WO2011070405A1 - An expression vector carrying scaffold/matrix attachment region(s) - Google Patents
An expression vector carrying scaffold/matrix attachment region(s) Download PDFInfo
- Publication number
- WO2011070405A1 WO2011070405A1 PCT/IB2010/000484 IB2010000484W WO2011070405A1 WO 2011070405 A1 WO2011070405 A1 WO 2011070405A1 IB 2010000484 W IB2010000484 W IB 2010000484W WO 2011070405 A1 WO2011070405 A1 WO 2011070405A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- expression vector
- expression
- vector
- darbepoetin alfa
- recombinant
- Prior art date
Links
- 239000013604 expression vector Substances 0.000 title claims abstract description 26
- 239000011159 matrix material Substances 0.000 title claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 108010019673 Darbepoetin alfa Proteins 0.000 claims description 30
- 229960005029 darbepoetin alfa Drugs 0.000 claims description 23
- 239000013598 vector Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 8
- 125000000837 carbohydrate group Chemical group 0.000 claims 1
- 230000014509 gene expression Effects 0.000 description 28
- 108090000623 proteins and genes Proteins 0.000 description 21
- 108010077544 Chromatin Proteins 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- 210000003483 chromatin Anatomy 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 210000004962 mammalian cell Anatomy 0.000 description 8
- 108700019146 Transgenes Proteins 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 208000020832 chronic kidney disease Diseases 0.000 description 6
- 230000010354 integration Effects 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 4
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 4
- 208000007502 anemia Diseases 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 102000044890 human EPO Human genes 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 210000000299 nuclear matrix Anatomy 0.000 description 4
- 230000004481 post-translational protein modification Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 230000003442 weekly effect Effects 0.000 description 4
- 102100032610 Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 241000699800 Cricetinae Species 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 102000008297 Nuclear Matrix-Associated Proteins Human genes 0.000 description 2
- 108010035916 Nuclear Matrix-Associated Proteins Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 208000028208 end stage renal disease Diseases 0.000 description 2
- 201000000523 end stage renal failure Diseases 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000005534 hematocrit Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000012212 insulator Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 230000007067 DNA methylation Effects 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 101001094545 Homo sapiens Retrotransposon-like protein 1 Proteins 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101000689689 Oryzias latipes Alpha-1A adrenergic receptor Proteins 0.000 description 1
- 102100035123 Retrotransposon-like protein 1 Human genes 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 230000000739 chaotic effect Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 229940037201 oris Drugs 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000011165 process development Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
Definitions
- the present invention relates to a eukaryotic expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR). It relates to the use of production of recombinant derbapoietin alpha in increased quantity.
- S/MAR Scaffold/Matrix Attachment Region
- tPA human tissue plasminogen activator
- mammalian cells In 1986, FDA approved human tissue plasminogen activator (tPA; Genentech, CA, USA) protein from mammalian cells to be used for therapeutic purpose. It was the beginning. Purrently, there are many more monoclonal antibodies, which got the regulatory approval. Moreover, several hundreds are in pipeline. Like tPA, most of these proteins are expressed immortalized Chinese hamster ovary (CHO) cells, but other cell lines, such as mouse myeloma (NSO), baby hamster kidney (BHK), human embryo kidney (HEK-293) have also been approved for recombinant protein production. There are two critical issues during the production of therapeutics (a) time taken to provide the material (b) lowering the price of the material to the common user. Therefore, industry continues to look at new technologies and process development strategies that will reduce timelines and also will help in reducing the cost.
- NSO mouse myeloma
- BHK baby hamster kidney
- mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post-translational modifications.
- mammalian cell expression systems are now available for expression of proteins.
- Generally expression vectors use a strong viral or cellular promoter/enhancer to drive the expression of recombinant gene.
- the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
- Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects.
- r-HuEPO recombinant human erythropoietin
- HuEPO HuEPO erythropoiesis stimulating protein
- the main object of the present invention is to construct an eukaryotic expression vector that comprise the Recombinant Darbepoetin alfa, encoding DNA.
- Another object of the present invention is to use the eukaryotic expression vector to drive expression of soluble Darbepoetin alfa when transfected into an appropriate cell line.
- Yet another object of the present invention is to obtain recombinant- Darbepoetin alfa using this expression vector.
- the present invention relates to constructing a novel eukaryotic expression vector that comprises novel DNA compounds, which encode recombinant Darbepoetin alfa (novel erythropoiesis stimulating protein) activity.
