WO2011113841A1

WO2011113841A1 - Eukaryotic vector

Info

Publication number: WO2011113841A1
Application number: PCT/EP2011/053910
Authority: WO
Inventors: Robert Steinfeld
Original assignee: Robert Steinfeld
Priority date: 2010-03-16
Filing date: 2011-03-15
Publication date: 2011-09-22

Abstract

The invention relates to an expression vector for an animal cell comprising, (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed and, (iii) a first selection gene, (b) a second transcription unit with, (iv) a second promoter, (v) a second gene to be expressed and (vi) a second selection gene, (c) wherein said first and second selection genes differ and (d) wherein an internal ribosome binding entry site (IRES) is located between at least one of said genes to be expressed and said selection genes.

Description

EUKARYOTIC VECTOR

FIELD OF THE INVENTION

The present invention is in the field of molecular biology, more particularly in the field of molecular cloning techniques and especially in the field of vectors for transfer of genes into an organism and expression thereof. BACKGROUND

Expression of human proteins, for therapeutic applications in particular, requires the protein to have the native form or a form that comes as close as possible to the native form. Frequently, Chinese hamster ovary (CHO) cells are used. This is a cell line derived from the ovary of the Chinese hamster. They are often used in biological and medical research and commercially in the production of therapeutic proteins. They were introduced in the 1960s and grow as a cultured monolayer. They require the amino acid proline in their culture medium. CHO cells are used in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. Today, CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics. However, these cells do not produce a native protein as a human cell line would.

In contrast, Human Embryonic Kidney 293 cells, also often referred to as HEK 293, 293 cells, or less precisely as HEK cells are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK 293 cells are easy to grow and transfect readily and have been widely-used in cell biology research for many years. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. The widespread use of this cell line is due to its extreme transfectability by the various techniques, including calcium phosphate method, achieving efficiencies approaching 100%. An important variant of this cell line is the 293T cell line that contains, in addition, the SV40 Large T-antigen, that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of the desired gene products. However, long-term expression in HEK 293 cells usually requires repetitive transient transfections that are influenced by the used DNA as well as by the condition of the cells. Hence, there is a lack of continuous high expression systems to produce proteins reliably in HEK 293 cells. Further, there is a need for a vector that will enable rapid and stable overexpression of the gene of interest in eukaryotic/mammalian cells without the necessary requirement of time-consuming sub-cloning.

SUMMARY OF THE INVENTION

The invention relates to an expression vector for an animal cells comprising, (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed and, (iii) a first selection gene, (b) a second transcription unit with, (iv) a second promoter, (v) a second gene to be expressed and (vi) a second selection gene, (c) wherein said first and second selection genes differ and (d) wherein an internal ribosome binding entry site (IRES) is located between at least one of said genes to be expressed and said selection genes.

The invention also relates to an expression vector for an animal cells comprising, (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed, and (iii) a first selection gene, (b) a second transcription unit with (iv) a second promoter, and (v) a second gene to be expressed, and (c) a third transcription unit with (vi) a third promoter, and (vii) a second selection gene, wherein said first and second selection genes differ; wherein an internal ribosome entry site (IRES) is located between said first gene to be expressed (ii) and said first selection gene (iii).

Furthermore, the present invention relates to a host cell comprising a vector according to the present invention, preferably a nucleic acid expression vector for an animal cell, said vector comprising, (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed, and (iii) a first selection gene, (b) a second transcription unit with (iv) a second promoter, and (v) a second gene to be expressed, and (c) a third transcription unit with (vi) a third promoter, and (vii) a second selection gene, wherein said first and second selection genes differ; wherein an internal ribosome entry site (IRES) is located between said first gene to be expressed (ii) and said first selection gene (iii); and wherein the host cell is a HEK293 cell. The invention also relates to a method of expressing and isolating one or more proteins in a eukaryotic cell, wherein (a) cells are transfected with a vector according to the invention, (b) cells comprising the vector are selected with the aid of one or both of the resistance genes, (c) transcription of the one or more genes of interest is initiated and (d) the expressed protein is isolated.

