRNA sequence specifically reduces or disrupts the expression of a target gene by aligning a single strand of RNA (eg, an antisense strand in a siRNA) with RNA, particularly host cell mRNA. The degree of complementarity between two strands of nucleic acid can vary from complete complementarity (each nucleotide opposite its opposite) to partial complementarity (50%, 60%, 70%, 80%, 90% or 95%). The degree of complementarity determines the stability of the complex and thus how successfully a gene can, for example, be knocked out. Thus, complete or at least 95% complementarity is preferred.

The RNAi activity of siRNA molecules largely depends on their ability to bind to the RNA-inducible silencing complex (RISC). Binding of the siRNA duplex to RISC is followed by unfolding and cleavage of the sense strand by endonucleases. The remaining antisense strand-RISC complex can then bind to target mRNAs to initiate transcriptional silencing.

In the context of the present invention, transgenes, as defined above, typically express proteins that are desirable to be produced in large quantities, for example, for pharmaceutical use, while the sequences encoding TEP proteins/functional TEP RNAs, or the functional RNA molecules themselves, designed to aid the expression of such transgenes either directly or indirectly. A "typical list of TEP proteins expressed using transposon-carrying vectors" is given in Table A. It will be apparent to one skilled in the art that a large number of such proteins have been described in the art and are presented in Table A as NCBI sequence identification numbers for the corresponding proteins, and the nucleic acid sequences encoding them. The last column lists the sequence identifiers for some of these sequences. It will be apparent to one skilled in the art that protein variants, as well as sequences that are more than 80%, 90%, 95% or 98% identical, are within the scope of the present invention.

A "typical list of shRNAs expressed using, for example, certain vectors carrying piggybac transposons" is shown in Table B. It will be apparent to one skilled in the art that such shRNAs can be easily constructed once the target gene is selected. For example, any of the known recombination pathway genes is a ready-made target gene. However, other genes, such as the protein-coding genes shown in Table A, may represent ready targets for siRNAs derived from such shRNAs. Table C is a list of examples of miRNAs (sense strand) and examples of shRNAs derived from the corresponding miRNAs. The antisense strand of siRNA is ultimately used to block and/or cause degradation of cellular mRNA. This typically results in a decrease in the levels of the protein encoded by said mRNA.

Identity refers to the degree of relatedness between two nucleotide sequences, which is determined by the identity of pairs between two strands of such sequences, for example, a whole sequence and a complete sequence. Identity can be easily calculated. Although there are many ways to measure identity between two nucleotide sequences, the term "identity" is well known to those skilled in the art (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., eds., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Human Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Methods commonly used to determine identity between two sequences include, but are not limited to, those described in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman , D., SIAM J Applied Math. 48: 1073 (1988). Preferred methods for determining identity have been developed to obtain the highest matches between two sequences of interest. Such methods are encoded in computer programs. Preferred computer program-based methods for determining identity between two sequences include, but are not limited to, the GCG (Genetics Computer Group, Madison, Wis.) software package (Devereux, J., et al., Nucleic Acids Research 12(1). 387 (1984 )), BLASTP, BLASTN, FASTA (Altschul et al. (1990); Altschul et al. (1997)). The well-known Smith Waterman algorithm can also be used to determine identity.

As an example, a nucleic acid comprising a nucleotide sequence that is at least 95% identical to, for example, a reference nucleotide sequence means that the nucleic acid nucleotide sequence is identical to the reference sequence except that said nucleotide sequence may contain up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide, or a plurality of nucleotides constituting up to 5% of all nucleotides in the reference sequence, can be inserted into the reference sequence. These reference sequence mutations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, either scattered individually among the nucleotides in the reference sequence or located in one or more contiguous groups within the reference sequence. Sequence identities of greater than about 60%, about 70%, about 75%, about 85%, or about 90% to any sequence described herein (e.g., by SEQ ID and/or accession number) are also included in the scope of the present invention.

A nucleic acid sequence substantially identical to another nucleic acid sequence refers to a sequence containing point mutations, deletions or insertions in the sequence that have no or negligible effect on the respective method described and are often represented by one, two, three or four mutations per 100 By.

The present invention relates to both polynucleotide and polypeptide variants. "Variant" refers to a polynucleotide or polypeptide that differs from the described polynucleotide or polypeptide but retains its essential properties. Typically, the variants are generally very similar and in many areas identical to the polynucleotide or polypeptide of the present invention. These variations may include changes in coding regions, non-coding regions, or both. Particularly preferred are polynucleotide variants comprising changes that cause silent substitutions, insertions or deletions but do not alter the properties or activity of the encoded polypeptide. Nucleotide variants obtained by silent substitutions, due to the degeneracy of the genetic code, are preferred. Moreover, variants in which 5-10, 1-5 or 1-2 amino acids described herein are replaced, deleted or inserted in any combination are also preferred. The present invention also includes allelic variants of these polynucleotides. An allelic variant refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation in nature occurs as a result of mutation and can lead to polymorphism within a population. Mutations in a gene may be silent (without changes in the encoded polypeptide) or may encode polypeptides whose amino acid sequences are changed. An allelic variant polypeptide is a polypeptide encoded by an allelic variant of a gene. Variants of any of the nucleic acid molecules described in this application are included in the scope of the present invention.

A promoter sequence, or the promoter itself, is a nucleic acid sequence that is recognized by a host cell to express a specific nucleic acid sequence. The promoter sequence includes transcription control sequences that regulate the expression of the specified polynucleotide. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides, either homologous or heterologous to the host cell. The promoters of the present invention include inducible and non-inducible promoters. A nucleic acid sequence is under the control of a promoter when the promoter performs its function on said nucleic acid.

CGAPDH (also referred to herein as C_GAPDH) is an enhancer-promoter fusion that includes the human GAPDH promoter and the human CMV immediate-early gene enhancer. In one embodiment, to obtain it, the human GAPDH promoter and its 5' untranslated region (UTR) were amplified by PCR from genomic DNA of human HEK293 cells. The resulting product was placed in the 5'-3' direction of the human CMV immediate-early gene enhancer. See the example sequence in SEQ ID NO: 11. Sequences that are at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 11 are also within the scope of the present invention. Other desirable promoter(s) and/or enhancer(s) or fusion products thereof include, but are not limited to: human CMV immediate early gene enhancer, human GAPDH promoter, human EFI-alpha promoter, CMV promoter, SV40 promoter, Actb CHO or HspaS CHO promoter. These elements are well known in the art, and example sequences are listed in SEQ ID NOs: 110-116. It will be apparent to one skilled in the art that variants thereof are also within the scope of the present invention, as is an element which is at least 80%, 90%, 95%, 98%, 99% or 100% sequence identical to any from SEQ ID NO: 110-116.

A "transposon" is a mobile genetic element that moves efficiently between vectors and chromosomes using a "cut and paste" or "copy and paste" mechanism. During the process of transposition, a transposase (for example, transposase PB in the PiggyBac transposon system) recognizes transposon-specific inverted terminal repeat (ITR) sequences located at both ends of the transposon (there are 5' and 3' ITRs in any transposon system), moves the contents from source sites and inserts them into chromosomal sites, such as the TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system allows genes of interest located between two ITRs to be easily moved into target genomes. The PiggyBac transposon system is described, for example, in 2010/0154070, which is incorporated herein by reference in its entirety.

MAR elements (MAR constructs, MAR sequences, S/MAR, or simply MAR) belong to a broader group of epigenetic regulatory elements that also include boundary or insulator elements such as cHS4, loci regulatory regions (LCR), stabilizing and antirepressor (STAR) regions. elements, universal chromatin opening elements (UCOE), or histone modifiers such as histone deacetylase (HDAC).

MAR elements can be defined based on the identified MAR on which they are primarily based: a MAR S4 construct is accordingly MAR elements whose majority of nucleotides (50% or more, preferably 60%, 70% or 80%) are based on MAR S4 . Some simple sequence motifs, such as A- and T-rich motifs, are often found within MAR elements. Other common motifs are A-box, T-box, DNA unwinding motifs, SATB1 binding sites (H-box, A/T/C25) and vertebrate or Drosophila topoisomerase II consensus sites.

MARs are generally described as sequences in the DNA of eukaryotic chromosomes to which the nuclear matrix is attached. The properties of MARs are only partly determined by their primary structure. For example, it is known that the typical primary structure found in MAR elements, such as AT-rich regions, leads to the formation of tertiary structures, namely some of the bends, which determine MAR function. Thus, MARs are often defined not only by their primary structure, but also by their secondary, tertiary structure, such as their degree of curvature and/or physical properties such as melting point.

The AT/TA dinucleotide-rich DNA bend region (hereinafter referred to as the “AT-rich region”), found in most MAR elements, is a DNA bend region containing large amounts of A and T, particularly in the form of AT and TA dinucleotides . In a preferred embodiment, it contains at least 10% TA dinucleotide and/or at least 12% AT dinucleotide over a region of 100 contiguous base pairs, preferably at least 33% TA dinucleotide and/or at least 33% AT dinucleotide over a region of 100 contiguous base pairs (or a corresponding shorter region if the AT-rich region is shorter), and has a curved secondary structure. However, the "AT-rich regions" can be as short as about 30 nucleotides or less, but preferably their length is about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150, about 200, about 250, about 300 , about 350 or about 400 nucleotides or more.

Some binding sites also often have relatively high A and T content, such as SATB1 binding sites (H-box, A/T/C25) and topoisomerase II consensus sites for vertebrates (RNYNNCNNGYNGKTNYNY) or Drosophila (GTNWAYATTNATNNR). However, the binding site region (module), in particular the TFBS region, which contains a cluster of binding sites, can be easily distinguished from regions enriched in AT and TA dinucleotides ("AT-rich regions"), from MAR elements rich in A content and T, by comparing the curvature pattern of the specified areas. For example, the average degree of curvature of a human MAR 1_68 may be greater than about 3.8 or about 4.0, whereas the average degree of curvature of a TFBS region may be less than about 3.5 or about 3.3. The areas of identified MAR can also be confirmed using alternative methods, such as, but not limited to, determination of relative melting temperatures described elsewhere in this application. However, such values are species specific and therefore may vary from species to species and may, for example, be lower. Thus, the degrees of curvature of the corresponding AT and TA dinucleotide-rich regions may be lower, for example, from about 3.2 to about 3.4, or from about 3.4 to about 3.6, or from about 3.6 to about 3.8, and the degrees of curvature of the TFBS regions may be proportionally lower, for example, lower than about 2.7, lower than about 2.9, lower than about 3.1, lower than about 3.3. A qualified technician will select correspondingly smaller window sizes in the SMAR Scan II program.

A MAR element, MAR construct, MAR sequence, S/MAR, or simply MAR according to the present invention is a sequence of nucleotides having one or more (e.g., two, three, or four) properties, such as those described above, similar to those found in nature." SAR" or "MAR". Preferably, such a MAR element, MAR construct, MAR sequence, S/MAR, or simply MAR has at least one property that promotes protein expression of any gene affected by said MAR. The MAR element typically also has the property of an isolated and/or purified nucleic acid, preferably exhibiting MAR activity, in particular exhibiting transcription modulation activity, preferably transcription enhancement activity, but also exhibiting, for example, expression stabilizing activity and/or other activities.

The terms MAR element, MAR construct, MAR sequence, S/MAR, or simply MAR also include, in some embodiments, improved MAR constructs that have properties that provide an improvement over a naturally occurring and/or identified MAR that may be based on the MAR design according to the present invention. Such properties include, but are not limited to: reduced length compared to full-length naturally occurring and/or discovered MAR, increased gene/transcription expression, increased expression stability, tissue specificity, inducibility, or a combination of these properties. Accordingly, the MAR element that has been improved, for example, contains less than about 90%, preferably less than about 80%, even more preferably less than about 70%, less than about 60%, or less than about 50% of the number of nucleotides identified MAR sequences. The MAR element can increase gene expression and/or transcription of the transgene after transformation of a suitable cell with the specified construct.