- a novel eukaryotic expression vector has been constructed that comprise the recombinant Darbepoetin alfa (novel erythropoiesis stimulating protein) activity-encoding DNA and drive expression of Darbepoetin alfa activity when transfected into an appropriate cell line.
- the novel expression vector can be used to produce soluble Darbepoetin alfa.
- the recombinant-produced Darbepoetin alfa activity is useful in the treatment of anemia associated with chronic renal failure (CRF). It can also be used for treatment of anemia associated with cancer, HIV infection, and use in surgical setting to decrease the need for allogenic blood transfusions
- Figure 1 illustrates the construct of plasmid vector pCDNA3.1/ Darbepoetin Alfa
- Figure 2 illustrates the construct of plasmid vector pCDNA3.1/MAR1/ Darbepoetin Alfa
- Prokaryotic expression systems were part of the early repertoire of research tools in molecular biology. The de novo synthesis of recombinant eukaryotic proteins in a prokaryotic system imposed a number of problems on the eukaryotic gene product. Among the two most critical were improper protein folding and assembly, and the lack of posttranslational modification, principally glycosylation and phosphorylation. Prokaryotic systems do not possess all the appropriate protein synthesizing machinery to produce a structural and/or catalytically functional eukaryotic protein. Therefore, Mammalian expression system is generally preferred for manufacturing of therapeutic proteins, for simple reason that as post-translational modifications required will be addressed by the system.
- CHO SES Chinese hamster ovary
- HEK human embryonic kidney
- myeloma cell lines like J558L and Sp2/0, etc. are also employed as hosts for the establishment of stable transfectants.
- the integration of foreign DNA into the genome of a host cell is a chaotic and typically random process. It has been well documented that the transgene expression is highly variable among cell lines and its integration may cause unexpected changes in the phenotype. Reasons underlying the large variability in clonal expression levels include differing plasmid copy numbers and a phenomenon known as the position effect, which was initially described in Drosophila melanogaster as position-effect variegation.
- the position of integration can influence transgene expression through at least three mechanisms: the activity of local regulatory elements, the local chromatin structure and the local state of DNA methylation. Two common approaches can be used to protect DNA from negative position effects or integration-dependent repression.
- One approach will be to direct transgene integration into a predetermined site that is transcriptionally active using site- specific recombination methods. Another method is to simply incorporate into the expression vector DNA sequence elements found in chromatin border regions, such that regardless of the integration site the gene will be protected from surrounding chromatin influences. For recombinant protein expression, sequences that behave as chromatin borders and protect transfected genes from surrounding , chromatin influences include insulator sequences and scaffold/matrix-attachment regions (S/MARs).
- S/MARs are DNA sequences that bind isolated nuclear scaffolds or nuclear matrices in vitro with high affinity. Expression studies suggested that flanking transgene with insulator could reduce the position effect thus suppressing clonal expression variability. S/MARs are relatively short (100-1000 bp long) sequences that anchor the chromatin loops to the nuclear matrix. MARs often include the origins of replication (ORI) and can possess a concentrated area of transcription factor binding sites. Approximately 100 000 matrix attachment sites are believed to exist in the mammalian nucleus of which 30 000-40 000 serve as ORIs. MARs have been observed to flank the ends of domains encompassing various transcriptional units.
- ORI origins of replication
- MARs bring together the transcriptionally active regions of chromatin such that the transcription is initiated in the region of the chromosome that coincides with the surface of nuclear matrix. As such, they may define boundaries of independent chromatin domains, such that , only the encompassing c/s-regulatory elements control the expression of the genes within the domain.
- S/MARs which include forming boundaries of chromatin domains, changing of chromatin conformations, participating in initiation of DNA replication and organizing the chromatin structure of a chromosome.
- S/MARs are common in centromere-associated DNA and telomeric arrays, and appear to be important in mitotic chromosome assembly and maintenance of chromosome shape during metaphase.
- S/MARs are involved in multiple independent processes during different stages of the cell cycle.
- the chicken lysozyme 5' MAR was identified as one of the most active sequence in a study that compared the effect of various chromatin structure regulatory elements on transgene expression. It had also shown to increase the levels of regulated or constitutive transgene expression in various mammalian cell lines. Recently, inclusion of this MAR sequence increased overall expression of transgene when transfected into CHO cell line.
- mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post- translational modifications.