The invention also relates to a kit comprising vectors according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a nucleic acid expression vector for an animal cell comprising, (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed and, (iii) a first selection gene, (b) a second transcription unit with, (iv) a second promoter, (v) a second gene to be expressed and (vi) a second selection gene, (c) wherein said first and second selection genes differ and (d) wherein an internal ribosome binding entry site (IRES) is located between at least one of said genes to be expressed and said selection genes.

Furthermore, the present invention relates to a host cell comprising a vector according to the present invention, preferably a nucleic acid expression vector for an animal cell, said vector comprising, (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed, and (iii) a first selection gene, (b) a second transcription unit with (iv) a second promoter, and (v) a second gene to be expressed, and (c) a third transcription unit with (vi) a third promoter, and (vii) a second selection gene, wherein said first and second selection genes differ; wherein an internal ribosome entry site (IRES) is located between said first gene to be expressed (ii) and said first selection gene (iii); and wherein the host cell is a HEK293 cell.

Herein, a transcription unit comprises a promoter, a gene to be expressed and a selection gene. The gene to be expressed may be from any organism, preferably eukaryotic, more preferably human or mammalian. In one embodiment of the present invention the first and/or second promoter and/or third promoter are independently selected from the group consisting of SV 40 early promoter, CMV-derived promoter and elongation factor 1 alpha (EF-l a)-derived. In a further preferred embodiment the first promoter is an EF- la-derived promoter and the second promoter is a CMV-derived promoter and the third promoter is a SV40 early promoter.

Preferably, the first and/or second selection gene is a resistance gene.

Preferably, the first and second genes to be expressed are the same genes. In such a setup expression results are very good.

Preferably, the vector is a plasmid and additionally comprises (i) a bacterial origin of replication, (ii) a bacterial selection gene and/or the lac Z gene. Upon transfection into a eukaryotic cell, chromosomal integration ideally occurs. In a preferred embodiment the bacterial origin of replication is a pUC 18 derived origin (pUC ori). In a further preferred embodiment the bacterial selection gene is an ampicillin resistance gene (AmpR).

The first and/or second resistance genes are preferably selected from the group of resistance genes comprising dihydrofolate reductase (DHFR), zeozin-, puromycin- (Pac), neomycin-, hygromycin-, and histidinol-resistance gene. DHFR and Pac-resistance are very preferred. First and second resistance gene are preferably different. In a further preferred embodiment the first resistance gene is the PacR-gene and the second resistance gene is a neomycin-resistance gene. Preferably, the first and/or second promoter is selected from the group of SV 40 early promoter, CMV-derived promoter and elongation factor 1 alpha. Others as listed in the "Eukaryotic Promoter Database" (www.epd.isb-sib.ch/^'). In one embodiment in each transcription unit the gene to be expressed is located up-stream of the resistance gene. In a further preferred embodiment in the first transcription unit the first gene to be expressed is located up-stream of the first resistance gene, and the second transcription unit is located up-stream of the third transcription unit.

In one embodiment the first and second transcription unit comprise a transcription terminator such selected from the group of human growth hormone polyadenylation signal, bovine growth hormone polyadenylation signal (bGH PA) and SV40 virus polyadenylation signal(SV40 AP). In a preferred embodiment the first and second and third transcription unit comprise a transcription terminator independently selected from the group comprising human growth hormone polyadenylation signal, bovine growth hormone polyadenylation signal (bGH PA) and SV40 virus polyadenylation signal (SV40 AP). In a preferred embodiment of the present invention the first and the third transcription unit comprise each a SV40 virus polyadenylation signal (SV40 AP) and the second transcription unit comprises a bovine growth hormone polyadenylation signal (bGH PA).