The MAR element is preferably inserted in the 3'-5' direction of the promoter region to which the gene of interest is or can be operably linked. However, in some embodiments, it is preferred that the MAR element be located in both the 3'-5' direction and the 5'-3' direction, or only in the 5'-3' direction, of the gene/nucleic acid sequence of interest . Various other cis and/or trans MAR arrangements are also within the scope of the present invention.

The term “synthetic,” when used in the context of a MAR element, refers to a MAR whose development involved more changes than simple rearrangement, duplication and/or deletion of sequences/regions or portions of regions of identified MARs or MARs based thereon. In particular, synthetic MARs/MAR elements typically include one or more, preferably one, region of an identified MAR that may, in some embodiment, however, have been synthesized or modified, as well as specially designed, well-characterized elements, such as a single TFBS or a series of TFBS, which, in a preferred embodiment, are obtained synthetically. These designed elements are, in many embodiments, relatively short, particularly being typically no more than about 300 bp in length, preferably no more than about 100, about 50, about 40, about 30, about 20, or about 10 p.o. These elements, in some embodiments, may be multimerized. Such synthetic MAR elements are also included within the scope of the present invention, and it should be apparent that, in general, throughout the present specification, it is to be understood that whatever is stated to apply to a “MAR element” is equally applicable to a synthetic MAR element.

Functional nucleotide sequence fragments of discovered MAR elements are also included provided that they retain the functions of the MAR element described above.

Some preferred identified MAR elements include, but are not limited to: MAR 1_68, MAR X_29, MAR 1_6, MAR S4, MAR S46, including all permutations thereof described in WO 2005040377 and US Patent Publication 20070178469, which are specifically incorporated herein by reference to describe data sequences and other MAR elements. Chicken lysozyme MAR is also a preferred embodiment (see US Pat. No. 7,129,062, which is also specifically incorporated herein to describe MAR elements).

If a vector is specified to contain a single MAR, this means that within the specified vector there is a single MAR and no other MARs of either the same or a different type or structure.

In some embodiments of the present invention, multiple MARs are present, which may be of the same or different type or structure, all of which may be located in the 5'-3' direction of the gene of interest. This is called a single MAR cluster.

If something, such as the number of cells stably expressing a polypeptide, is stated to be “independent” of the presence of, for example, a sequence, then said sequence does not have an effect (for example, the number of cells stably expressing the polypeptide) to any statistically significant extent .

A transgene or sequence encoding a transgene expression/processing protein or functional RNA according to the present invention is often part of a vector.

A vector of the present invention is a nucleic acid molecule capable of carrying another nucleic acid, such as a transgene, to be expressed by a given vector to which it has been linked, typically into which it has been inserted. For example, a plasmid is a type of vector, a retrovirus or lentivirus is another type of vector. In a preferred embodiment of the present invention, the vector is linearized before transfection. An expression vector contains or is under the control of regulatory elements that are designed to facilitate the transcription and/or expression of the nucleic acid sequence carried by the expression vector. Regulatory elements include enhancers and/or promoters, but also various other elements described in this application (see also section "Vector Development").

The vector sequence is the DNA or RNA sequence of said vector, excluding any "other" nucleic acids such as transgenes, as well as genetic elements such as MAR elements.

A eukaryotic cell, including a mammalian cell, such as a recombinant mammalian cell/eukaryotic host cell, according to the present invention can be maintained under cell culture conditions. Some examples of this cell type are non-primate eukaryotic host cells, such as Chinese hamster ovary (CHO) cells and baby hamster kidney (BHK, ATCC CCL 10) cells. Primate eukaryotic host cells include, for example, human cervical carcinoma cells (HELA, ATCC CCL 2) and the SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL-1587). Recombinant eukaryotic host cell or recombinant mammalian cell means a cell that has been modified, for example, by transfection, for example, with a transgenic sequence, and/or by mutation. Eukaryotic host cells or recombinant mammalian cells are capable of carrying out post-transcriptional modifications of proteins expressed by these cells. In some embodiments of the present invention, the cellular counterpart in the eukaryotic (eg, non-primate) host cell is fully functional, i.e., has not, for example, been inactivated by mutation. Preferably, the transgenic sequence (eg, primate) is expressed in addition to its cellular counterpart (eg, non-primate).

Transfection according to the present invention is the introduction of a nucleic acid into a recipient eukaryotic cell, for example, but not limited to, by electroporation, lipofection, typically using a non-viral vector (vector-mediated transfection) or by chemical methods, including the use of polycationic lipids. Non-vector-mediated transfection involves, for example, direct introduction of isolated TEP siRNAs into the cell. In a transiently transfected cell, for example, siRNA is retained only temporarily. In the context of the present invention, it is possible to carry out a first transfection with at least one nucleic acid molecule with a sequence encoding a transgene expression/processing protein (TEP) or a functional TEP RNA, or, alternatively, a functional TEP RNA itself (for example, siRNA), and a second subsequent transfection with a nucleic acid encoding the transgene. Both the first and second transfections can be repeated. For example, siRNA introduced during the first transfection acts, specifically inhibits, a recombination protein (a protein that is involved in recombination events in the transfected cell). The transgene is then introduced in a second subsequent transfection.

Transcription means the synthesis of RNA from a DNA template. "Transcriptionally active" refers, for example, to a transgene that is transcribed. Translation is the process by which proteins are synthesized from RNA.

The improvement in secretion is measured in comparison with the value obtained for a control cell that does not contain the corresponding transgene sequence. Any statistically significant improvement over the control value is called stimulation.

A selection marker is a nucleic acid that contains a gene whose product confers resistance to an antibiotic selection agent (e.g., chloramphenicol, ampicillin, gentamicin, streptomycin, tetracycline, kanamycin, neomycin, puromycin) or the ability to grow on selective media (e.g., DHFR (dihydrofolate reductase)).

The class of proteins known as chaperones are defined as proteins that bind to and stabilize an otherwise unstable conformational isomer of another protein and, through controlled binding and release, promote its correct fate in vivo, whether folding, oligomer assembly, or transport to a specific cellular compartment. or disposal by degradation. BiP (also known as GRP78, Ig heavy chain binding protein, and Kar2p in yeast) is a common approximately 70 kDa chaperone of the hsp 70 family located in the endoplasmic reticulum (ER) that, among other functions, promotes secretory transport. system and promotes protein folding. Protein disulfide isomerase (PDI) is an ER-located chaperone protein that is involved in catalyzing disulfide bond formation during post-translational processing of proteins.

CELLULAR METABOLIC ENGINEERING

In cellular metabolic engineering, for example, processes inherent in the expressing cell are modified. For example, some secretory pathway proteins are overexpressed, for example. Alternatively, recombination events are altered by influencing recombination pathways.

PROTEIN SECRETION PATHWAY

Protein secretion is a process common to organisms from all three kingdoms. This complex secretion pathway first requires the movement of protein from the cytosol through the cytoplasmic membrane of the cell. Several steps and various factors are required for a protein to reach its final destination. In mammalian cells, this secretion pathway includes two main macromolecular assemblies: the signal recognition particle (SRP) and the secretory complex (Sec complex or translocon). SRP consists of six proteins with masses of 9, 14, 19, 54, 68 and 72 kDa and 7S RNA, and the translocon is a toroidal particle consisting of Sec61αβγ, Sec62 and Sec63. The accession numbers (in parentheses) provided for the human variant of some of these proteins are described below: hSRP14 (accession number X73459.1); hSRP9 (NM_001130440); hSRP54 (NM_003136); hSRPRα (NM_003139); hSRPRβ (NM_021203); hSEC61α1 (NM_013336); hSEC61 β (L25085.1); hSEC61 γ (AK311845.1).

The first step of protein secretion depends on signal peptides, which contain a specific peptide sequence at the amino terminus of the polypeptide that mediates the movement of the nascent protein across the membrane and into the lumen of the endoplasmic reticulum (ER). At this stage, the signal peptide that exits the leading translation ribosome interacts with a subunit of the SRP particle that recognizes the signal peptide, namely SRP54. Binding of SRP to the signal peptide blocks further elongation of the resulting polypeptide, resulting in translation arrest. SRP-9 and -14 proteins are required for elongation arrest (Walter and Blobel 1981). In the second step, the ribosome-nascent polypeptide-SRP complex docks with the ER membrane through the interaction of SRP54 with the SRP receptor (SR) (Gilmore, Blobel et al. 1982; Pool, Stumm et al. 2002). SR is a heterodimeric complex containing two proteins, SRα and SRβ, which exhibit GTPase activity (Gilmore, Walter et al. 1982). The interaction of SR with SRP54 is dependent on GTP binding (Connolly, Rapiejko et al. 1991). SR coordinates the release of SRP from the ribosome-nascent polypeptide complex and the association of the ribosome exit site with the Sec61 complex (translocon). The growing nascent polypeptide enters the ER through the translocon channel, and translation resumes at normal rates. The ribosome remains bound on the cytoplasmic side of the translocon until translation is complete. In addition to ribosomes, translocons are closely associated with ribophorin on the cytoplasmic side and with chaperones such as calreticulin and calnexin, and protein disulfide isomerases (PDI) and oligosaccharide transferase on the lumenal side of the ER. After insertion of the growing nascent polypeptide into the lumen of the ER, the signal peptide is cleaved from the pre-protein by an enzyme called signal peptidase, resulting in the release of the mature protein into the ER. After post-translational modification, proper folding and multimerization, proteins leave the ER and migrate to the Golgi apparatus and then to secretory vesicles. Fusion of secretory vesicles with the plasma membrane results in the release of the vesicle contents into the extracellular space.

Surprisingly, secreted proteins have evolved to acquire specific signal sequences that are well suited for their movement across the cell membrane. Various sequences found as distinct signal peptides may interact in unique ways with the secretory apparatus. Signal sequences are predominantly hydrophobic in nature, and this property may facilitate the production of a secretory protein from the resulting peptide. In addition to the hydrophobic amino acid region, the sequences of most mammalian secretion signals share many properties. Different signal peptides differ in the efficiency with which they direct the secretion of heterologous proteins, but several secretion signal peptides (i.e., signal sequences from interleukin, immunoglobulin, histocompatibility receptor, etc.) have been discovered that can be used to direct secretion heterologous recombinant proteins. Despite the similarities, these sequences are not optimal for promoting efficient secretion of some proteins that are difficult to express, due to the fact that the native signal peptide cannot function properly outside the native context, or due to differences associated with the host cell or secretion process. The selection of an appropriate signal sequence for efficient secretion of a heterologous protein can be further complicated by interactions of sequences within the cleaved signal peptide with other regions of the mature protein (Johansson, Nilsson et al. 1993).

RECOMBINATION PATHWAYS

Recombination pathways, also known as DNA recombination pathways, are cellular pathways that lead to the repair of DNA damage, such as joining the ends of a DNA molecule after chromosomal double-strand breaks, and the exchange or fusion of DNA sequences between chromosomal and nonchromosomal DNA molecules, such as , when chromosomes cross over during meiosis or rearrangement of immunoglobulin genes in lymphocytic cells. There are three main recombination pathways: the homologous recombination (HR) pathway, the non-homologous end-joining (NHJ) pathway, the microhomology-mediated end joining (OMJC) and alternative end joining (Alt-end joining) pathways.

MECHANISMS OF HOMOLOGY RECOMBINATION (HR), NON-HOMOLOGY END JOINING (HJJ), AND MICROHOMOLOGY-MEDIATED END JOINING (OMJC)

Transgenes use the recombination machinery to insert into a double-strand break in the host genome,

Double-strand breaks (DSBs) are the most biologically destructive type of genomic damage, potentially leading to cell death or a wide variety of genetic rearrangements. Accurate repair is essential for the successful maintenance and transmission of genetic information.

There are two main mechanisms of DNR repair: non-homologous end joining (HJJ) and homologous recombination (HR). A third mechanism, called microhomology-mediated end joining (MJK), often comes into play when the two main DNR repair mechanisms fail. Homologous recombination is the process of genetic exchange between DNA sequences that are homologous to each other, and acts primarily during the S/G2 phases of the cell cycle, whereas HSCT simply joins two broken ends of DNA, usually in the absence of sequence homology, and acts during all phases of the cell cycle. cycle, but is of particular importance during G0 - G1 and early S phase of mitotic cells (Wong and Capecchi, 1985; Delacote and Lopez, 2008). In vertebrates, GRs, NGSCs, and OMSCs are involved in DNR repair in different ways, depending on the nature of the DNR and the phase of the cell cycle (Takata et al., 1998).

