RU2425882C1 - Method of creating transgene mammal cell lines with stable and high level of transgene protein expression - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: mammal cells are transformed by an expression vector containing a reporter gene, a cytomegalovirus promotor and a transcription terminator built above a sequence of the cytomegalovirus promotor.

EFFECT: method can be used for producing mammal cell cultures with a high and stable level of target protein lifelength for large-scale production of recombinant proteins which can be used for the medical and research purposes.

1 dwg, 2 tbl, 3 ex

Description

The present invention relates to the field of biotechnology, in particular to methods of production in bioreactors based on cell cultures and animals of higher eukaryotes, new generation drugs and biological additives, such as antibodies and complex protein molecules.

In the biotechnology industry, more and more drugs and biological additives are produced in bioreactors based on higher eukaryotic cell cultures. Bioreactors for the production of proteins can be any living organism - bacteria, fungi, plants, animals and cell cultures. In modern production, bacteria and yeast are most widely used, which can be efficiently and economically propagated in industrial biotechnological plants. However, for the normal functioning of many human proteins, those changes that occur at the post-translational level are very important: glycosylation, acetylation, phosphorylation, carboxylation and some other transformations. Currently, many antibodies to proteins that disrupt the proper development of cells are used to treat diseases associated with such disorders. Most antibodies are produced in cell cultures. Therefore, for the production of many proteins, mammalian cell cultures are used in which the correct modifications of proteins occur. However, the low level of productivity, difficulties in organizing industrial production and the high cost of production limit the spread of this method.

Over the past 10 years, transgenic animals have often been used as bioreactors in which a foreign gene is expressed in mammary epithelial cells and its product is secreted into milk. This gene can be expressed only in the mammary gland and only during lactation, without having side effects on the body of the transgenic animal. In addition, in the mammary gland of transgenic animals, the majority of post-translational modifications of recombinant proteins are performed and the correct folding of proteins is necessary for the manifestation of their biological activity. Currently, permission has been given for the production of the first two products synthesized in the mammary gland: human antithrombin III derived from transgenic goats (GTC Biotherapeutics) and recombinant C1 inhibitor (Pharming Group NV) produced by transgenic rabbits.

There are several basic schemes for producing stable transgenic cultures of cells and transgenic animals. For the transformation of cells (upon receipt of cell cultures) or for microinjection of DNA into a fertilized egg (upon receipt of transgenic animals), linear DNA containing the gene construct is used. This method allows embedding large DNA fragments larger than 20 kb in the genome, which is of great importance, since for the efficient expression of many genes, in addition to coding regions, the presence of introns and extended 5'- and 3'-untranslated regions . However, this method has several disadvantages. When transgenes are obtained using linear DNA, multicopy tandem integrations of constructs into the genome are usually observed, which leads to the formation of regions containing multiple repeats of the construct. It is known that repetitive DNA sequences often form inactive chromatin, the negative effect of which is amplified over several generations. As a result, after several generations after receiving the original transgenic animal or cell line, the expression of transgenes can attenuate. At the same time, the formation of a heterochromatin zone on repeats of transgenes can adversely affect the activity of host genes located nearby, which with a high degree of probability will lead to a decrease in producer survival. Reduced survival is a particularly significant negative factor when large transgenic animals are used as bioreactors, the cost of obtaining which is extremely high (from $ 20,000 to $ 300,000) and takes several years.