- a variety of mammalian cell expression systems are now available for expression of proteins.
- the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
- Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects.
- the aim of inventing this new molecular analogue of recombinant human erythropoietin (r-HuEPO) is to obtain a therapeutic with a longer biological half-life compared to r-HuEPO, allowing a reduction of the frequency of injections necessary to maintain a desired level of systemic haemoglobin and haematocrit.
- CRF chronic respiratory disease
- r-HuEPO chronic respiratory disease 2019
- Studies on HuEPO have confirmed direct relationship between the silalic acid containing carbohydrate content of the molecule and its serum half-life and biological activity; therefore, later hyperglycosylated rHuEPO analogues were developed.
- Darbepoetin alfa novel erythropoiesis stimulating protein, NESP
- NESP was generated to contain five N-linked carbohydrate chains (two more rHuEPO). Due to increased sialic acid containing carbohydrate content, NESP has longer serum half-life, greater in vivo potency and can be administered less frequently to obtain same biological response.
- Present invention relates to a novel expression vector using the above- mentioned S/MAR to produce Darbepoetin alfa in larger quantity.
- products of expression of the DNA sequence display the biological activities of Darbepoetin alfa.
- Vector development, cloning and sub-cloning, transfection, fermentation and purification strategies are disclosed.
- pCDNA3.1 vector the gene of interest is regulated by Human cytomegalovirus (CMV) immediate-early promoter/enhancer. It permits efficient, high level expression of the recombinant protein.
- CMV Human cytomegalovirus
- the gene of interest, Darbepoetin Alfa was cloned into pCDNA3.1 vector using restriction enzymes BamHI and Xhol. The positive transformants were verified by appropriate restriction enzymes. It was double digested using BamHI and Xhol that gave the expected pattern. It was also confirmed by using the restriction enzymes PstI, which clearly demonstrated the expected pattern. The inserts were later sequence verified.
- S/MARs Isolated scaffold/matrix-attachment regions
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a eukaryotic expression vector carryin Scaffold/Matrix Attachment Region(s) (S/MAR). It relates to the use of production of recombinant derbapoietin alpha in increased quantity.
Description
"AN EXPRESSION VECTOR CARRYING SCAFFOLD/ MATRIX ATTACHMENT REGION (S)"
FIELD OF THE INVENTION
The present invention relates to a eukaryotic expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR). It relates to the use of production of recombinant derbapoietin alpha in increased quantity.
BACKGROUND AND PRIOR ART OF THE INVENTION
In 1986, FDA approved human tissue plasminogen activator (tPA; Genentech, CA, USA) protein from mammalian cells to be used for therapeutic purpose. It was the beginning. Purrently, there are many more monoclonal antibodies, which got the regulatory approval. Moreover, several hundreds are in pipeline. Like tPA, most of these proteins are expressed immortalized Chinese hamster ovary (CHO) cells, but other cell lines, such as mouse myeloma (NSO), baby hamster kidney (BHK), human embryo kidney (HEK-293) have also been approved for recombinant protein production. There are two critical issues during the production of therapeutics (a) time taken to provide the material (b) lowering the price of the material to the common user. Therefore, industry continues to look at new technologies and process development strategies that will reduce timelines and also will help in reducing the cost.
As mentioned, mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post-translational modifications. A variety of mammalian cell expression systems are now available for expression of proteins. Generally expression vectors use a strong viral or cellular promoter/enhancer to drive the expression of recombinant gene. However, the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects. The aim of inventing this new molecular analogue of recombinant human erythropoietin (r-HuEPO) is to obtain a therapeutic with a longer biological half-life compared to r-HuEPO, allowing a reduction of the frequency of injections necessary to maintain a desired level of systemic haemoglobin and haematocrit. The chronic nature of CRF (unless a subject receives a kidney transplant) means that treatment may continue for a long part of the subject's life and multiple weekly injections of r-HuEPO can have a major impact on subjects and care givers. Studies on HuEPO have confirmed direct relationship between the silalic acid containing carbohydrate content of the molecule and its serum half-life and biological activity; therefore, later hyperglycosylated rHuEPO analogues were developed. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP) containing more carbohydrate content has greater in vivo potency.
In order to facilitate production of large quantities of Darbepoetin alfa from cell culture, a novel expression vector has been developed with genetic compounds. Use of this expression vector has been shown to increase the expression of therapeutic protein. The cloning, expression and purification of Darbepoetin alfa have been mentioned in this application.