NGSC: basic mechanisms

Essentially, the molecular mechanism of the NGSC process appears simple: 1) a set of enzymes capture the broken DNA molecule, 2) a molecular bridge is formed that brings the two ends of the DNA closer together, and 3) the broken molecules are religated. The NGSC apparatus in mammalian cells includes two protein complexes for carrying out such reactions: the Ku80/Ku70 heterodimer associated with DNA-PKx (the catalytic subunit of DNA-dependent protein kinase), and DNA ligase IV with its cofactor XRCC4 (complementing X-ray damage repair gene 4 in Chinese hamster) and a variety of protein factors such as Artemis and XLF (XRCC4-like factor; or Cernunnos) (Delacote et al., 2002). NGSC is often considered an error-prone DNR repair because it simply joins two broken ends of DNA, usually in the absence of sequence homology, and generates small insertions and deletions (Moore and Haber, 1996; Wilson et al., 1999). NGSC provides a mechanism for DNR repair throughout the cell cycle, but is of particular importance during G0 - G1 and early S phase of mitotic cells (Takata et al., 1998; Delacote and Lopez, 2008). DNR repair through NGSC is observed in organisms ranging from bacteria to mammals, which indicates its conservative nature in the process of evolution.

After DNR formation, a key step in the NGSC repair pathway is the physical bringing together of broken DNA ends. NGSC is triggered by the interaction of the Ku70/80 heterodimeric protein complex with both ends of a broken DNA molecule to capture, bring together the ends, and create a scaffold for the assembly of other key NGSC factors. The DNA-bound heterodimeric Ku complex recruits DNA-PKx, a 460 kDa protein belonging to the PIKK family (Gottlieb and Jackson, 1993), to the DNR site and activates its serine/threonine kinase function ( Yaneva et al., 1997). Two DNA-PKx molecules from the two ends of the DNR interact, thus forming a molecular bridge between both broken DNA ends and inhibiting their degradation (DeFazio et al., 2002). The DNA ends can then be directly ligated, although most ends resulting from DNR must be properly processed before ligation (Nikjoo et al., 1998). Depending on the nature of the break, different combinations of processing enzymes may be required to produce compatible overhangs by filling the gaps, removing damaged DNA, or removing secondary structures surrounding the break. This step in the NGSC process is thought to be responsible for the random loss of nucleotides associated with NGSC repair. One key end-processing enzyme in mammalian NHSC is Artemis, a member of the metallo-β-lactamase superfamily of enzymes, the gene of which is found to be mutated in the majority of patients with radiosensitive severe combined immunodeficiency (SCID) (Moshous et al., 2001). Artemis has both 5'→3' exonuclease activity and DNA-PKx-dependent endonuclease activity towards DNA containing d-on junctions (two-strand to single-strand transitions) and DNA hairpins (Ma et al., 2002). . Its activity is also regulated by the protein kinase ATM. Thus, it appears that Artemis is likely involved in multiple DNA damage responses. However, it appears that Artemis is involved in the repair of only a subset of DNA damage, as no significant defect in DNR repair was observed in cells lacking Artemis (Wang et al., 2005, Darroudi et al., 2007).

Gaps in DNA must be filled for repair to be possible. The addition of nucleotides to DNRs can only be accomplished by polymerases μ and λ (Lee et al., 2004; Sarr et al., 2007). Through interaction with XRCC4, polynucleotide kinase (PNK) is also recruited to DNA ends to allow polymerization as well as DNA ligation (Koch et al., 2004). Finally, NGSC is completed by DNA end ligation, a step carried out by a complex containing XRCC4, DNA ligase IV and XLF (Grawunder et al., 1997). Other ligases may partially replace DNA ligase IV, since NGSC can occur in the absence of XRCC4 and ligase IV (Yan et al., 2008). Moreover, studies have shown that XRCC4 and ligase IV do not play a role outside of NGSC, whereas, on the contrary, KU is involved in other processes such as transcription, apoptosis and responses to the microenvironment (Monferran et al., 2004; Müller et al. , 2005; Downs and Jackson, 2004).

NGSC can be attenuated or terminated in a variety of ways, many of which directly affect the proteins mentioned above (eg, Ku80/Ku70 heterodimer, DNA-PKx, but especially DNA ligase IV, XRCC4, Artemis, and XLF (XRCC4-like factor; or Cernunnos), PIKK (phosphoinositide 3-kinase-like protein kinase family).

GR: basic mechanisms

Homologous recombination (HR) is a very precise repair mechanism. The homologous chromatid serves as a template for the repair of the broken strand. GR occurs in the S and G2 phases of the cell cycle when sister chromatids become accessible. Classical GR is mainly characterized by three stages: 1) removal of 5' nucleotides from broken ends, 2) strand insertion and exchange with a homologous DNA duplex, and 3) divergence of recombination intermediates. DNR repair can be completed in a variety of ways, depending on the ability to carry out strand insertion, which include the DNA synthesis-dependent strand annealing (DSA) pathway, classical double-strand break repair (CDBR) (Szostak et al., 1983), break-induced replication (BRR) and, alternatively, the single-strand annealing (SEA) pathway. All GR mechanisms are interconnected and involve many common enzymatic steps.

The first step of all GR reactions corresponds to the removal of the 5' end of the broken DNA strand by nucleases using the MRN complex (MRE11, RAD50, NBN (formerly NBS1, Nijmegen break syndrome 1)) and CtIP (CtBP-interacting protein) (Sun et al., 1991; White and Haber, 1990). The resulting set of sequences with 3' single-stranded DNRs can search for a homologous sequence. Insertion of the homologous duplex is accomplished using a nucleofilament consisting of 3'-ssDNA coated with the protein recombinase RAD51 (Benson et al., 1994). Assembly of the RAD51 filament requires the recruitment of replica protein A (RPA), a heterotrimeric ssDNA-binding protein involved in ssDNA-related DNA metabolic processes in eukaryotes (Wold, 1997, Song and Sung, 2000). RAD51 then interacts with RAD52, which has a ring-like structure (Shen et al., 1996), to displace RPA molecules and promote RAD51 loading (Song and Sung, 2000). Rad52 is important for recombination processes in yeast (Symington, 2002). However, in vertebrates, BRCA2 (breast cancer susceptibility protein type 2) rather than RAD52 appears to play an important role in strand insertion and turnover (Davies and Pellegrini, 2007; Esashi et al., 2007). The RAD51/RAD52 interaction is stabilized by binding to RAD54. RAD54 also plays a role in the maturation of recombination intermediates after D-loop formation (Bugreev et al., 2007). On the other hand, BRCA1 (breast cancer 1) interacts with BARD1 (BRCA1-associated RING domain 1) and BACH1 (BTB and CNC homolog 1) to mediate ligase and helicase DNR repair activities, respectively (Greenberg et al., 2006 ). BRCA1 also interacts with CtIP in a CDK-dependent manner and is ubiquitinated in response to DNA damage (Limbo et al., 2007). Therefore, BRCA1, CtIP and the MRN complex play a role in the activation of GR-mediated DNA repair in the S and G2 phases of the cell cycle.

Nucleofilament insertion results in the formation of a heteroduplex called a displacement loop (D-loop) and involves the replacement of one strand of the duplex by the invading strand and its pairing with another strand of the duplex. Multiple GR pathways can then complete repair by using the homologous sequence as a template to replace the sequence surrounding the DNR. Depending on the mechanism used, crossovers between the homologous template and the broken DNA molecule may or may not be associated with GR repair. Crossing overs can lead to significant genetic consequences, such as genomic rearrangements or loss of heterozygosity.

Five Rad51 paralogs are also involved in homologous recombination: Xrcc2, Xrcc3, Rad51B, Rad51C, Rad51D (Suwaki et al., 2011). Rad51 paralogs form two types of complexes: one, called BCDX2, contains Rad51B, Rad51C, Rad51D, and Xrcc2; the other contains Rad51C and Xrcc3 (CX3) (Masson et al., 2001). The first complex has been proposed to be involved in the formation and/or stabilization of the Rad51-DNA complex (Masson et al., 2001). The role of the second complex appears to be branch migration and splitting of the Holliday structure (Liu et al., 2007).

It has previously been reported that increasing GR compared to NGSC (see US Patent Publication 20120231449, which is incorporated herein by reference in its entirety) can be used to enhance and/or promote transgene expression.

The present invention is directed to reducing or switching off GR. GR can be reduced or switched off in a variety of ways, many of which directly affect the proteins mentioned above (eg, MRN complex proteins (MRE11, RAD50, NBN (formerly NBS1, Nijmegen gap syndrome 1)) and CtIP (CtBP-interacting protein), RAD51 protein replication A (RPA), Rad52, BRCA2 (breast cancer susceptibility protein type 2), RAD54, BRCA1 (breast cancer 1), interacting with BARD1 (BRCA1 associated RING domain 1), BACH1 (BTB and CNC homolog 1 )). The present invention focuses on the production of RNA molecules, such as siRNA, to achieve this goal.

Microhomology-mediated end joining (OMJK)

When other recombination pathways fail or are inactive, DNRs can undergo repair by another error-prone repair mechanism called microhomology-mediated end joining (OMJK). This pathway remains to be fully characterized and is sometimes referred to as alternative end joining (alt-end joining), although it is unclear whether the two processes actually underlie the same mechanism. The most distinctive property of this pathway, which differs from NGSC, is the use of microhomology of 5-25 bp. during the process of straightening broken DNA strands (McVey and Lee, 2008).

OCSC can occur at any stage of the cell cycle and does not depend on the main factors of NCSC and GR, i.e. Ku70, ligase IV and Rad52 genes (Boboila et al., 2010; Yu and McVey, 2010; Lee and Lee, 2007; Ma et al., 2003). Instead of using its own set of proteins for OSC initiation, the most important proteins used for initiation are components of the MRN complex (MRX in yeast), containing Mre11, Rad50 and Nbs1 (Xrs2 in yeast), also involved in the first stages of GR (Ma et al. ., 2003). Besides the MRN complex, many other factors have been proposed as participants in OMCs, such as CTP-interacting protein (CtIP; Yun and Hiom, 2009), poly(ADP-ribose) polymerase 1 (PARP1), ligase III/Xrcc1 complex, ligase I (Audebert et al., 2004), DNA polymerase θ (Yu and McVey, 2010) and the ERCC1/XPF complex (Ma et al., 2003). However, many other proteins are involved in this process.

It has been proposed that in the absence of other DNA end-binding proteins (such as Ku or Rad51), DNRs are recognized by PARP1, which then initiates their repair by OMCs (McVey and Lee, 2008). The repair process, similar to GR, begins with the removal of nucleotides from the 5' to 3' end, which exposes short regions of homology on each side of the break. This processing step is carried out by the MRN complex and is regulated by CtIP (Mladenov and IIakis, 2011). Complementary regions (present in 3′ ssDNA fragments) are paired and non-complementary fragments (flaps) are removed (Yu and McVey, 2010), probably by the ERCC1/XPF complex. The gaps (if any) are then filled by a polymerase (e.g. DNA polymerase θ or δ (Yu and McVey, 2010; Lee and Lee, 2007)) and the breaks are bridged by ligase I or the ligase III/Xrcc1 complex.

In the absence of areas of direct microhomology at the ends of DNA, which is most common, a more distant fragment of the repaired molecule can be copied using a precise DNA polymerase (for example, polymerase 6). This duplicated region then participates in the alignment of DNA ends, resulting in the formation of an insertion at the junction. This more complex version of microhomology-mediated repair has been called synthesis-dependent OMS (SD-OMS) (Yu and McVey, 2010).

Although OMC has been considered an alternative pathway for recombinational repair, it has been shown to be very efficient in the process of IgH class switch recombination in B cells (Boboila et al., 2010), suggesting that it may play a greater role than simply being a backup mechanism. It is also possible that some DNRs, such as incompatible protrusions or blunt ends (which are difficult to repair by NGSCs and/or GR), may be more efficiently repaired by OMSCs (Zhang and Paull, 2005).