In recent years, a new scheme for producing transgenic animals and cell cultures using retroviruses, primarily lentiviruses, has appeared. One of the advantages of retroviral vectors is a significant increase in the efficiency of obtaining transgenic cell lines and animals. When using retroviral vectors, single or low-copy integrations into genome regions transcribed or containing active chromatin are observed. As a result, this positively affects the level of transgene transcription. However, on the other hand, the low copy number of the transgene does not allow to obtain a significant level of expression. In addition, even with only two or three copies of the transgene, the effect of co-repression is often observed, which leads to gradual methylation of DNA and modifications of the histones characteristic of inactive chromatin and, as a consequence, repression of the transgene (Yao et al., 2004 [4] ) It should be noted that the capacity of retroviral vectors is only 7–9 kb, which is determined by the size of the viral particle into which the infectious DNA is packed. Thus, the viral vector allows you to use mainly only cDNA genes that are expressed much less efficiently than genes containing introns. In addition, great restrictions are placed on the size of regulatory elements that can be used to increase the level of transgene expression.

Research on the improvement of transgenic vectors is carried out in many laboratories and firms in the world, especially in Western Europe and the USA. Recently, work on the creation of new vectors began to be actively conducted in China and India. In the Russian Federation, there is still no developed biotechnological industry and, therefore, the development of such vectors is almost not carried out. An exception may be work on order from foreign biotechnology companies. It is undoubted that the improvement of vectors can reduce the cost of creating an effective producer and industrial production of the product by orders of magnitude.

The main problem in achieving effective expression of proteins in mammalian cell and mammary gland culture is the unstable, low expression of the transgene when it is integrated into the recipient's genome. Such repression is a consequence of the negative influence of surrounding chromatin and regulatory elements, which in general can be considered a consequence of the genome's protective reaction to the integration of foreign information, the expression of which must be suppressed. Thus, it is extremely urgent to use regulatory elements in expression vectors that can support the efficient operation of the gene encoding the target protein.

In order to increase the efficiency of transformation and increase the stability of transgene expression since the beginning of the 90s, DNA sequences, usually A / T-rich, which, as shown in in vitro experiments, interact with the nuclear matrix fraction (MAR, matrix attachment region) are widely used . The existing model suggests that MAR, interacting with proteins of the nuclear skeleton, reduces the dependence of the expression of genes flanked by MAR on the negative effect of surrounding chromatin.

Also known insulators are used to protect transgene transcription from repression and the negative effect of the surrounding genome (Recillas-Targa et al., 2004 [3]; Kwaks and Otte, 2006 [2]). So, the Invitrogen company used a chicken HS4 insulator (1.2 kb), found at the boundary of the β-globin domain. Typically, two copies of the HS4 insulator are placed in front of the gene. In some cases, combinations of the known MAR and HS4 insulator or multimerization of its core sequence (500 bp) are used (Recillas-Targa et al., 2004). Such elements increased both the efficiency of transgene production and the level of expression of the transgene itself. However, far from all studied cell cultures and HS4 organisms, the insulator works effectively.

Millipore-Bioprocess has developed regulatory elements from promoters of “housekeeping” genes, which should work efficiently at all stages of development and in all cells of the body. It was shown that such elements improve the efficiency of obtaining transgenic cell cultures and make the level of transgene expression more stable. However, the size of such elements is quite large and can reach 2.5 kb, which reduces the efficiency of their use. In addition, it is not yet known how effective these elements are in various cell cultures and organisms.

Finally, using an original test system, elements ranging in size from 500 bp were detected. up to 2000 bp named STAR, which had the ability to block the distribution of heterochromatin (Kwaks et al., 2003 [1]). However, there is no information on further investigation of the elements found to protect transgenes.