References:
1. Yin, J. et. al. Journal of Biotech. 127, 335-347 (2007)
2. Geisse, S. et. al. Protein Express. Purif. 8, 271-282 (1996)
3. Henikoff, S. Curr. Opin.Genet. Dev. 2, 907-912 (1992)
4. Kleinjan, DJ. et. al. Hum.Mol.Genet. 7, 1611-1618 (1998)
5. Allen, GC. et. al. Plant Mol. Biol. 43, 361-76 (2000)
6. Girod, PA. and Mermod, N. Gene Transfer and Expression in
Mammalian Cells, Elsevier Sciences, 359-379 (2003)
7. Boulikas, T. Int. Rev. Cytol. 162A, 279-388 (1995)
8. kalos, M. and Fournier, REK. Mol. Cell. iol. 15, 198-207 (1995)
9. Phi-Val, L. et. al. Mo/. Cell. Biol. 10, 2302-2307 (1990)
10. Zahn-Zabal, M. et. al. Journal of Biotech. 87, 29-42 (2001)
11. Girod, PA. et. al. Biotechnol. Bioeng. 91, 1-11 (2005)
12. Singh, GB.et. al. NAR. 25, 1419-1425 (1997)
13. Frish,, M. et. al. Genom. Biol. 12, 349-354 (2002)
14. Egrie, JC and Browne, JK. Nephrol Dial Transplant. 16, 3-13
(2001)
15. Macdougall IC. Nephrol Dial Transplant. 16, 14-21 (2001) OBJECTIVES OF THE INVENTION
The main object of the present invention is to construct an eukaryotic expression vector that comprise the Recombinant Darbepoetin alfa, encoding DNA.
Another object of the present invention is to use the eukaryotic expression vector to drive expression of soluble Darbepoetin alfa when transfected into an appropriate cell line.
Yet another object of the present invention is to obtain recombinant- Darbepoetin alfa using this expression vector.
SUMMARY OF THE INVENTION
The present invention relates to constructing a novel eukaryotic expression vector that comprises novel DNA compounds, which encode recombinant Darbepoetin alfa (novel erythropoiesis stimulating protein) activity. A novel eukaryotic expression vector has been constructed that comprise the recombinant Darbepoetin alfa (novel erythropoiesis stimulating protein) activity-encoding DNA and drive expression of Darbepoetin alfa activity when transfected into an appropriate cell line. The novel expression vector can be used to produce soluble Darbepoetin alfa. The recombinant-produced Darbepoetin alfa activity is useful in the treatment of anemia associated with chronic renal failure (CRF). It can also be used for treatment of anemia
associated with cancer, HIV infection, and use in surgical setting to decrease the need for allogenic blood transfusions
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is further explained with reference to the drawings in which:
Figure 1 illustrates the construct of plasmid vector pCDNA3.1/ Darbepoetin Alfa;
Figure 2 illustrates the construct of plasmid vector pCDNA3.1/MAR1/ Darbepoetin Alfa;
DETAILED DESCRIPTION OF THE INVENTION
Prokaryotic expression systems were part of the early repertoire of research tools in molecular biology. The de novo synthesis of recombinant eukaryotic proteins in a prokaryotic system imposed a number of problems on the eukaryotic gene product. Among the two most critical were improper protein folding and assembly, and the lack of posttranslational modification, principally glycosylation and phosphorylation. Prokaryotic systems do not possess all the appropriate protein synthesizing machinery to produce a structural and/or catalytically functional eukaryotic protein. Therefore, Mammalian expression system is generally preferred for manufacturing of therapeutic proteins, for simple reason that as post-translational modifications required will be addressed by the system. A variety of mammalian cell expression systems are now available for either the transient expression of recombinant genes or stably transfected ones. Generally, Chinese hamster ovary (CHO) cell stable expression systems (CHO SES) are used for this purpose to express recombinant genes. Moreover, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) 293 cells, mouse L-cells, and myeloma cell lines like J558L and Sp2/0, etc., are also employed as hosts for the establishment of stable transfectants.
