# US 11,898,154 B2 · Enhanced transgene expression and processing

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- Number: US 11,898,154 B2
- Document ID: US11898154B2
- Status: Granted
- Kind: B2
- Application number: 14/761,460
- Filed: 2014-02-01
- Published: 2015-12-17
- Granted: 2024-02-13
- Earliest claimed: 2013-02-01
- Assignees: Selexis S.A.
- Applicants: Selexis S.A.
- Inventors: Valerie Le Fourn; Nicolas Mermod; Alexandre Regamey; Montse Buceta; Deborah Ley; Niamh Harraghy; Kaja Kostyrko; Pierre-Alain Giro; David Calabrese
- Web page: https://patentgrep.com/patent/US11898154B2

## CPC

- C12N 15/85: for animal cells
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/241: {Tumor Necrosis Factors}
- C07K 16/32: against translation products of oncogenes
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C07K 2317/10: characterized by their source of isolation or production
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C12N 2800/107: for mammalian
- C12N 2800/90: Vectors containing a transposable element
- C12N 2830/46: elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Disclosed are constructs and methods for expressing DNAs of interest in particular in non-primate eukaryotic host cells that display advantages with regard quantity and quality of expression including high stability of expression and, if appropriate, transport of the expression product out of the cell.

## Claims

### Claim 1

A recombinant nucleic acid molecule that is part of a transposon vector comprising:
a) a 5′ transposon-specific inverted terminal repeat (ITR) and a 3′ ITR, and
b) located between the 5′ ITR and the 3′ ITR:
(i) at least one nucleic acid sequence encoding a transgene expression processing (TEP) protein under the control of a promoter,
wherein the TEP is selected from the group consisting of human Signal Recognition Particle 14 (hSRP14), human Protein Transport Protein 61α1 (hSec61α1), human Protein Transport Protein 61β (hSec61β), human Protein Transport Protein 61γ (hSec61γ), human Signal Recognition Particle 54 (hSRP54), human Signal Recognition Particle 9 (hSRP9), human Signal Recognition Particle Receptor α (hSRPRα), human Signal Recognition Particle β (hSRPβ), human Calnexin (hCANX), and combinations thereof, and
(ii) a matrix attachment region (MAR) element,
wherein the transposon vector increases an expression of a transgene encoding a product of interest in a mammalian cell by at least 10% relative to a plasmid vector comprising the TEP protein.

### Claim 2 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein said MAR is a singular MAR element and is selected from SEQ ID Nos: 1 (MAR 1-68), or 3 (MAR X_S29), an engineered counterpart thereof, or has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with any one of SEQ ID Nos: 1 or 3.

### Claim 3 (depends on 2)

The recombinant nucleic acid molecule according to claim 2, wherein said singular MAR element is directly upstream of the 3′ ITR.

### Claim 4 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein said nucleic acid molecule is at least 5000, 6000, 7000, 8000, 90000 or 10000 base pairs (bps) long.

### Claim 5 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein the transposon vector further comprises, between the 5′ ITR and the 3′ ITR (iii) an expression cassette, wherein the MAR element is located downstream of the expression cassette but not upstream of the expression cassette.

### Claim 6 (depends on 5)

The recombinant nucleic acid molecule according to claim 5, wherein the expression cassette comprises a transgene promoter.

### Claim 7 (depends on 6)

The recombinant nucleic acid molecule according to claim 6, further comprising, located between the 5′ ITR and the 3′ ITR at least one transgene encoding a product of interest under the control of the transgene promoter.

### Claim 8 (depends on 7)

The recombinant nucleic acid molecule according to claim 7, wherein the MAR element is a singular MAR element.

### Claim 9 (depends on 7)

The recombinant acid molecule of claim 7, wherein the product of interest is an antibiotic resistance protein or an immunoglobulin.