Known methods for producing transgenes: for the transformation of cells in the production of cell cultures using linear DNA containing a gene construct. This method allows large DNA fragments to be inserted into the genome larger than 20 kb in size. However, when transgenes are obtained using linear DNA, multiple tandem integrations of constructs into the genome occur, which leads to the formation of regions containing multiple construct repeats. Such repeating DNA sequences often form inactive chromatin, the negative effect of which is amplified over several generations. As a result, several generations after the transgenic cell line is obtained, the transgene expression fades. At the same time, the formation of a heterochromatin zone can adversely affect the activity of genes located nearby, which with a high degree of probability will lead to a decrease in producer survival. There is a scheme for producing transgenic cell cultures using lentiviral vectors (RU 2305708, publ. 10.09.2007). One of the advantages of such vectors is a significant increase in the efficiency of obtaining transgenic cell lines and animals. However, when they are used, single integrations take place in genome regions that are transcribed or contain active chromatin. In addition, activating regulatory sequences that increase the level of transgene expression are usually inserted in the promoter region of the vector itself. As a result, this has a positive effect on the level of heterologous protein production. However, on the one hand, the low copy number of the transgene does not allow to obtain a significant yield of the product. On the other hand, even with an initial high level of transgene expression, the effect of co-repression is subsequently observed, caused by transcription from the active regions of the genome through the transgenic construct integrated into the genome, which leads to a gradual repression of the transgene. Thus, the main problem in achieving effective protein expression in cell culture — unstable, low transgene expression when integrated into the recipient’s genome — remains unsolved,

An extremely urgent task is the use of regulatory elements in expression vectors that can support the efficient operation of the gene encoding the target protein. To date, there is no known universal regulatory element that would work effectively in a variety of cell cultures, with different types of promoters, etc. The described foreign analogues are created from insulator regulatory elements (US 5610053, publ. 11.03.1997) or from activator elements that promote the discovery of chromatin at the transgene integration point (US 6949361, publ. 09/27/2005). However, the ability of some insulators to interact with the nuclear matrix is not needed to create transcriptionally independent domains and to form a barrier between active and inactive chromatin. At the same time, often the repression effects during transgene insertion are associated with transcription passing through the transgene. Insulators and activator regulatory elements are not able to stop such transcription. Thus, the use of transcription terminators in expression vectors should help protect the transgene from the negative transcriptional effects of the genome.

Expression constructs, including the green fluorescent protein gene in plasmid pEGFP-N 1 (Clontech), the mammalian CMV transcription promoter (cytomegalovirus promoter), are often used to transfect mammalian cells (RU 2234530, publ. 08.20.2004). However, all of these plasmids provide the expression of the reporter gene at a high level only during short-term cultivation, and during long-term cultivation, the transgene expression is rapidly attenuated.

Thus, it is an object of the present invention to provide expression vectors for mammalian cell cultures in which transcription terminators terminating genomic transcripts at the sites where the transgene is inserted into the genome will be used to maintain a high and stable level of production of the target protein.

The problem is solved by the creation of transgenic mammalian cell lines producing a protein with a stable and high level of expression, including the transformation of mammalian cells by an expression vector containing a reporter gene, a cytomegalovirus promoter and a transcription terminator inserted above the sequences of the cytomegalovirus promoter.

The present invention can be used to construct transgenic constructs containing efficient regulatory elements in biotechnology, molecular biology, and is of interest for large-scale production of recombinant proteins that can be used for medical and research purposes.

The essence of the described invention is illustrated by the drawing, which shows the design diagrams for testing DNA elements in mammalian cell cultures, where

A - control pEGFP_N1, in which the tested DNA elements are absent;

B - control 2xIns_pEGFP, in which an insulator from the β-globin chicken locus is inserted above the cytomegalovirus promoter;

C, D - constructions of SV-40_pEGFP and β_pEGFP with DNA elements - transcription terminators built up above the cytomegalovirus promoter.

Example 1. Construction of recombinant plasmids.

The gene of the green fluorescent protein EGFP (Enhanced Green Fluorescent Protein), controlled by the cytomegalovirus promoter, was used as the reporter gene. It is known that this promoter provides a high level of transcription of the target gene in most known mammalian cell cultures. DNA elements capable of terminating transcription (terminators) were placed in front of the gene promoter.

To evaluate the effectiveness of the use of transcription terminators to maintain a stable and high level of production of the target protein, the following plasmids were used (see drawing).