However, the integration of foreign DNA into the genome of a host cell is a chaotic and typically random process. It has been well documented that the transgene expression is highly variable among cell lines and its integration
may cause unexpected changes in the phenotype. Reasons underlying the large variability in clonal expression levels include differing plasmid copy numbers and a phenomenon known as the position effect, which was initially described in Drosophila melanogaster as position-effect variegation. The position of integration can influence transgene expression through at least three mechanisms: the activity of local regulatory elements, the local chromatin structure and the local state of DNA methylation. Two common approaches can be used to protect DNA from negative position effects or integration-dependent repression. One approach will be to direct transgene integration into a predetermined site that is transcriptionally active using site- specific recombination methods. Another method is to simply incorporate into the expression vector DNA sequence elements found in chromatin border regions, such that regardless of the integration site the gene will be protected from surrounding chromatin influences. For recombinant protein expression, sequences that behave as chromatin borders and protect transfected genes from surrounding , chromatin influences include insulator sequences and scaffold/matrix-attachment regions (S/MARs).
S/MARs are DNA sequences that bind isolated nuclear scaffolds or nuclear matrices in vitro with high affinity. Expression studies suggested that flanking transgene with insulator could reduce the position effect thus suppressing clonal expression variability. S/MARs are relatively short (100-1000 bp long) sequences that anchor the chromatin loops to the nuclear matrix. MARs often include the origins of replication (ORI) and can possess a concentrated area of transcription factor binding sites. Approximately 100 000 matrix attachment sites are believed to exist in the mammalian nucleus of which 30 000-40 000 serve as ORIs. MARs have been observed to flank the ends of domains encompassing various transcriptional units. It has also been shown that MARs bring together the transcriptionally active regions of chromatin such that the transcription is initiated in the region of the chromosome that coincides with the surface of nuclear matrix.
As such, they may define boundaries of independent chromatin domains, such that , only the encompassing c/s-regulatory elements control the expression of the genes within the domain. A number of possible functions have been discussed earlier for S/MARs, which include forming boundaries of chromatin domains, changing of chromatin conformations, participating in initiation of DNA replication and organizing the chromatin structure of a chromosome. S/MARs are common in centromere-associated DNA and telomeric arrays, and appear to be important in mitotic chromosome assembly and maintenance of chromosome shape during metaphase. Thus, S/MARs are involved in multiple independent processes during different stages of the cell cycle. The chicken lysozyme 5' MAR was identified as one of the most active sequence in a study that compared the effect of various chromatin structure regulatory elements on transgene expression. It had also shown to increase the levels of regulated or constitutive transgene expression in various mammalian cell lines. Recently, inclusion of this MAR sequence increased overall expression of transgene when transfected into CHO cell line.
As previously mentioned, mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post- translational modifications. A variety of mammalian cell expression systems are now available for expression of proteins. However, the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects. The aim of inventing this new molecular analogue of recombinant human erythropoietin (r-HuEPO) is to obtain a therapeutic with a longer biological half-life compared to r-HuEPO, allowing a reduction of the frequency of injections necessary to maintain a desired level of systemic haemoglobin and haematocrit. The chronic nature of CRF (unless a subject receives a kidney transplant) means that treatment may continue for
a long part of the subject's life and multiple weekly injections of r-HuEPO can have a major impact on subjects and care givers. Studies on HuEPO have confirmed direct relationship between the silalic acid containing carbohydrate content of the molecule and its serum half-life and biological activity; therefore, later hyperglycosylated rHuEPO analogues were developed. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP) was generated to contain five N-linked carbohydrate chains (two more rHuEPO). Due to increased sialic acid containing carbohydrate content, NESP has longer serum half-life, greater in vivo potency and can be administered less frequently to obtain same biological response.
Present invention relates to a novel expression vector using the above- mentioned S/MAR to produce Darbepoetin alfa in larger quantity. Upon isolation from culture media, products of expression of the DNA sequence display the biological activities of Darbepoetin alfa. Vector development, cloning and sub-cloning, transfection, fermentation and purification strategies are disclosed.
Cloning and Construction of Expression Vector
In pCDNA3.1 vector, the gene of interest is regulated by Human cytomegalovirus (CMV) immediate-early promoter/enhancer. It permits efficient, high level expression of the recombinant protein. The gene of interest, Darbepoetin Alfa was cloned into pCDNA3.1 vector using restriction enzymes BamHI and Xhol. The positive transformants were verified by appropriate restriction enzymes. It was double digested using BamHI and Xhol that gave the expected pattern. It was also confirmed by using the restriction enzymes PstI, which clearly demonstrated the expected pattern. The inserts were later sequence verified.