TABLE D lists some of the key genes in each of the three pathways, which are therefore also key targets for influence on each of the three pathways (see also US Patent Publication 20120231449, which is incorporated herein by reference in its entirety). This table also includes proteins involved in DNA repair, such as MDC1 and MHS2. MDC1 is required for activation in response to DNA damage of the S phase checkpoint and G2/M phase checkpoint of the cell cycle. However, MDC1 also functions in Rad51-mediated homologous recombination by retaining Rad51 in chromatin.

"Knockdown" in the context of the present invention means that the expression of the target gene is reduced, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Complete suppression means that there is no longer detectable expression of the target gene. Table D also shows the results obtained for some knockdown targets. It will be apparent to one skilled in the art that variations exist in the nucleic acid sequences of these targets and therefore gene variants, particularly variants that are 80%, 90% or 95% sequence identical to the target sequences, are within the scope of the present invention.

PROTEIN PROCESSING AND METABOLIC PROTEINS

This category of proteins that can be used for cellular metabolic engineering does not belong to either the protein secretion or recombination pathway, but otherwise influences the processes inherent in the expressing cell.

Protein processing proteins or metabolic proteins are often enzymes such as chaperones (see definitions of chaperones above), protein isomerases, sugar adding enzymes (eg sialyl or glycosylate enzymes) or phosphatases, or proteins that control cellular energy levels or mitochondrial functions.

TABLE A provides a list of proteins that were expressed (exp.) and/or whose expression was “suppressed” (knockdown, ND), under the subtitle “Protein Processing and Metabolic Proteins.”

VECTOR DEVELOPMENT

Among nonviral vectors, transposons are particularly attractive due to their ability to insert single copies of DNA sequences at high frequency into multiple loci within the host genome. It has been reported that, unlike viral vectors, some transposons do not preferentially insert close to cellular genes, and they are thus less likely to lead to damaging mutations. Moreover, transposons are easy to obtain and handle, typically comprising a transposon-carrying donor plasmid that contains transposable DNA flanked by inverted repeat sequences and a transposase-expressing helper plasmid or mRNA. Several transposon systems have been developed to move DNA in different cell lines without interfering with endogenous copies of the transposon. For example, the PiggyBac (PB) transposon, originally isolated from metallic sulfur, efficiently transfers target DNA into various mammalian cells.

Epigenetic regulatory elements can be used to protect translocated DNA from unwanted epigenetic effects when placed adjacent to a transgene in plasmid vectors. For example, elements called matrix attachment regions (MARs) have been proposed to enhance genomic incorporation and transcription of translocated DNA while preventing heterochromatin silencing, exemplified by the efficient human MAR 1-68. They can also act as insulators and thereby prevent the activation of neighboring cellular genes. MAR elements are thus used to mediate high levels of long-lasting expression in the context of plasmid or viral vectors.

This application demonstrates that, with proper vector design, the beneficial properties of epigenetic regulators, in particular MAR elements, can be combined with those of transposon-based vectors.

Transposons and transposon-based vectors according to the present invention can be used in cellular metabolic engineering, for example, for the expression of secretory proteins from various secretory pathways described in this application. They are also particularly useful when multiple rounds of insertion of translocated DNA are required. This was confirmed when multiple proteins in the cell's secretory pathway were tested, where transfection with multiple vectors and/or multiple successive rounds of transfection could exhaust the available antibiotic or interfere with other selection methods. The ability to rapidly express therapeutic proteins without the need for antibiotic selection is also of particular interest, for example when multiple candidate therapeutic proteins need to be expressed for screening purposes, since significant quantities of proteins can be recovered from unselected cell populations 2-3 weeks after transfection. In particular, MAR-containing transposon-carrying vectors thus represent a promising addition to the currently available arsenal of expression vectors.

The experimental approaches chosen allowed (as opposed to approaches that rely on antibiotic-based assays) to distinguish between effects based on (1) copy number of translocated DNA and effects based on (2) expression levels of translocated DNA.

MAR 1-68 was particularly effective when it was located midway between the ITRs of the PiggyBac transposon, as it did not reduce transposition efficiency. MAR X-29 also worked well at transposon edges without reducing transposition or expression efficiency.

Notably, the extent of MAR-mediated activation of translocated genes was less than that for spontaneous plasmid insertion. Moreover, the level of expression normalized, for example, to the transgene copy, was higher using transposons than that obtained by spontaneous insertion of plasmids in the absence of transposase. This effect was observed regardless of the size of the constructs, the presence of MAR, or promoter strength. This would be expected since transposition can often occur at genomic loci that are relatively permissive of expression, for example because chromatin structures may be more accessible to both transposase and transcription factors. In this regard, previous studies have suggested that transposons may preferentially insert within gene introns, promoters, or genomic loci with lower silencing capacity, although this remains a topic of debate. Alternatively, the co-incorporation of multiple copies of a plasmid into the same genomic locus, which occurs as a result of spontaneous insertion events, may result in the formation of heterochromatin and silencing of repeated sequences, which would be prevented by MAR, whereas the insertion of a single copy of a transposon may be less likely to occur. extent to contribute to such chromatin-mediated silencing. In addition, the insertion of transposons into multiple independent genomic loci increases the likelihood that at least one copy has inserted into a favorable genomic environment and is expressed, whereas plasmid insertion has been found to occur predominantly at only one genomic locus.

The highest levels of expression of translocated DNA, such as transgene/TER, were obtained for the MAR-containing transposon when it was associated with a strong promoter. Surprisingly, it has been discovered that high levels of expression can be obtained by moving a small number of DNA copies, for example, no more than 20, 15, 10 or 5. If, however, high productivity can be achieved, a smaller number of inserted, for example, transferred DNA copies is an advantage because it reduces the likelihood of point mutations occurring in one or a subset of these transgenes resulting from spontaneous mutagenesis events. In addition, transposase-mediated insertion events are less mutagenic than the DNA repair and recombination mechanisms involved in spontaneous plasmid insertion, which can result in the insertion of incomplete or rearranged copies of the transgene.

The high genomic insertion efficiency of the piggyBac (PB) transposon is also useful when the number of target cells is limited, for example, for non-viral transfer of therapeutic genes into primary stem cells to generate clonal populations, for example, for cell-based therapies or regenerative medicine. In this context, physiological levels of expression from multiple transposed DNA copies and the frequent occurrence of transposition events, thus obviating the need for antibiotic selection, are an advantage since the use of antibiotic resistance genes and/or unreliable, for example, transgene expression may raise safety concerns .

Effect of MAR inclusion on transposition efficiency

Since antibiotic resistance does not necessarily reflect efficient transgene expression, green fluorescent protein (GFP) expressed from a derivative of the strong cellular GAPDH promoter was used as an indicator. To test whether the addition of a MAR element to the PB transposon could affect transposition efficiency and transgene expression, and to evaluate whether the location of the MAR in the construct had any influence on these effects, a series of donor constructs carrying transposons containing GFP were developed and the puromycin resistance gene (Puro), into which MAR 1-68 or a control neutral spacer DNA sequence was inserted at various positions in the plasmid (Figure 1). The original transposon-carrying Puro-GFP plasmid without the insert was used as a transposition control to distinguish the contribution of increased transposon size from the effect of the addition of a MAR or spacer sequence.

In the presence of transposase, the highest level of GFP expression was observed in unselected cells when the MAR was positioned in the middle, but not when the MAR was placed 5'-3' of the GFP coding sequence, and not when it was inserted outside the transposase. sequences as expected (Figure 3A). Upon puromycin selection, MAR-mediated activation was reduced, both with and without transposase, whereas mean GFP expression values were increased by one order of magnitude (Figure 3B). The results confirmed that puromycin selection produced only a small number of cells in which expression levels were highest, as suggested by the above counts of transposition events. Additionally, it was shown that transposon-based vectors containing a centrally located MAR produced expression levels similar to those of the same plasmid transfected without transposases.

Effect of MAR inclusion on the copy number of an integrated transposon

Higher levels of GFP fluorescence may result from increased transcription of transgenes and/or insertion of more copies of the transgene. This was assessed by determining the number of transgene copies introduced into the genome using different types of vectors. Total genomic DNA was isolated from pooled cell populations, either after cytofluorimetric sorting of fluorescent cells from unselected populations or after selection for puromycin resistance. Transgene copy number was determined by quantitative polymerase chain reaction (qPCR) analysis of the GFP coding sequence compared with the cellular β2-microglobulin (B2M) gene. In the absence of antibiotic selection, the average number of transgenes inserted, either by transposase or cellular recombination enzymes, was similar, about 1–6 copies per genome, and was not significantly affected by MAR or control sequence (Fig. 4A). However, the lowest copy number was obtained when the MAR was inserted at the transposon boundary, supporting our earlier conclusion that it reduces transposition at this position. After puromycin selection of cells with high levels of expression, the number of transgenes transferred was in a similar range of 2-7 copies (Fig. 4B). However, the copy number of transgenes inserted in the absence of transposase was generally significantly higher, ranging from 6 to 14 copies. This can be easily explained by the fact that spontaneous insertion usually results in the insertion of concatemers of multiple copies of the plasmid into a single genomic locus (results not shown), and that higher copy numbers of transgenes should lead to higher expression levels if cells affected by silencing were removed by antibiotic selection. Together with the previous finding that antibiotic selection produces predominantly high-expressing cells, this also suggests that spontaneous plasmid insertion results in greater transgene copy number variability than with transposon-based vectors.

The level of GFP expression was then normalized to the gene copy number to assess the intrinsic expression potential of these vectors, regardless of their ability to integrate into the genome. In general, lower levels of expression per transgene copy were obtained for unselected cells or for antibiotic-selected cells transfected without transposase or with MAR at the central position, indicating that transgene expression is affected by both the inclusion of an epigenetic regulatory element and and a method for inserting the transgene (Fig. 5). Expression levels per gene copy were generally increased by transposase when different vectors and element combinations were evaluated, and this effect was observed with and without antibiotic selection. The highest levels of expression per transgene copy were obtained after antibiotic selection of cells into which a vector carrying a transposon containing a MAR element in the central position was introduced, in the presence of a transposase. Incorporation of a MAR immediately 5'-3' of the GFP coding sequence did not significantly increase transgene expression, as previously noted for absolute expression levels.

Finally, it was assessed whether the beneficial effect of MAR 1-68 on expression could be limited to the use of the strong human GAPDH promoter used here, or would also occur with other promoters. Thus, we replaced the human GAPDH promoter, which drives GFP expression, with the weaker simian virus 40 (SV40) early promoter. Use of a weaker promoter produced comparable numbers of GFP-positive cells and inserted transgenes, indicating that transgene expression did not alter transposition efficiency (results not shown, Fig. 2A and Fig. 4B). However, absolute expression levels were lower when using the SV40 promoter (results not shown compared with Fig. 3B). In addition, the expression level normalized to transposon copy number was reduced by 4.6-fold when using the SV40 promoter in the absence of MAR and by 3.1-fold in the presence of MAR 1-68 (results not shown). This suggests that MAR could partially, but not completely, prevent the reduction in expression resulting from the use of a weaker promoter, even in the presence of the transposase. Overall, it can be shown that a few inserted copies are sufficient to produce high levels of transgene expression by transposons, and that the highest level of expression per transgene was obtained when, in this context, MAR-68 was placed in the 3'-5' direction of the strong promoter

CHO-M cells were electroporated once or twice with a single transposon-based vector containing the MAR X_29 transgene. Transposition efficiency was highest after electroporation (stable expression was observed in 30% - 45% of cells). However, transgene expression levels were similar to those observed with chemical transfection, in which fewer positive cells and therefore lower transposition efficiency were observed. In fig. 7 shows results for the light and heavy chains of therapeutic immunoglobulins inserted in the 3'-5' direction of MAR X_29, and titers ranging from 1 to 8 μg/ml were obtained. These levels increased further to 23-55 μg/ml after sorting the expressing cells (Fig. 7C).