A) As a negative control, the commercial vector pEGFP_N1 (Clontech) was used, containing the gene for the green fluorescent protein EGFP (Enhanced Green Fluorescent Protein), which is controlled by the cytomegalovirus promoter. This plasmid contains the neomycin resistance gene, which was used as a marker for subsequent selection of recombinant cells.

B) To compare the efficiency of using transcription terminators with the currently known elements capable of maintaining high level transcription of the gene, the 2xIns_pEGFP plasmid was constructed.

To obtain this plasmid into the pEGFP_N1 vector at the Pci I restriction site located in front of the EGFP gene above the cytomegalovirus promoter at a distance of 640 bp from the transcription start point, a 2427 bp sequence was inserted that contained two tandem copies of the insulator from the beta-globin chicken locus. This sequence was obtained from the commercial plasmid pBC 1 (Invitrogen). This element is currently widely used to increase the level of expression of the target gene, since it is believed that it has the ability to isolate the transgene from the influence of surrounding cis-regulatory sequences.

C) The plasmid SV-40_pEGFP, containing the sequence of the transcription terminator of monkey virus 40 (SV-40, simian virus 40) before the EGFP gene above the cytomegalovirus promoter, was obtained by inserting this DNA fragment 868 bp long at the Pci I restriction site.

D) The plasmid β_pEGFP containing the transcription terminator sequence from the human β-globin locus in front of the EGFP gene above the cytomegalovirus promoter was obtained by inserting this DNA fragment 868 bp long. restriction site Pci I.

Example 2. Obtaining transfected cell lines.

Analysis of the expression of the reporter gene was carried out on a culture of Chinese hamster ovary cells (CHO-K1), as the most widely used in biotechnological practice to produce target proteins.

The CHO-K1 cell line was cultured on DMEM medium containing 10% inactivated fetal calf serum, 2 mM L-glutamine, 35 mg / L L-proline, as well as commercial antibiotic Mycokill-AB (PAA Laboratories) at a working concentration. Reseeding of cells was performed 1 time in 5 days at a cell concentration of 10 ⁵ cells per 1 cm ² , with a dilution of 1:20. Cells were cultured at a temperature of 37 ° C, in an atmosphere of 5% CO ₂ and high humidity.

1. Transfection of cells.

This cell culture was transfected with the obtained recombinant plasmids. For more efficient integration of the transgenic construct into the genome, plasmids were linearized using the restriction enzyme ApaLI. Transfection was performed according to the following protocol.

A) Cells in the amount of 70-80% of the monolayer (8 * 10 ⁴ cells per 1 cm ² ) were washed with a culture medium containing no serum.

B) A transfection mixture was prepared: 3-4 μg of linearized plasmid DNA was mixed with 375 μl of serum-free culture medium. In a separate tube with the same amount of a similar medium, commercial Lipofectamine 2000 transfection reagent was mixed, the amount of which was determined by the ratio of 3 μl of reagent per 1 μg of plasmid DNA. After that, the resulting solutions were combined and incubated at room temperature for 30-40 minutes.

B) The resulting transfection mixture was layered on the washed cells.

D) 4-6 hours after the start of transfection, the transfection mixture was replaced with serum DMEM culture medium.

Analysis of the expression level of the reporter gene was assessed by fluorescence intensity on the second day after transfection (36-48 hours) using flow cytofluorimetry. As a negative control, nontransfected CHO-K1 cells were used.

2. Cytofluorimetric analysis.

For cytofluorimetric analysis, the cells were washed with phosphate-buffered saline, trypsinized and removed from Petri dishes. Then washed twice from trypsin and carefully resuspended in phosphate-buffered saline. The resulting suspension (10 ⁶ cells in 1 ml of buffer) was transferred into 5 ml round-bottom tubes.

The voltage of the channels of the flow cytofluorimeter was selected so that the autofluorescence of non-transfected cells was not taken into account during measurements. For this, the voltage in the FL1 channels (in which eGFP fluorescence is detected) and FL2 (which was used as the control empty channel) were changed so that during the measurements the vast majority of cells that did not contain the eGFP gene turned out to be in the region not exceeding the value “10 »On the logarithmic scale, delayed for channels FL1 and FL2.