In a preferred embodiment the vector has the elements shown in Fig. 3 more preferably even in said orientation. The invention also relates to a method of expressing and isolating one or more proteins in a eukaryotic cell, wherein (a) cells are transfected with a vector according to the invention, (b) cells comprising the vector are selected with the aid of at least one of the resistance genes, (c) transcription of the one or more genes of interest is initiated and (d) the expressed protein is isolated. Preferably, the transfected cells are eukaryotic, more preferably of human origin. One example are HEK 293 cells. Preferably the cells are selected with the aid of both genes. Preferably these differ.

Furthermore, the present invention relates to a method of expressing and isolating one or more proteins in a host cell according to the present invention, the method comprising the steps of: (a) providing a host cell according to the present invention, (b) selecting the host cells with the aid of at least one of the resistance genes, and (c) initiating transcription and expression of the one or more genes of interest, and (d) isolating the expressed protein. SEQ ID NO. 1 discloses the sequence of the original pEF-Tl vector sequence. SEQ ID NO. 2 discloses the pIRES-T3 vector sequence. SEQ ID NO. 3 discloses the pEF-TT vector sequence variant with two Notl sites (8664 bp). SEQ ID NO. 4 discloses the pEF- TT vector sequence variant with one Notl site (8645 bp). SEQ ID NO. 3 and 4 are preferred sequences of nucleic acids according to the invention.

Ideally, the transfected cell is a eukaryotic cell. Preferably, the cell is selected from the group comprising human embryonic kidney cells, such as HE 293, Hela cells, CHO cells, BHK cells, COS cells, and others see ATCC catalogue:

(www, atcc. org CulturesandProducts/CellBiolo gv/CellLinesandH ybridomas/

In a very preferred embodiment of the present invention the host cell is a HEK 293 cell.

The invention also relates to a kit comprising a vector according to the invention. Furthermore, the invention relates to a kit comprising a host cell according to the present invention, i.e. comprising a vector according to the present invention.

The invention also relates to the use of the vector according to the invention for expressing mammalian proteins, preferably human proteins. The invention also relates to the use of the host cell according to the invention for expressing mammalian proteins, preferably human proteins.

The invention furthermore relates to the use of vector in a method for expressing one or more genes of interest, said vector comprising: (a) a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed, and (iii) a first selection gene, (b) a second transcription unit with (iv) a second promoter, and (v) a second gene to be expressed, and (c) a third transcription unit with (vi) a third promoter, and (vii) a second selection gene, wherein said first and second selection genes differ; wherein an internal ribosome entry site (IRES) is located between said first gene to be expressed (ii) and said first selection gene (iii).

In a preferred embodiment of the use according to the present invention the expression of the one or more genes of interest are performed in a host cell, preferably in a host cell selected from the group comprising of human embryonic kidney cells, such as HEK 293, Hela cells, CHO cells, BHK cells, COS cells, and others see ATCC catalogue:

(www.atcc.org/CulturesandProducts/CellBiology/CellLinesandHvbridomas/V In a very preferred embodiment of the use according to the present invention the host cell is a HEK 293 cell.

The embodiments of the vector and its components as outlined above apply to the embodiment of the vector of the use according to the present invention. FIGURE CAPTIONS

FIG. 1

The pEF-Tl (4445 bp) vector comprises the following features, a human elongation factor 1 alpha promoter (EF-la promoter (212-1387 bp)), an intron (1423-1556 bp), a multiple cloning site (MCS) from Nhel to Notl (1619-1704 / 1723 bp), an origin of replication derived from the pUC18 plasmid (pUC ori) (2634-3304 bp), a gene for ampicillin resistance (AmpR) (3449-4350 bp). The two Notl sites within the MCS are 19 bp apart and flank a Xhol and a Xbal site. The usage of the Xhol or the Xbal site for subcloning of a gene of interest will increase the length of the construct by 19 bp difference.