The expression of transgenes can also be significantly increased, often independently of the use of transposons, by designing a specific vector, in particular using specific MAR element(s) at specific positions relative to the transgene and, preferably, a combination of these MAR element(s) with promoters, enhancers or products of their fusion.

A suitable vector may contain MAR elements that flank the transgene expression cassette. For example, said vector may contain, for example, one or more MAR elements located in the 3'-5' direction, and one or more MAR elements located in the 5'-3' direction, for example, one MAR located in the 3'-5' direction from the transgene expression cassette, and one MAR located in the 5'-3' direction from it (Fig. 18A, Fig. 18B, Fig. 19). Said vector may contain an integrated puromycin resistance gene under the control of the SV40 promoter. The transgene may be under the control of a human GAPDH promoter fused to a human cytomegalovirus (CMV) immediate-early gene enhancer (specifically the CGAPD promoter fusion discussed above).

The highest percentile of cells with high and very high production (% M3/M2), as assessed by GFP fluorescence using FACS analysis and the lowest variability, could be obtained using 1_6R2, 1_68R2 and X_29R3 as MARs located in the 3'- 5' direction (over 80%, 80% and over 80%). Thus, percentiles of cells with high and very high production levels (% M3/M2) of greater than 70%, greater than 75%, or greater than 80% are within the scope of the present invention. It will be apparent to one skilled in the art that some deviations from the defined sequence of these MAR elements are acceptable. Accordingly, vectors containing nucleic acid sequences more than 80%, 85%, 90%, 95% identical to the sequences of SEQ ID NO: 6, 7, 8, 9 and 10 are within the scope of the present invention (Fig. 18A, Fig. .18V).

Attenuation of expression in a bioreactor and/or in the absence of selection pressure often limits production of the protein of interest. Vectors containing derivatives of 1_68R2, 1_6R2 and X_29R3 MAR as MARs located in the 3'-5' direction were examined over a culture period of 5 weeks in the absence of selection, and GFP fluorescence was assessed weekly during this period. When considering the percentile of the M3 subpopulation, element 1_6R2 as a MAR located in the 3'-5' direction and unrearranged MAR 1-68 as a MAR located in the 5'-3' direction were found to be the best combination tested in vectors with at least one MAR element located in the 3'-5' direction and one MAR element located in the 5'-3' direction (well above 80% after more than 2, 3, 4 weeks) (see Fig. 19) .

A similarly designed vector may also contain, for example, only one or more MAR elements located in the 5'-3' direction, for example, one MAR located in the 5'-3' direction of the transgene expression cassette (Fig. 20A, 20B, 21 and 22), and do not contain MAR located in the 3'-5' direction. Said vector may also, in this case, contain an integrated puromycin resistance gene under the control of the SV40 promoter. The transgene may be under the control of the human GAPDH promoter fused to the human cytomegalovirus (CMV) immediate-early gene enhancer. See, for example, SEQ ID NO: 11 and other sequences that are more than 80%, 85%, 90% or 95% identical to the specified sequence. In this combination with a single MAR, where X_29 acted as the MAR, excellent results were achieved. The percentile of cells with high levels of GFP expression (determined as described above) as well as the stability of expression over time (determined as described above) were better than, for example, those for high expression vectors in which MAR elements flanked the cassette expression of the transgene, namely, for a vector containing MAR 1_6R2 located in the 3'-5' direction, and unrearranged MAR 1-68 located in the 5'-3' direction (see Fig. 22). This finding contrasts with the generally accepted assumption that MAR elements are most effective when they flank a transgene (see US Pat. No. 5,731,178). Stability of expression means that the DNA of interest, such as a transgene, is expressed by a population of cells even after a period of time, for example, after more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks, at a level comparable (no more than 20%, 10 or 5% less) and, especially, even higher than within two weeks from the start of expression. Often, stable expression is associated with a high percentile (eg, greater than 80%) of cell subpopulations with high levels of expression.

CELLULAR METABOLIC ENGINEERING: SECRETORY PROTEINS

Secretion of heterologous proteins such as IgG is reduced by improper polypeptide processing and low IgG production in cultured cells such as CHO cells.

It was observed that the expression of stress-induced chaperones such as BiP was induced and that the chaperone was correctly localized to the ER and was able to interact with IgG precursor chains. However, IgG containing certain variable sequences, such as those found in infliximab, were however not processed and collected correctly, resulting in low secretion. Consequently, activation of the unfolded protein response (UPR) in these cells remained ineffective to restore significant levels of immunoglobulin.

SRP14 has been shown to be involved in a molecular step of the secretory pathway that is rate-limiting in CHO cells overexpressing exogenous protein. Notably, this rate-limiting step is also observed for the easily expressed trastuzumab, which easily leads to the production of clones with high levels of expression. This conclusion followed the discovery that expression of human SRP14 readily restored expression of LP clones, but it also increased secretion of readily expressed IgG. Expression of SRP14 was found to enhance the processing and availability of LC and HC precursors and produce comparable levels of secretion of both types of IgG. Overall, it has been demonstrated that SRP14 may be generally limiting when secreted proteins such as IgG are overexpressed in CHO cells.

The strong effect of SRP14 expression in CHO cells observed in this study was unexpected and suggested that SRP14 causes a prolonged delay in LC elongation in clones that have difficulty producing IgG (Fig. 12B, point 1).

Previous research has shown that the signal peptide that exits the translating ribosome first interacts with the SRP54 subunit of the SRP particle, whereas association with SRP9 and SRP14 can block further elongation of the nascent polypeptide, resulting in translation arrest (Walter and Blobel 1981). In the second step, the ribosome-nascent polypeptide-SRP complex docks with the ER membrane through interaction with the SR receptor (Gilmore et al., 1982; Walter et al., 1982). The SR can then coordinate the release of SRP from the ribosome-nascent polypeptide complex and the association of the ribosome exit site with the translocon channel through which the nascent nascent polypeptide enters the ER (Lakkaraju et al., 2008). Translation-coupled movement can then resume, resulting in removal of the signal peptide and the synthesis of properly processed and secreted polypeptides.

Proper processing of difficult-to-express IgGs may require an unusually long translational pause if ER docking kinetics may be slower for particular combinations of IgG variable domain and signal peptide sequences due to unfavorable structures of the resulting peptide. Thus, it was believed that, in turn, modulation of translational arrest kinetics by expression of exogenous human SRP14 component improves proper ER docking and relocation of pre-LC to the ER and thus restores efficient signal peptide processing (Fig. 12B, point 2 ). Accordingly, reduction of SRP14 levels in human cells results in a lack of delay in translation elongation in polysomes, which may result in the length of nascent polypeptides being excessively increased beyond a critical value, after which SRP can no longer correctly target the secreted protein to the ER.

Thus, the affinity of SRP14 and possibly also SRP54 in CHO cells for the signal sequences of the heterologous human infliximab protein will be reduced, leading to incorrect docking with the ER and/or elongation of the resulting peptide before proper docking occurs. This will be corrected by overexpressing human SRP14, increasing the period of time during which the stalled ribosome-SRP complex can search for a properly organized docking site on the ER, despite the 'molecular congestion' of overexpressed IgG proteins occurring at the entrance to the ER.

Accordingly, overexpression of SR and translocon, which can increase the capacity of the ER for translocation, also resulted in improved secretion, even in the absence of overexpression of human SRP14. Finally, it was demonstrated that metabolic engineering of the secretory pathway, through coexpression of combinations of human SRP, translocon, and SR subunits, results in further improvements in the cell's ability to secrete the protein, allowing even higher levels of secretion to be obtained. Overall, it is concluded that SRP proteins, their receptors and the translocon may be generally limiting when secreted proteins, such as human immunoglobulins, are overexpressed in CHO cells.

Little is known about the relative abundance of SRP and ER membrane components compared to secreted proteins and ribosomes in different cell types, but translocation defects can potentially occur in cells expressing large amounts of recombinant protein. For example, the SR and/or translocon may become limiting when secreted proteins are expressed at abnormally high levels, or SRP14 may occur in CHO cells at substoichiometric levels compared to other SRP subunits. Accordingly, SRP9 and SRP14 are present in 20-fold excess over other SRP proteins in primate cells but not in mouse cells, and overexpression of human SRP14 in normal human cells does not increase the efficiency of alkaline phosphatase secretion. Moreover, human SRP14 is larger than rodent SRP14 because it contains an alanine-rich tail at the C terminus, which is not found in rodent SRP14. Thus, incorporation of the larger human SRP14 into CHO SRP may result in the formation of a functional SRP chimera with higher activity, with a dominant-positive effect.

The discovery that expression of cytosolic components of SRP, such as SRP14, leads to efficient processing and secretion of overexpressed proteins in CHO cells indicates a bottleneck that can be exploited to increase recombinant protein yield. This difficulty limits the expression of various and unrelated IgGs and possibly also a variety of other monoclonal antibodies and derivatives that constitute by far the most common class of recombinant therapeutic proteins.

The analysis of secretion intermediates and possible cellular stress responses, followed by a systematic search for the upstream limiting acting factors that cause this stress response, and then ultimately the development of the secretion metabolism of CHO cells, led to a better understanding of the metabolic limitations in these cells and how to circumvent them .

Heterologous expression of SRP14 restores LC secretion and processing

IgG clones HP (high production) and LP (low production) were cotransfected with a vector encoding the SRP14 component of the SRP and a neomycin resistance plasmid. Individual cells from the neomycin-resistant cell population were separated using the serial dilution method and then examined for growth and immunoglobulin secretion in a batch of shaken culture dishes. SRP14-expressing LP clone-derived subclones secreted significantly higher amounts of antibody than the parent clones in the entire culture and produced immunoglobulin titers similar to those of the SRP14-expressing HP subclones (Fig. 8A and 8B, left panels).

Expression of SRP14 had no effect on cell viability, but it appeared to slow and prolong HP cell culture growth to similar cell densities (Figures 8A and 8B, right panels). Culture supernatants of various subclones were collected and their antibody concentrations were analyzed. In fig. Figure 8C shows that expression of SRP14 increased secretion from LP cells, resulting in a 7-fold increase in specific IgG productivity. Moreover, exogenous expression of SRP14 also improved IgG secretion from HP subclones, resulting in a 30% increase in specific productivity. Notably, individual SRP14-expressing subclones secreted hard- and easily-expressed IgG at essentially identical mean levels, with median specific productivity values exceeding 30 picograms per cell per day (pcd). These very high levels of IgG secretion were maintained over more than 6 months of culture, indicating that this is a permanent property of SRP14-expressing cells.

To further investigate the relationship between SRP14 expression and IgG productivity, SRP14 mRNA levels in 5 individual SRP14-expressing LP subclones were analyzed by comparative qPCR. In fig. 8D shows that the subclones overexpressed SRP14 at levels ranging from 50- to nearly 200-fold compared to those of endogenous SRP14 mRNA in CHO cells. This was accompanied by a 4-6-fold improvement in IgG secretion compared to the LP control cell clone. It is noteworthy that the highest specific productivity was obtained in a subclone with an intermediate level of increase in SRP14 expression, approximately 100 times relative to the level of endogenous SRP14 mRNA in CHO cells (SRP14-LP subclone E, results not shown). This implies a relationship between the level of SRP14 overexpression and the specific productivity of IgG up to a threshold level of SRP14 corresponding to a 100-fold increase relative to the endogenous expression level. This suggested that other components of the secretory pathway may in turn become limiting at very high levels of SRP14, and that balanced expression of components of this pathway may be required for optimal IgG expression.

To test whether the increased specific productivity obtained during clonal cell line evaluation could be applied to the production process, the best HP- and LP-expressing subclones of SRP14 were examined in shaken culture dishes under fed-batch culture conditions (i.e. LP subclone E and HP subclone B, see Fig. 8). The SRP14 LP-expressing subclone produced large numbers of viable cells and immunoglobulin titers similar to the SRP14 HP-expressing subclone, with a maximum cell concentration of 8 x 10 ⁶ cells/ml and a maximum titer above 2 g per liter at the end of the production run (Fig. 9A and B).