### Claim 10 (depends on 7)

The recombinant nucleic acid molecule according to claim 7, further comprising a polyadenylation signal downstream of the transgene, and the MAR element is a singular MAR element downstream of the polyadenylation signal.

### Claim 11 (depends on 8)

The recombinant nucleic acid molecule according to claim 8, wherein the singular MAR is a MAR 1-68 or a MAR X-S29 element, a MAR element that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID No: 1, or a MAR element that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID No: 3.

### Claim 12 (depends on 8)

The recombinant nucleic acid molecule according to claim 8, wherein said product of interest is a therapeutic protein.

### Claim 13 (depends on 8)

The recombinant nucleic acid molecule of claim 8, wherein the TEP protein corresponds to one or more of the following amino acid sequences: hSRP14 having SEQ ID NO: 13, hSec61α1 having SEQ ID NO:15, hSec61β having SEQ ID NO: 17, hSec61γ having SEQ ID NO: 19, hSRP54 having SEQ ID NO: 21, hSRP9 having SEQ ID NO: 23, hSRPRα having SEQ ID NO: 25, hSRPβ having SEQ ID NO: 27, and hCANX having SEQ ID NO: 29, or amino acid sequences having more than 80%, 90%, 95% or 98% sequence identity with said sequences.

### Claim 14 (depends on 11)

The recombinant nucleic acid molecule according to claim 11, wherein the singular MAR element has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NOs: 6, 7, 9 or 10.

### Claim 15 (depends on 11)

The recombinant nucleic acid molecule according to claim 11, wherein the singular MAR element is a MAR X-S29 element or a MAR element that has at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO. 3.

### Claim 16 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein the promoter of b) is a fusion promoter or a combination of promoters and enhancers, wherein the promoter or promoters are selected from the group consisting of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cytosolic Glyceraldehyde 3-phosphate dehydrogenase (cGAPDH), Simian vacuolating virus 40 promoter (SV40p), Cytomegalovirus promoter (CMVp), Chinese Hamster Ovary elongation factor 1 α (CHO EF1α), Chinese Hamster Ovary Actin β (CHO Actβ), and Chinese Hamster Ovary Heat shock protein family A member 5 (CHO Hspa5), and are optionally followed by a Bovine Growth Hormone polyadenylation (BGH polyA) signal.

### Claim 17 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein the promoter is a fusion promoter or a combination of promoters and enhancers, wherein the promoter or promoters are selected from the group consisting of GAPDH, cGAPDH, CHO Actβ, and CHO Hspa5.

### Claim 18 (depends on 1)

The recombinant nucleic acid molecule of claim 1, wherein the TEP protein corresponds to one or more of the following amino acid sequences: hSRP14 having SEQ ID NO: 13, hSec61α1 having SEQ ID NO: 15, hSec61β having SEQ ID NO: 17, hSec61γ having SEQ ID NO: 19, hSRP54 having SEQ ID NO: 21, hSRP9 having SEQ ID NO: 23, hSRPRα having SEQ ID NO: 25, hSRPβ having SEQ ID NO: 27, and hCANX having SEQ ID NO: 29, or amino acid sequences having more than 80%, 90%, 95% or 98% sequence identity with said sequences.

### Claim 19 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein said MAR is a singular MAR element.

### Claim 20 (depends on 1)

The recombinant nucleic acid molecule according to claim 1, wherein the TEP protein is hSPR14.

### Claim 21 (depends on 20)

A recombinant mammalian cell comprising:
a) at least one first recombinant nucleic acid sequence encoding a TEP protein as part of the vector of claim 20, and
b) a second recombinant nucleic acid molecule comprising:
(i) at least one transgene of interest, and
(ii) optionally, a MAR element.

### Claim 22 (depends on 21)

The recombinant mammalian cell of claim 21, wherein the TEP protein is at least one TEP, wherein the TEP is one or more of hSRP14, hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ, and hCANX.