FIG. 2

The pIRES-T3 (6830 bp) vector has the following features, an internal ribosomal entry site (IRES): 23-582 bp a gene for puromycin acyl-tranferase (PacR) (601-1200 bp), a SV40 polyadenylation site (SV40 PA) (1439-1569 bp), a cytomegalovirus promoter (CMV promoter) (1789-2376 bp), a multiple cloning site (MCS) from Nhel to Pmel site (2453- 2530 bp), a bovine growth hormone polyadenylation site (bGH PA) (2652-2876 bp), an SV40 promoter (SV40 prom.) (3255-3598 bp), a gene for neomycin resistance (NeomycinR) (3660-4454 bp), an SV40 polyadenylation site (SV40 PA) (4628-4758 bp), an origin of replication derived from the pUC18 plasmid (pUC ori) (5141-5811 bp), a gene for ampicillin resistance (AmpR) (5956-6830 bp).

FIG. 3

The pEF-TT vector exists in two variants that differ by 19 bp lengths (total 8645 bp or 8664 bp). The two Notl sites within the MCS1 are 19 bp apart and flank a Xhol and a^¾ l site. The usage of the Xhol or the Xbal site for subcloning of a gene of interest will increase the length of the construct by 19 bp difference. For the description of the features of pEF-TT the sequence ranges are given for both variants and are separated by "/". The pEF-TT vector comprises a human elongation factor 1 alpha promoter (EF-la promoter) (212-1387 bp), an intron (1423-1556 bp), a multiple cloning site 1 (MCS1) from Nhel to NotI site (1619-1704 / 1723 bp), an internal ribosomal entry site (IRES) (1717-2276 / 1736-2295 bp), a gene for puromycin acyl-tranferase (PacR) (2294-2893 / 2313-2912 bp), an SV40 polyadenylation site (SV40 PA) (3132-3262 / 3151-3281 bp), a cytomegalovirus promoter (CMV promoter) (3482-4069 / 3501-4088 bp), a multiple cloning site 2 (MCS2) from Nhel to Pmel site (4146-4223 / 4165-4242 bp), a bovine growth hormone polyadenylation site (bGH PA) (4345-4569 / 4364-4588 bp), an SV40 promoter (SV40 pro.) (4948-5291 / 4967-5310 bp), a gene for neomycin resistance (NeomycinR) (5353- 6147 / 5372-6166 bp), an SV40 polyadenylation site (SV40 PA) (6322-6452 / 6341-6471 bp), an origin of replication derived from the pUC18 plasmid (pUC ori) (6834-7504 / 6853-7523 bp) and a gene for ampicillin resistance (AmpR) (7649-8523 / 7668-8542 bp).

FIG. 4

TPP1 activity of HEK 293 cells transfected with the pEF-TT [2xTPPl] construct. Wild- type (none), vector-transfected (vector) and TPP1 -transfected HEK 293 cells were collected, sonicated and cleared by centrifugation. TPP1 activity was measured in cell lysates of wild-type, vector-transfected and mixed TPP1 -transfected or 17 isolated TPP1- transfected clones. The measurements were done at least in duplicates. All transfected clones showed a significant increase in TPP1 activity when compared to wild-type or vector-transfected HEK 293 cells. Results are given as mean (column) and standard deviations (error bars).

EXAMPLES

Creating pEF-TT from pEF-Tl and pIRES-T3

The vector system according to the invention comprises for example the two vectors pEF- Tl (Fig. 1) and pIRES-T3 (Fig. 2) that are ligated together to form the vector pEF-TT (Fig. 3). From the vector pEF-Tl the DNA fragment from the Pvul site to either of the two Noil sites comprising part of the ampicillin resistance, the human elongation factor 1 alpha promoter (EF- la promoter), the intron, and the multiple cloning site (MCS) is inserted into the vector pIRES-T3 using the Pvul and the Notl site. Prior to this ligation either two different genes (e.g. gene X and gene Y) or two inserts of the same gene of interest are cloned into the multiple cloning sites of pEF-Tl and pIRES-T3, respectively. Both constructs, pEF-Tl and pIRES-T3, are digested with the two restriction enzymes Notl and Pvul and the resulting fragments are separated by standard agarose gel electrophoresis. The two largest fragments of each digestion are ligated together to form the construct pEF-TT containing two independent transcription units that are located consecutively.