The contribution of SRP14 overexpression to immunoglobulin synthesis was then examined for these two subclones. The results showed that expression of human SRP14 in the LP-derived subclone produced a normally processed and mature LC capable of IgG folding and assembly (Fig. 10A, LP, lane S versus lane -). The migration of free LC was unaffected, indicating that SRP14 expression acted in particular on the misprocessed LC of a difficult to express protein. Surprisingly, expression of SRP14 completely impaired the accumulation of aggregated LC in the Triton X-insoluble fraction (Fig. 9A, lower panel). Expression of the control protein GFP did not improve protein solubility, nor did it restore proper LC processing (Fig. 10A, lane G for LP cells). Expression of SRP14 had no effect on the HC and LC migration pattern obtained for the HP subclone, and little effect was observed on the amount of free chains and fully assembled IgG compared to controls (Fig. 10A, lane G for HP cells).

Cycloheximide-based displacement assays were performed to examine the kinetics of IgG folding and assembly, as well as the fate of IgG aggregates in the SRP14-expressing infliximab-producing subclone. In contrast to parental LP cells containing aggregated LCs unable to participate in IgG assembly, the SRP14-expressing LP subclone no longer accumulated Tx-100-insoluble LCs (Fig. 9B, lower panel). However, free LC remained in small amounts compared to free HC, which was also observed in HP cells, indicating that it was rapidly incorporated into HC-LC dimers and mature IgG and that it may have limited IgG assembly (Figs. 9A and 9B). Taken together, these results suggest that SRP14 may play an important role in LC processing in LP cells and that additional expression of SRP protein may improve the secretion of difficult-to-express and easily-expressible IgG to similar levels.

ER translocation engineering improves recombinant IgG secretion

Because overexpression of heterologous SRP14 increased IgG secretion to this threshold, it was concluded that other components of the secretory pathway that interact directly or indirectly with SRP14 may become limiting in the SRP14-LP subclones. Therefore, we examined whether overexpression of other components of the secretory pathway could also improve IgG expression, either alone or in combination with SRP14. These components included the human SRP9 and SRP54 proteins, which form part of the SRP complex along with SRP14, the SRP receptor (SR) subunits, and the Sec61α, β, and γ translocon subunits.

In the first series of experiments, the best LP clone, namely LP clone E in FIG. 1 were transfected with expression vectors encoding SRP proteins or translocon proteins, alone or in combination. IgG production was then assessed in the resulting polyclonal populations of LP cells during batch culture. Expression of SRP components or translocon proteins increased immunoglobulin secretion by these retransfected polyclonal LP cell populations (Fig. 11A and results not shown). Compared with expressing SRP14 alone, overexpression of combinations of SRP or translocon proteins improved the specific productivity of transfected polyclonal LP cell populations by an additional 20% to 40% (Fig. 11A, comparison of median values). The results clearly indicate that certain combinations were more effective in restoring infliximab secretion than SRP14 expression alone, such as combinations consisting of expression of three SRP polypeptides and its receptor (SR), or coexpression of SR and translocon (Fig. 11A). .

It was also assessed whether the SRP14-expressing LP subclone E could be further optimized by expressing combinations of SR and/or translocon. Compared to 30 pcd for SRP14-LP subclone E, polyclonal cell populations selected after transfection with SR and translocon proteins yielded specific productivity above 60 pcd for the difficult-to-express immunoglobulin (Fig. 10B). It was concluded that vectors expressing the SR and translocon proteins could also be used to produce clones with increased specific productivity compared to SRP14-LP clone E, and that an approach based on a series of sequential rounds of transfection and selection could be used successfully.

CELLULAR METABOLIC ENGINEERING: suppression of the recombination pathway and expression of TEP and functional TEP RNA

To influence recombination, important DNA recombination genes can be silenced. The knockdown targets are genes known from the literature to be important for certain recombinational repair pathways in mammals (Table D). Since most of these genes are essential for cell survival and development, persistent silencing of them can lead to decreased cell viability. Therefore, temporary silencing of these target genes using RNA interference (RNAi) is preferable.

In cells treated with a mixture of siRNAs against factors involved in the first stages of NGSC, i.e. Ku 70, Ku 80, and DNA-PKx (one siRNA duplex per protein), the frequency of NGSC events was significantly lower than in untreated cells. Instead, in these cells, GR was more effective, resulting in a decrease in the ratio of NGSC to GR (from 2.7:1 to 1.3:1). In contrast, in cells treated with siRNA directed against the essential GR factor Rad51, the GR-dependent restoration of GFP levels was almost completely abolished, which increased the NGSC:GR ratio to 5:1. These results appear to indicate that the GR and NGSC reporter assay is sensitive enough to be used in an extrachromosomal form. They also further question the reliability of previously used mutant CHO cell lines (namely, 51D1 cells, originally published as GR-negative cells but apparently capable of GR by this assay; results not shown).

Increased expression and incorporation of GFP in the presence of MAR. In parallel experiments, CHO cells treated with various siRNAs directed against GR or against NGSC were transfected with plasmids containing GFP or MAR-GFP. After two weeks of antibiotic selection, GFP expression and incorporation in these cells was analyzed by FACS and qPCR, respectively. Under all conditions tested, addition of MAR resulted in a 4- to 5-fold increase in GFP expression compared to cells transfected with a plasmid without MAR (Figure 13, Figure 14A), which was consistent with previous reports (Grandjean et al., 2011). This increase was accompanied by an approximately 3-fold increase in the copy number of the integrated GFP in cells transfected with the MAR-GFP plasmid compared with cells that received the vector without MAR (Fig. 13, Fig. 14B). A 2-fold increase in average GFP fluorescence per gene copy was also observed (Fig. 13, Fig. 14C), possibly due to a more favorable localization of the insertion site (eg, in a euchromatin-rich region). Therefore, it can be hypothesized that MAR elements have the ability to target genes to genomic loci that allow gene expression. Combining anti-GR siRNA with MAR (siRNA: RAD51, Rad51C, and Brca1) resulted in a greater than 11-fold change in average GFP expression, whereas anti-GR siRNA alone (siRNA: Rad51) resulted in a less than 9-fold change. Other combinations, such as combining anti-OCSC siRNA with MAR, also resulted in improved expression compared with siRNA alone (results not shown). Accordingly, the combination of different miRNAs (2, 3, 4, 5, 6, 8, 9, 10) to targets in the same or different metabolic pathways, in particular the recombination pathway, is within the scope of the present invention.

No effect of NGSC gene suppression on transgene expression and integration.

Treatment with siRNA against NGSC proteins did not appear to significantly affect stable transgene expression. There was also no significant change in genomic GFP copy number compared to untreated cells (except for cells treated with siRNA against 53BP1, but only in the absence of MAR, possibly indicating an effect not inherent to recombination through the NGSC pathway).

Increased expression and integration of the transgene in the absence of GR factors. In contrast to the downregulation of NGSC factors, the presence of siRNAs against GR proteins often resulted in a significant increase in levels of stable GFP expression compared to untreated cells (with the exception of Brca2, which showed a significant decrease in levels, but again only in the absence of MAR) (Fig. 15A). As with NGSC gene silencing, the presence of MAR resulted in increased levels of stable GFP expression and incorporation, as well as increased expression levels per gene copy. This time, however, silencing of GR factors increased this effect by 5-7 times (the most striking was the suppression of Rad51, resulting in GFP expression levels 7.4 times greater) (Fig. 15A), which may indicate that that GR proteins counteract the positive effect of the MAR element on transgene expression. In the absence of MAR, increased GFP expression correlated with increased GFP copy number in the genome (results not shown). Surprisingly, this was not observed in the presence of MAR, indicating that GR protein silencing did not affect MAR-mediated copy number expansion (except for Rad51D siRNA). This appears to suggest that the absence of a functional GR repair pathway does not increase the number of recombination events driven by MAR. Instead, it stimulates the insertion of the MAR and transgene into a more favorable locus, allowing it to be expressed more efficiently. This view is also supported by the increased expression of individual copies of GFP in the presence of MAR in cells treated with siRNA against GR (Fig. 14C). Another possibility is that the number of plasmid copies integrated into the genome is already at its maximum level in control cells (at the amount of plasmid DNA used here) and cannot be further increased even under conditions more favorable to MAR.

Taken together, these results suggest that the process of MAR-mediated transgene insertion is predominantly mediated by a pathway opposite to homologous recombination, although probably not by the HSCT pathway, since silencing its components did not affect insertion or expression. One might speculate that this alternative pathway is less active in the presence of a functional GR pathway, but becomes more important if the GR is not functioning.

EXPRESSION OF SHRNA AGAINST GR TO INCREASE THE EXPRESSION OF THERAPEUTIC PROTEINS

Three siRNAs targeting Rad51 were converted into shRNA sequences that can form hairpin structures, and the shRNA coding sequence was inserted into a vector carrying the piggyBac transposon under the control of a GAPDH enhancer-CMV promoter fusion followed by MAR X-29, but without antibiotic selection gene. Suspension-adapted CHO-M cells were transfected three times with a donor transposon-carrying plasmid and a transposase expression vector, and 30 individual cell clones were randomly collected using a ClonePix device. The parent cells, as well as the shRNA-expressing cell population and clones, were re-transfected with a GFP-expressing plasmid (namely, the Puro-GFP-MAR X_29 construct), and then puromycin selection was performed on polytonal populations of GFP-expressing cells. Comparison of GFP fluorescence profiles indicated that a higher proportion of cells with medium to high levels of fluorescence (M2 cell population) or very high levels of fluorescence (M3 cell population) were obtained from the shRNA vector-transfected cell population compared to parental cells SNO-M (Fig. 17A).

Several shRNA-expressing clones mediated very high levels of GFP, with more than 80% of the antibiotic-resistant cells being in the M3 subpopulation with high levels of fluorescence 10 days after transfection, such as clone 16 and clone 26 (Fig. 17A and B). High levels of GFP fluorescence were maintained in these two clones after 35 days of additional culture without selection (Fig. 17C). In contrast, clone 17 did not express very high levels of GFP at day 10, and GFP expression appeared to be unstable (Fig. 17B and C). Intermediate levels of expression and intermediate stability were obtained for clone 8 and clone 22. These clones were also transfected with expression plasmids encoding the light and heavy chain of the difficult to express therapeutic antibody infliximab. Still, clones 16 and 26 produced the highest levels of antibody, followed by clones 8 and 22, while clone 17 expressed amounts of immunoglobulin that were close to the 0.5-1 pcd obtained for the parental CHO-M cells (Fig. 17D and results not presented). Cell clones exhibiting the most significantly reduced levels of Rad51 mRNA exhibited high levels of expression of both GFP and the infliximab immunoglobulin, indicating that increased transgene expression resulted from decreased expression of the recombination protein (results not shown).

Thus, higher levels and more stable production of secreted therapeutic proteins, such as infliximab, can be achieved by cells expressing shRNA targeting Rad51 or by cells transiently transfected with siRNA, such as siRNA targeting Rad51.

MATERIALS AND METHODS

Plasmids and DNA vectors

The expression vector carrying the PB transposase, pCS2+U5V5PBU3, contains the coding sequence for the PB transposase flanked by the 5' and 3' untranslated terminal regions (UTRs) of the Xenopus laevis β-globin gene. This plasmid was constructed as described below: a 3'-UTR fragment of 317 bp. from pBSSK/SB10 (kindly provided by Dr. S. Ivies) was inserted into pCS2+U5 (Invitrogen/life Technologies, Paisley, UK) to generate pCS2+U5U3. The PB transposase coding sequence (2067 bp, GenBank accession number: EF587698) was synthesized at ATG:biosynthetic (Merzhausen, Germany) and cloned into the pCS2+U5U3 scaffold between two UTRs. The control vector PB corresponds to the unmodified plasmid pCS2+U5 (Figure 1, left panel).

Vectors carrying the various transposons used in this study were generated by inserting 235-bp PB inverted terminal repeats (ITRs) of 3′ and 5′. and 310 bp, respectively, synthesized at ATG:biosynthetic (Merzhausen, Germany), into the plasmid pBluescript SK (pBSK ITR3'-ITR5', Figure 1, right panel). The puromycin resistance gene (PuroR) was then inserted under the control of the SV40 promoter from plasmid pRc/RSV (Invitrogen/Life Technologies) between the two indicated ITRs. The MAR 1-68 and MAR X-29 elements, puromycin resistance genes, and GFP used in this study were as previously described. Immunoglobulin expression vectors and sequences encoding SRP9, SRP14, SRP54, SRPR-alpha, SRPR-beta, SEC61A1, SEC61B and SEC61G were as described in Le Fourn et al. (Metab, Eng., Epub, February 1, 2013 .)