After calibration, all samples were measured at constant voltage values along the channels FL1 and FL2. So, if eGFP fluorescence was detected in the cells, the appearance of cells in the region exceeding the value “10” was recorded on a logarithmic scale on the FL1 channel, and all quantitative values of the gene expression level were measured in this region.

3. Maintenance of pools of transfected cells.

After transfection, the resulting pools were cultured according to the protocol described above.

In order to get rid of cells that did not contain a transgene, the obtained pools of transfected cells were selected using the commercial antibiotic Geneticin (Invitrogen), administered at a concentration of 800 μg / ml. Since, as a result of suppression of the transcriptional activity of the transgene, the production of the antibiotic resistance gene product decreases over time, after 77 days of cultivation, the antibiotic concentration was gradually reduced to 200 μg / ml.

4. The results of the analysis of the temporal profile of the expression of the EGFP gene in pools of transfected cells.

To determine the degree of transgene suppression in the pools of cells transfected with control (pEGFP_N1 and 2xIns_pEGFP) and experimental (SV-40_pEGFP, β_pEGFP) constructs, the level of reporter gene expression was analyzed every 10-14 days using flow cytofluorimetry.

The average level of EGFP gene expression in a pool of cells at a certain point in time was estimated by the percentage of cells characterized by a fluorescence intensity value greater than "10" on a logarithmic scale, that is, producing a significant amount of this protein.

The ratio of the number of cells expressing EGFP in the pool of cells carrying the construct with this element to the number of expressing EGFP cells with a control plasmid (pEGFP_N1) served as an indicator of the efficiency of using the DNA element inserted before the gene for a high level of protein production.

The results of the analysis of the temporal profile of EGFP gene expression in pools of transfected cells are shown in table 1.

Table 1 The cultivation time, days 2 16 27 39 56 68 77 91 103 114 128 design 2xIns_pEGFP 0.49 2.17 1.43 1.27 1.75 1,50 1.37 1.83 2,36 2.61 4.78 SV-40_pEGFP 0.69 2.24 1.36 1.42 1.66 1.22 1.21 1.78 1.32 1.99 3.16 β_peGFP 0.73 2.86 1.43 1.74 1.77 1.39 1,61 2,56 1.06 2.13 5.83

The results of the analysis of the level of EGFP expression during 128 days of culturing the total population of transfected cells (which corresponds to approximately 30 passages) indicate that the use of the transcription terminator sequence in front of the promoter of the target gene significantly increases the efficiency of protein production in cell culture during long-term cultivation - more than three times at the end of the test period.

Example 3. Obtaining and analysis of stable cell lines.

Analysis of heterogeneous cell pools can lead to deviations due to the fact that the transgene during lipofection integrates multiple copies into different parts of the genome, as a result of which the cells may have different viability, division rate, antibiotic resistance and other characteristics that are important for efficient transgene expression.

Therefore, in practice, the production of recombinant proteins is carried out by selecting stable cell lines obtained from one transfected cell and, thus, characterized by a homogeneous genotype, which eliminates the influence of the above factors and selects the most effective clone.

Individual cell clones were obtained from total cell populations by the method of limiting dilutions after 50 and 75 days of cultivation.

1. Obtaining individual clones.

Cultivated pools were removed from the tablets and diluted in 10 ml of culture medium. After this, the cell concentration was calculated using the Garyaev chamber and additional dilutions were made so that the cell concentration was 2-3 cells per milliliter of medium. The resulting suspension was transferred into 24-well plates at 1 ml per well.

After 2 weeks of cultivation on DMEM containing Geneticin antibiotic at a concentration of 800 μg / μl, a cytofluorometric analysis of the surviving clones was performed. Further maintenance of the obtained lines and analysis of the level of EGFP expression was performed according to the procedure described for pools of transfected cells (see example 2).