Application of the pEF-TT vector according to the invention for the expression of the complete human TPP1 (tripeptidyl peptidase 1)

The cDNA of the complete human TPP1 (tripeptidyl peptidase 1) coding sequence was cloned into the vectors pEF-Tl and pIRES-T3. Both constructs were digested with Notl and Pvul and the largest fragment of both restriction digests were ligated together resulting in the construct pEF-TT[2xrPPi]. This plasmid and the vector pEF-TT itself were linearized with Pvul and purified before lipofection with HEK 293 cells was performed using the QIAGEN Effectene system according to the supplier's protocol. Selection was started 48h after transfection by addition of ^g/mL of puromycin plus 500 μg/mL of G418 final concentration, respectively. Stable colonies were picked after 12 days of selection and further expanded before analysis by enzymatic testing with the fluorogenic probe Ala-Ala-Phe-amido-methyl-coumarine (AAF-AMC) as described by Vines and Warburton, Biochim Biophys Acta. 1998, 1384(2):233-342. The remaining colonies were expanded as mixture of transfected cells and also tested for enzymatic activity. TPP1 activity of isolated clones overexpressing TPP1 in HEK cells varied between 14- to 32-fold of wild-type or vector transfected HEK 293 cells (Figure 4). The mixture of transfected HEK 293 cells showed a 22-fold higher TPPl activity than wild-type or vector transfected HEK 293 cells. These results prove that this novel expression system enables rapid and stable over expression of the gene of interest in eukaryotic cells without the requirement of time-consuming subcloning.

Claims

A host cell comprising a nucleic acid expression vector for an animal cell, said vector comprising,

a. a first transcription unit with, (i) a first promoter, (ii) a first gene to be expressed, and (iii) a first selection gene,

b. a second transcription unit with (iv) a second promoter, and (v) a second gene to be expressed, and

c. a third transcription unit with (vi) a third promoter, and (vii) a second selection gene,

wherein said first and second selection genes differ;

wherein an internal ribosome entry site (IRES) is located between said first gene to be expressed (ii) and said first selection gene (iii); and

wherein the host cell is a HEK293 cell.

The host cell according to claim 1, wherein the first and/or second selection gene is a resistance gene.

The host cell according to claims 1 or 2, wherein the first and second genes are to be expressed are the same genes.

The host cell according to any of claims 1 to 3, wherein the vector is a plasmid and additionally comprises (i) a bacterial origin of replication, (ii) a bacterial selection gene and/or optionally the lac Z gene.

The host cell according to any of claims 1 to 4, wherein the first and/or second resistance genes are selected from the group of dihydrofolate reductase (dhfr), zeozin, puromycin, neomycin, hygromycin, histidinol.

The host cell according to any of claims 1 to 5, wherein the first and/or second promoter and/or third promoter are independently selected from the group consisting of SV 40 early promoter, CMV-derived promoter and elongation factor 1 alpha (EF- la)-derived promoter. The host cell according to any of claims 1 to 6, wherein in the first transcription unit the gene to be expressed is located up-stream of the resistance gene.

The host cell according to any of the above claims, wherein the first and second and the third transcription unit comprise a transcription terminator selected from the group of human growth hormone polyadenylation signal, bovine growth hormone polyadenylation signal and SV40 virus polyadenylation signal.

Method of expressing and isolating one or more proteins in a host cell according to any one of claims 1 to 8, the method comprising the steps of:

a. providing a host cell according to any one of claims 1 to 8,

b. selecting the host cells with the aid of at least one of the resistance genes, and c. initiating transcription and expression of the one or more genes of interest, and d. isolating the expressed protein.

A kit comprising a host cell according to any one of claims 1 to 8.

Use of vector in a method for expressing one or more genes of interest, said vector comprising:

wherein said first and second selection genes differ;

wherein an internal ribosome entry site (IRES) is located between said first gene to be expressed (ii) and said first selection gene (iii). 12. The use according to claim 11, wherein the expression of the gene of interest is performed in a host cell, said host cell being a HE 293 cell.