GFP, immunoglobulin, or secretory proteins were expressed using a eukaryotic expression cassette consisting of the human CMV enhancer and the human GAPDH promoter located 3'-5' of the coding sequence, followed by the SV40 polyadenylation signal, the human gastrin terminator and the SV40 enhancer (see Le Fourn et al., 2013). Expression cassettes and/or MAR elements were inserted between ITR sequences or into a bacterial vector backbone as shown in Figure 1 and the figure legend using standard cloning techniques.

Cell culture and transfection assay

The DG44 CHO cell line was cultured in DMEM:F12 (Gibco) supplemented with hypoxanthine/thymidine (HT, Gibco) and 10% fetal bovine serum (FBS, Gibco). Transfection was performed using PEI (JetPRIME, Polyplus Transfection) following the manufacturer's instructions. Cells were transfected with varying amounts of plasmid DNA containing the PB transposase (ranging from 0 to 1500 ng) for titration experiments or cotransfected with an optimal ratio of 300 ng of plasmid expressing the PB transposase and 300 ng of donor plasmid carrying the transposon. Two days after transfection, cells were transferred to several Petri dishes depending on the experiment. For analysis of unselected transfected CHO cells, cells were subcultured without antibiotic selection for 3 weeks and the percentage of fluorescent cells and the fluorescence intensity of GFP-positive cells were determined by FACS analysis using a CyAn ADP flow cytometer (Beckman Coulter). To analyze gene copy number in unselected cells, stable GFP-positive CHO cells were sorted using FACSArial. For quantitative analysis of antibiotic-resistant colonies, 50,000 transfected cells were plated in 100-mm dishes and subjected to selection with 5 μg/ml puromycin for 2 weeks. Resistant colonies were then fixed and stained with 0.7% methylene blue in 70% EtOH for 10 min, and colonies >0.5 mm in diameter were counted. To examine GFP expression, cells were gated for two weeks before FACS analysis of GFP fluorescence described above.

CHO-M cells were maintained in suspension culture in serum-free SFM4CHO Hyclone medium (ThermoScientific) supplemented with L-glutamine (PAA, Austria) and HT supplement (Gibco, Invitrogen life sciences), at 37°C, 5% CO2 in humidified air. Transposon-carrying donor plasmids were transferred into these cells by electroporation according to the manufacturer's recommendations (Neon devices, Invitrogen). Quantitative analysis of immunoglobulin secretion by batch cultures was performed as described previously (see Le Fourn et al., 2013). Briefly, cell populations expressing immunoglobulins were assessed by batch culture in 50 ml minibioreaction tubes (TPP, Switzerland) at 37°C in a humidified chamber with 5% CO2 for 7 days. Immunoglobulin concentrations in culture supernatants were measured using a sandwich enzyme-linked immunosorbent assay (ELISA).

Gene copy number assays using quantitative PCR (qPCR)

Total DNA was isolated from stable CHO cell populations after transposition assays using the DNeasy Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The copy number of genome-integrated transgenes was assessed using 6 ng of genomic DNA by quantitative PCR using a SYBR Green-Taq polymerase kit from Eurogentec Inc and an ABI Prism 7700 PCR device (Applied Biosystems). Primers GFP-forward: ACATTATGCCGGACAAAGCC and GFP-reverse: TTGTTTGGTAATGATCAGCAAGTTG were used to quantify the GFP gene, while primers B2M-forward: ACCACTCTGAAGGAGCCCA and B2M-reverse: GGAAGCTCTATCTGTGTCAA were used to amplify the beta-2 microglobulin gene. For the amplicon generated with the B2M primers, one hit per haploid CHO genome was found after alignment with our CHO genome assembly using NCBI BLAST software. Since CHOs are nearly diploid cells, it is estimated that B2M is present in 2 copies per genome. Copy number ratios of the GFP target gene were calculated relative to those of the B2M reference gene as previously described.

Sorting and analysis of immunoglobulin-expressing cells

For magnetic sorting of IgG-expressing cells, transfected CHO-M cells were seeded at a cell concentration of 3 x 10 ⁵ cells per ml in SFM4CHO medium (Thermo Scientific) supplemented with 8 mM L-glutamine and 1 x HT supplement (both from Gibco), called full environment. After 4 days in culture, 2 x 10 ⁶ cells were washed, resuspended in PBS and incubated with biotinylated human IgG (KPL216-1006) at a final concentration of 3 μg/ml along with 30 μl of pre-washed streptavidin-coated MyOne T1 Dynabeads (Invitrogen), on on a rotating wheel for 30 minutes at room temperature. The mixture of cells and beads was then placed on a magnet to separate labeled cells from unlabeled cells. The beads were washed 4 times with phosphate-buffered saline (PBS). After a final wash with PBS, beads and cells were resuspended in 500 μl of prewarmed complete medium, transferred to a 24-well plate, and incubated at 37°C with 5% CO2. After 24 h, the polyclonal cells separated by magnetic sorting were separated from the beads and incubation continued until the cells reached a sufficient density for their proliferation in 50 ml TPP centrifugal bioreactors (TECHNO PLASTIC PRODUCTS AG, Switzerland).

Alternatively, two clones were isolated from unsorted and unselected populations expressing each of the three IgGs using the clonePix device. Briefly, semisolid media were used to immobilize single cells, and colonies secreting large amounts of IgG were collected ten days after embedding. These cell lines were subcultured every 3-4 days into centrifugal bioreactors at a density of 3×10 ⁵ cells/ml in peptone-containing growth medium (SFM4CHO, supplemented with 8 mM glutamine, from Hyclone) and incubated in a humidified chamber at 37°C and 5% CO2 when rotating at 180 rpm.

When assessing polyclonal cell populations, IgG titers were determined for cells that were seeded at a concentration of 1×10 ⁵ cells per ml and grown for 6 days in 5 ml of complete medium in 50 ml centrifugal bioreactors. Alternatively, shake flask cultures of clonal populations were inoculated at a density of 3 x 10 ⁵ cells/ml in SFM4CHO medium to begin the fed-batch production process. Fed-batch culture production was carried out in 25 ml culture in 125 ml shake flasks or in 5 ml culture in 50 ml TPP culture tubes in humidified chambers at 37°C and 5% CO2 with shaking at 150 rpm (shaking flasks and centrifuge tubes with a volume of 125 ml). Production was carried out for ten days with feeding 16% of the initial volume of the culture of concentrated feeding with a certain chemical composition (Hyclone, Cell Boost 5, 52 g/l) on days zero, three and six to eight. Glutamine and glucose were not fed during the cultivation process. Viability and viable cell concentration (VCD) in the culture were measured daily using a GUAVA device (Millipore). A dual sandwich ELISA assay was used to determine the concentrations of MAbs secreted into the culture media.

Plasmids and relative quantitation PCR analysis

The cloning vectors used in this study were mammalian expression vectors SLXplasmid_082 from Selexis. The luciferase sequence of the control vector pGL3 (Promega) was replaced with a eukaryotic expression cassette consisting of the human CMV enhancer and the human GAPDH promoter, located 3′-5′ of the EGFP coding sequence, followed by the SV40 polyadenylation signal, the human gastrin terminator and enhancer SV40. Two human MAR-derived genetic elements flanked the expression cassette, and the puromycin resistance gene was expressed under the control of the SV40 promoter, with SLXplasmid_082 differing in the type of MAR element located 3'-5' from the expression cassette (hMAR 1-68 and hMAR X-29; Girod et al., 2007).

Trastuzumab and infliximab heavy and light chain cDNAs were cloned into an expression vector to replace EGFP. A vector carrying both the heavy and light chain expression cassettes of each IgG was prepared by combining the heavy and light chain expression cassettes on a single plasmid vector. The sequences of signal peptides from all heavy and light chains, as well as the constant regions of the light chains, are identical. Heavy chain constant regions differ at several amino acid positions (DEL versus EEM variants).

PCR amplification primers and GenBank accession numbers for the SRP9, SRP14, SRP54, SRPR-alpha, SRPR-beta, SEC61A1, and SEC61B cDNAs are listed elsewhere in this text. PCR products encoding secretory proteins were cloned into a vector to replace the EGFP sequence. When multiple secretory proteins were expressed together, the inverted tail sequences of the piggyBac transposon were inserted into vectors to enclose the expression cassette, and the resulting vectors were cotransfected with an expression vector carrying the piggyBac transposase to improve transgene integration and eliminate the need for antibiotic selection.

A conventional expression vector carrying the PB transposase, which was pCS2+U5V5PBU3 containing the PB transposase coding sequence flanked by the 5' and 3' untranslated terminal regions (UTRs) of the Xenopus laevis β-globin gene, was used in related experiments. This plasmid was constructed as described below: a 3'-UTR fragment of 317 bp. from pBSSK/SB10 was inserted into pCS2+U5 (Invitrogen/Life Technologies, Paisley, UK) to generate pCS2+U5U3. The PB transposase coding sequence (2067 bp, GenBank accession number: EF587698) was synthesized at ATG:biosynthetic (Merzhausen, Germany) and cloned into the pCS2+U5U3 scaffold between two UTRs. The control vector PB corresponded to the unmodified plasmid pCS2+U5.

Vectors carrying different transposons were generated by inserting 235-bp 3'- and 5'-inverted terminal repeats (ITRs) of PB. and 310 bp, respectively, synthesized at ATG:biosynthetic (Merzhausen, Germany), into the plasmid pBluescript SK (pBSK ITR3'-ITR5'). The neomycin phosphotransferase (NeoR) gene under the control of the SV40 promoter from plasmid pRc/RSV (Invitrogen/Life Technologies) was then inserted between the two indicated ITRs. The MAR 1-68 and MAR X-29 elements, puromycin resistance genes, and GFP used in this study were as described previously (Girod et al. 2007; Grandjean et al. 2011; Hart and Laemmli 1998). Immunoglobulin expression vectors and coding sequences for SRP9, SRP14, SRP54, SRPR-alpha, SRPR-beta, SEC61A1 and SEC61B are described herein. The secretory proteins were expressed using a eukaryotic expression cassette consisting of the human CMV enhancer and the human GAPDH promoter located 3'-5' of the coding sequence, followed by the SV40 polyadenylation signal, the human gastrin terminator and the SV40 enhancer. Expression cassettes and/or MAR elements were inserted between ITR sequences or into the backbone of a bacterial vector using standard cloning techniques.

PiggyBac transposon systems, including suitable 3' and 5' ITRs as well as transposase, for example, are available for purchase from SYSTEM BIOSCIENCE.

For relative qPCR analysis, total RNA was isolated from 1 x 10 ⁵ cells and reverse transcribed into cDNA using the FastLane Cell cDNA kit (Qiagen) following the manufacturer's instructions. SRP14 and GAPDH expression were quantified by qPCR using Rotor Gene Q (Qiagen) and LightCycler®480 SYBR Green I Master (Roche), using the primers listed in the accompanying Table S1. SRP14 messenger RNA levels were normalized to those of GAPDH using Rotor-Gene Q Series software (Qiagen).

Cell culture, stable transfection and subcloning of CHO cell lines

Suspension Chinese hamster ovary cells (CHO-K1) were maintained in serum-free SFM4CHO Hyclone medium (ThermoScientific) supplemented with L-glutamine (PAA, Austria) and HT supplement (Gibco, Invitrogen life sciences), at 37°C, 5% CO2 in humidified air. CHO-K1 cells were transfected with trastuzumab or infliximab heavy and light chain expression vectors carrying the puromycin resistance gene by electroporation according to the manufacturer's recommendations (Neon devices, Invitrogen). Two days later, the cells were transferred to T75 plates in medium containing 10 μg/ml puromycin, and the cells were further cultured under selection conditions for two weeks. Stable individual clones of cells expressing IgG trastuzumab and infliximab were then obtained using a serial dilution method, grown, and their growth efficiency and IgG production levels were analyzed. Trastuzumab and infliximab IgG-producing cell clones expressing the highest levels of IgG were selected for additional biochemical experiments. Some of these clones were then cotransfected by electroporation with a vector expressing SRP14 and a plasmid carrying the neomycin resistance gene. Cells were then cultured in medium containing 300 μg/ml G418 for two weeks as described above. Stable clones were isolated by limited dilution and SRP14 expression was confirmed by qPCR assays before growing in culture for biochemical analysis.