### Claim 23 (depends on 21)

The recombinant mammalian cell of claim 21, wherein the at least one transgene of interest within the second recombinant nucleic acid molecule encodes a therapeutic protein.

### Claim 24 (depends on 21)

The recombinant mammalian cell of claim 21, wherein the second recombinant nucleic acid molecule comprises a MAR element.

### Claim 25 (depends on 1)

A kit comprising in one container the at least one transposon vector comprising the recombinant nucleic acid molecule according to claim 1, wherein the MAR is selected from any one of SEQ ID Nos:1 or 3, and in the same or a second container, a vector comprising at least one transgene and, in a third optional container a vector encoding a compatible transposase binding to the ITRs and in a further container instruction of how to use the vector or vectors.

### Claim 26 (depends on 25)

The kit of claim 25, wherein more than one type of vector comprising the recombinant nucleic acid molecule is provided in one or more containers and wherein the TEP proteins of said more than one type of vector are at least two of the proteins selected from the group consisting of a chaperone, hSRP14, hSRP9, and hSRP54.

### Claim 27 (depends on 1)

A method for expressing a TEP comprising:
providing a recombinant mammalian cell comprising a transgene and a vector, and cultivating the mammalian cell,
wherein the vector is the transposon vector according to claim 1 which expresses the TEP protein.

### Claim 28 (depends on 27)

The method of claim 27, wherein the transposon vector comprises a singular MAR X-S29 element and/or a nucleic acid sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO. 3 and wherein, after more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks of cultivation, the TEP protein increases the expression of the transgene by at least 20%.

### Claim 29 (depends on 1)

A recombinant mammalian cell comprising not more than 20, 15, 10 or 5 of the recombinant nucleic acid molecules of claim 1,
wherein the recombinant nucleic acid molecules are optionally integrated into the genome of the cell as single copies and optionally, wherein the cell is a primary stem cell, a CHO cell, or a HEK293 cell.

### Claim 30 (depends on 29)

The recombinant mammalian cell of claim 29, wherein said recombinant nucleic acid sequence encoding the TEP protein within said cell comprises nucleic acid sequences encoding:
one or more of the following proteins of the protein secretion pathway hSRP14, hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ, and hCANX.

### Claim 31 (depends on 1)

A method for transfecting mammalian cells comprising:
transfecting said mammalian cells with
(i) at least one, at least two, at least three or at least four of said recombinant nucleic acid molecules of claim 1 each encoding a TEP protein; and
(ii) a nucleic acid molecule encoding at least one transgene; and
(iii) a nucleic acid molecule encoding a transposase; wherein the transposase recognizes the 5′ and the 3′ ITR.

### Claim 32 (depends on 31)

The method of claim 31, wherein the cell is transfected with at least two of said recombinant nucleic acid molecules encoding one, two or three of the TEP proteins selected from the group consisting of hSRP14, hSec61α1, hSec61β, hSec61γ, hSRP54, hSRP9, hSRPRα, hSRPβ, hCANX, and combinations thereof.

### Claim 33 (depends on 31)

The method of claim 31, wherein the vectors are co-transfected.

### Claim 34 (depends on 33)

The method of claim 33, wherein the recombinant nucleic acid molecules encoding the TEP protein encode TEP proteins hSRP14, hSRP9 and hSRP54.

### Claim 35 (depends on 31)

The method of claim 31, wherein said mammalian cells are transfected a second time.

### Claim 36 (depends on 31)

The method of claim 31, wherein at least 30%, 40% or 45% of said mammalian cells express said protein of interest following transfection.

### Claim 37 (depends on 31)

The method of claim 31, wherein said protein of interest is an immunoglobulin, hormone, cytokine or growth factor.

### Claim 38 (depends on 31)

The method of claim 31, wherein said recombinant nucleic acid molecules encoding a TEP protein of (i) further comprise a selection marker; and
wherein the nucleic acid molecule encoding at least one transgene of (ii) is expressed
a) without selection for said marker, or
b) with selection for said marker.