2. Results of analysis of the temporal profile of EGFP gene expression in individual cell clones.

The main quantitative indicator of the expression activity of the reporter gene in this case was the median of the distribution of cells by fluorescence intensities.

Initially, 10 individual clones with the pEGFP_N1 construct were obtained, 17 individual clones with the 2xIns_pEGFP construct and 10 clones with the SV-40_pEGFP construct. Of these, 2 clones with the pEGFP_N1 construct, 5 clones with the 2xIns_pEGFP construct, 5 clones with the SV-40_pEGFP construct and 6 clones with the β_peGFP construct were characterized by a fairly high level of EGFP expression.

It should be noted that clones with a control construct initially expressed EGFP protein significantly weaker than clones with constructs 2xIns_pEGFP, SV-40_pEGFP, β_peGFP (see table 2).

After two and a half months of cultivation, the level of EGFP expression in stable cell clones with pEGFP_N1 and 2xIns_pEGFP constructs decreased by about 10 times. Moreover, the expression activity of clones containing the constructs of SV-40_pEGFP and β_peGFP, averaged about 75% of the original.

table 2 Initial activity (median distribution) Activity after two months of cultivation (median distribution) Decreased expression activity Average value pEGFP_N1 # 6 23.71 2,35 0.1 0.10 pEGFP_N1 # 8 16.11 1.70 0.11 2xIns_pEGFP # 1 69.78 9.73 0.14 0.13 2xIns_pEGFP # 6 116.52 3,59 0,03 2xIns_pEGFP # 8 103.66 16.60 0.16 2xIns_pEGFP # 12 33.08 5.99 0.18 2xIns_pEGFP # 14 77.74 10.55 0.14 SV-40_pEGFP # 2 67.93 13.10 0.19 0.76 SV-40_pEGFP # 3 339.82 82.79 0.24 SV-40_pEGFP # 4 50.25 134.45 2.68 SV-40_pEGFP # 7 7.04 2,37 0.34 SV-40_pEGFP # 11 3.31 1.24 0.37 β_pEGFP # 1 78.44 15,54 0.2 1.76 β_pEGFP # 3 79.86 19.63 0.25 β_pEGFP # 4 76.35 14.46 0.19 β_pEGFP # 5 2.19 13.34 6.09 β_pEGFP # 10 15.75 56.74 3.6 β_pEGFP # 11 11.34 2.41 0.21

Thus, sequences of transcription terminators that terminate genomic transcripts and isolate the transgene from transcriptional signals have the ability to maintain the level of transgene transcription at a stable high level for a fairly long time. The results demonstrate that the terminator is more effective than the insulator in maintaining a stable level of transgene expression in clones, which indicates the possibility of using the terminator in expression vectors to protect the transgene from the negative effects of the genome.

Information sources

1. Kwaks, T. H. J., Bamett, P., Hemrika, W. et al. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nature Biotechnology, 2003, 21; 553-558.

2. Kwaks T.H.J. and Otte A.P. Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends in Biotechnology, 2006, 24: 137-142.

3. Recillas-Targa F., Valadez-Graham V., Farrell S.M. Prospects and implications of using chromatin insulators in gene therapy and transgenesis. BioEssays, 2004, 26: 796-807.

4. Yao S., Sukonnik T., Kean T., Bharadwaj R.R., Pasceri P., Ellis J. Retrovirus Silencing, Variegation, Extinction, and Memory Are Controlled by a Dynamic Interplay of Multiple Epigenetic Modifications. Mol ther. 10: 27-36.

Claims (1)

A method of creating transgenic mammalian cell lines producing a protein with a stable and high expression level, comprising transforming mammalian cells with an expression vector containing a reporter gene, a cytomegalovirus promoter, and a transcription terminator inserted above the sequence of the cytomegalovirus promoter.