Batch culture and fed-batch culture

The growth and production efficiency of individual clones expressing trastuzumab and infliximab was assessed by batch cultivation in a 50 ml minibioreactor (TPP, Switzerland) at 37°C in a humidified chamber with 5% CO2 for 7 days. At day 3, day 4, and day 7 of cell culture, cell concentration and viability were determined using a Guava EasyCyte flow cytometry system (Millipore). The IgG titer in culture supernatants was measured using sandwich ELISA. Cell density (Cv*ml-1) and IgG titer values (µg*ml-1) were plotted on the indicated sampling day during the experiment. The specific productivity of IgG clones expressing trastuzumab and infliximab was determined as the drop in IgG concentration depending on the total number of viable cells (IVCD), calculated on day 3 - day 7 (production phase), and expressed as pg per cell per day (pcd). .

For fed-batch cultures, cells were seeded at a density of 0.3 x 10 ⁶ cells/mL in 125 mL shake flasks in 25 mL of serum-free SFM4CHO Hyclone medium. Cultures were maintained at 37°C and 5% CO2 with shaking. Cultures were fed daily with commercially available Hyclone feed (ThermoScientific). Cell densities and IgG production were assessed daily.

Analysis of protein expression and aggregation

Soluble cytoplasmic proteins were extracted by permeabilizing cells with 1% Triton X-100 in PBS buffer in the presence of a protease inhibitor cocktail (Roche, inc). After incubation for 30 min on ice, cells were centrifuged for 10 min at 14,000 rpm. The supernatant was called the “soluble cytosolic and ER proteins” fraction. The precipitate was dissolved by sonication in Laemmli buffer with urea (62.5 mM Tris, 2% SDS, 8 M urea, 5% glycerol, bromophenol blue dye), which made it possible to obtain an insoluble fraction of aggregated and vesicular proteins. The soluble and insoluble fractions were then diluted with Laemmli buffer with or without 2-mercaptoethanol and boiled for 8 min at 95°C. Samples in reducing and non-reducing buffers were separated on 10% or 4-10% gradient acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively.

The proteins were then transferred to a nitrocellulose membrane. After blocking in 5% milk diluted in TBS-Tween (20 mM Tris, 0.5 M NaCl, 0.1% Tween 20), the membranes were assayed for various proteins using the following primary antibodies: donkey anti-IgG (H+ L) human, conjugated with horseradish peroxidase (JK immunoresearch, No. 709 035 149, 1:5000), rabbit polyclonal antibody against human BiP (Cell signaling, BiP, C50 B12, 1:2000), mouse monoclonal antibody against human CHOP (Cell signaling, CHOP, L63F7, 1:500), goat polyclonal antibody against human GAPDH. After overnight incubation at 4°C, each membrane was treated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Cell signaling, 1:20,000). Proteins specifically recognized by each antibody were detected using enhanced chemiluminescence (ECL) reagents and exposure to ECL film (Amersham Biosciences).

Cycloheximide-based protein displacement experiments

Cycloheximide-based displacement experiments were performed on high (HP) and low (LP) IgG production CHO-K1 clones. Equal numbers of cells were seeded into 6-well plates in complete culture medium supplemented with 100 μM cycloheximide (Sigma). At various time points, cells were harvested and lysed in PBS, 1% Triton X-100. Tx-soluble and insoluble fractions were then separated on a 4-10% acrylamide gel using SDS-PAGE under non-reducing conditions and immunoblotted with anti-human IgG antibody.

Differential detergent fractionation assays

Fractionation of cytosolic and membrane proteins was carried out by differential detergent extraction from cell pellets. Cells were first washed in 1 ml PBS, and plasma membranes of HP and LP cells were permeabilized in KHM buffer (110 mM KAc, 20 mM HEPES, 2 mM MgCl2, pH 7.2) containing 0.01% digitonin (Sigma) for 10 min with or without 1% Triton X-100. Semi-permeabilized cells were washed once in KHM buffer and trypsin was added to a final concentration of 50 μg/ml for 10 min at room temperature to digest soluble proteins. Trypsin digestion was stopped by adding 1 mM phenylmethylsulfonyl fluoride (PMSF) and 4 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). Cells were collected by centrifugation and soluble proteins were extracted in the presence of Triton X-100 and protease inhibitor as described in section 2.4. Laemmli reducing buffer containing 2-mercaptoethanol was added to the pellet and supernatant fractions, which were then subjected to 8% SDS-PAGE. Immunoblotting was performed to detect IgG and BiP proteins.

Protein cross-linking and Western blot analysis

Cells producing low levels of infliximab (LP) were washed once in PBS and incubated with or without 1 mM crosslinker dithiobis(succinimidylpropionate) (DSP) (ThermoScientific) for 30 min on ice. Cross-linking was quenched by incubation with 50 mM Tris-HCl (pH 7.4) for 10 min before protein extraction in PBS buffer containing 1% Triton X-100. After centrifugation for 10 min at 14,000 rpm in a microcentrifuge, the Triton X-100-insoluble fraction or whole protein extract was analyzed by EP-PAGE/SDS under reducing conditions, immunoblotted, and stained with antibodies against BiP and LC. Equal amounts of proteins from Tx-insoluble fractions were analyzed in parallel.

It should be obvious that the methods and compositions of the present invention may be included in various embodiments, only a small number of which are described in this application. It will be obvious to one skilled in the art that other embodiments exist that do not depart from the spirit of the present invention. Thus, the described embodiments are illustrative and should not be construed as limiting.

Claims (35)

1. A recombinant nucleic acid molecule for increasing the efficiency of production of a transgene expressed in a mammalian cell, containing: 5' and 3' transposon-specific inverted terminal repeats (ITRs); at least one nucleic acid sequence encoding a transgene expression/processing protein (TEP) located between said 5' and 3' ITRs and which is under the control of a promoter; And at least one matrix attachment region (MAR), which is located between the 5' and 3' ITR; wherein: said transgene expression/processing protein (TEP) is hSERCA2, hCALR, hSPCA1, hNRF2, hP4HA1, hP4HB, hCOSMC or hPDI; said matrix attachment site (MAR) is selected from SEQ ID NOs: 1 (MAR 1-68), 2 (MAR 1_6), 3 (MAR X_S29), 4 (MAR S4), 5 (chicken lysozyme MAR), or is a designed, specifically rearranged, analogue whose sequence is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences of SEQ ID NO: 1 to 5. 2. The recombinant nucleic acid molecule according to claim 1, which additionally contains at least one transgene located between the specified 5'- and 3'-ITRs, which is under the control of the transgene promoter. 3. An expression vector containing a recombinant nucleic acid molecule according to any one of paragraphs. 12. 4. The vector of claim 3, wherein said vector contains a single MAR element or two or more MAR elements, said element(s) located between said 5' and 3' ITRs. 5. The vector according to claim 3, characterized in that said vector contains two MAR elements, with the first MAR element located in the 3'-5' direction from the TEP, and the second MAR element located in the 5'-3' direction from the TEP, wherein said first MAR element includes a MAR 1_6 element or an element which is at least 80%, 90%, 95%, 98%, 99% or 100% sequence identical to the sequence of SEQ ID NO: 2, in particular, rearranged MARs at based on MARs 1-6, more specifically, elements whose sequences are at least 80%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 8 (MAR 1_6R2), and the second MAR element includes a MAR 1-68 element or an element that is at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identical to SEQ ID NO: 1. 6. The vector of claim 3, wherein said vector comprises a MAR unit, wherein said MAR unit is located in the 5'-3' direction from the TEP, and wherein the MAR unit is MAR 1-68, or a MAR X-29 element, or an element which is at least 80%, 90%, 95%, 98%, 99% or 100% sequence identical to SEQ ID NO: 1 or 3, particularly a rearranged MAR based on MAR 1-68 or MAR X-29, particularly an element which has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6, 7 or 10 (elements MAR 1_68R, 1_68R2 or X_29R3) or SEQ ID NO: 9, and is preferably a MAR X-29 element or an element that is at least 80%, 90%, 95%, 98%, 99% or 100% sequence identical sequence SEQ ID NO: 3. 7. A method for expressing a transgene and transgene expression/processing protein (TEP), including: providing a recombinant mammalian cell containing a transgene, and introducing a vector according to any one of claims. 3-6 into said recombinant mammalian cell to provide expression of said transgene and TEP in said cell, wherein expression of TEP using said vector can increase expression of the transgene in said mammalian cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70%. 8. The method of claim 7, wherein said vector contains a single MAR X-29 element or a nucleic acid sequence that is at least 80%, 90%, 95%, 98%, 99% or 100% identical to the sequence SEQ ID NO: 3, and wherein after more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks of culture, the TEP expressed from said vector increases expression of the gene of interest by at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70%. 9. A recombinant mammalian cell for transgene expression, containing no more than 20, 15, 10 or 5 recombinant nucleic acid molecules according to claim 1 or 2. 10. A recombinant cell according to claim 9, characterized in that said recombinant molecules are preferably integrated into the genome of said cell in the form of single copies. 11. Recombinant mammalian cell for transgene expression, containing: a) a recombinant nucleic acid molecule according to claim 1, where said nucleic acid molecule is integrated into the genome of said mammalian cell; And b) a recombinant nucleic acid molecule containing: (i) at least one transgene of interest; And (ii) a MAR element, where the specified MAR element is selected from SEQ ID NOs: 1 (MAR 1-68), 2 (MAR 1_6), 3 (MAR X_S29), 4 (MAR S4), 5 (chicken lysozyme MAR), or is an engineered, specifically rearranged, analogue that is at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identical to any of the sequences of SEQ ID NO: 1 to 5. 12. A method for transfecting a mammalian cell in vitro, including: transfecting said mammalian cell with at least one, at least two, at least three or at least four recombinant nucleic acid molecules according to any one of claims. 1 and 2. 13. The method according to claim 12, characterized in that transfection with at least one specified recombinant nucleic acid molecule is carried out as a first transfection, and at the same time, as a second subsequent transfection, transfection is carried out with a nucleic acid encoding the transgene. 14. The method according to claim 12 or 13, characterized in that said mammalian cell is a hamster cell. 15. A kit for performing cell transfection, which in one container contains at least one vector containing a recombinant nucleic acid molecule according to any one of paragraphs. 1 and 2, and in an additional container contains instructions for using the specified vector or vectors. 16. The set according to claim 15, characterized in that the said set, in addition to the said two containers, contains in another additional container a vector encoding a suitable transposase. 17. The use of recombinant nucleic acid according to any one of paragraphs. 1 or 2 and/or a recombinant mammalian cell according to any one of paragraphs. 9-11 to increase the integration of the transgene into the genome of the host cell and/or the expression of the transgene in vitro. 18. A recombinant mammalian cell for transgene expression that belongs to a cell line and contains: A) at least one recombinant nucleic acid molecule encoding a transgene expression/processing protein (TEP), wherein said TEP is hSERCA2, hSPCA1 or hNRF2; And B) a recombinant nucleic acid molecule containing: (i) at least one transgene of interest; And (ii) MAR element. 19. The recombinant mammalian cell of claim 18, wherein said at least one transgene expresses a therapeutic protein selected from an immunoglobulin, a hormone such as erythropoietin, or a growth factor, and wherein, optionally, in said recombinant mammalian cell integration and/or expression transgene is increased compared to a cell not containing the specified recombinant nucleic acid molecule(s). 20. Recombinant mammalian cell according to any one of paragraphs. 18, 19, wherein said cell is a primary stem cell, a hamster cell, such as a CHO cell, or a human cell, such as a HEK293 cell.

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