Isolated scaffold/matrix-attachment regions (S/MARs), here marked as S/MAR sequences were initially cloned into pGEMTeasy vector by PCR and confirmed by appropriate restriction enzyme analysis and sequencing. Subsequently, the cloned S/MARs were inserted upstream of CMV promoter in the pCDNA3.1 vector using restriction sites. The insertion was confirmed by restriction analysis.
Claims
We Claim:
1) An expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
2) The vector as claimed in claim 1, wherein the vector is a eukaryotic expression vector.
3) The vector as claimed in claim 1 , wherein the vector is used for production of Darbepoetin alfa.
4) The vector as claimed in claim 3, wherein the Darbepoetin alfa is a recombinant Darbepoetin alfa.
5) The vector as claimed in claim 4, wherein the recombinant Darbepoetin alfa contains 5 N linked carbohydrate chains..
6) A method for constructing an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR), said method comprising step of inserting the S/MAR into the expression vector.
7) The method as claimed in claim 6, wherein the vector is a eukaryotic expression vector.
8) A host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s).
9) A recombinant Darbepoetin alfa expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s).
10) An expression vector, a method of constructing, host cell and a recombinant Darbepoetin alfa as substantially herein described with accompanying examples and figures.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN3008CH2009 | 2009-12-07 | ||
| IN3008/CHE/2009 | 2009-12-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2011070405A1 true WO2011070405A1 (en) | 2011-06-16 |
Family
ID=44145148
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2010/000484 WO2011070405A1 (en) | 2009-12-07 | 2010-03-05 | An expression vector carrying scaffold/matrix attachment region(s) |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2011070405A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2819866C2 (en) * | 2022-04-21 | 2024-05-28 | Общество с ограниченной ответственностью "Протеиновый контур" | Expression vector, eukaryotic producer strain and method of producing darbepoetin alpha |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5985607A (en) * | 1994-12-19 | 1999-11-16 | Cangene Corporation | Recombinant DNA molecules and expression vectors for tissue plasminogen activator |
| WO2002048379A1 (en) * | 2000-12-15 | 2002-06-20 | Pangen Biotech Inc. | Expression vector for animal cell containing nuclear matrix attachment region fo interferon beta |
| US20020142393A1 (en) * | 1998-10-17 | 2002-10-03 | Armin Baiker | Episomally replicating vector, its preparation and use |
| US20030224477A1 (en) * | 2002-05-31 | 2003-12-04 | Heartlein Michael W. | Optimized promoter constructs |
| WO2005005644A1 (en) * | 2003-07-11 | 2005-01-20 | Cytos Biotechnology Ag | Gene expression system |
| WO2005040377A2 (en) * | 2003-10-24 | 2005-05-06 | Selexis S.A. | High efficiency gene transfer and expression in mammalian cells by a multiple transfection procedure of matrix attachment region sequences |
| US20070124829A1 (en) * | 2001-03-30 | 2007-05-31 | Rapp Jeffrey C | Avians containing a lysozyme promoter transgene |
-
2010
- 2010-03-05 WO PCT/IB2010/000484 patent/WO2011070405A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5985607A (en) * | 1994-12-19 | 1999-11-16 | Cangene Corporation | Recombinant DNA molecules and expression vectors for tissue plasminogen activator |
| US20020142393A1 (en) * | 1998-10-17 | 2002-10-03 | Armin Baiker | Episomally replicating vector, its preparation and use |
| WO2002048379A1 (en) * | 2000-12-15 | 2002-06-20 | Pangen Biotech Inc. | Expression vector for animal cell containing nuclear matrix attachment region fo interferon beta |
| US20070124829A1 (en) * | 2001-03-30 | 2007-05-31 | Rapp Jeffrey C | Avians containing a lysozyme promoter transgene |
| US20030224477A1 (en) * | 2002-05-31 | 2003-12-04 | Heartlein Michael W. | Optimized promoter constructs |
| WO2005005644A1 (en) * | 2003-07-11 | 2005-01-20 | Cytos Biotechnology Ag | Gene expression system |
| WO2005040377A2 (en) * | 2003-10-24 | 2005-05-06 | Selexis S.A. | High efficiency gene transfer and expression in mammalian cells by a multiple transfection procedure of matrix attachment region sequences |
Non-Patent Citations (1)
| Title |
|---|
| EGRIE J.C. ET AL: "Development and characterization of novel erythropoiesis stimulating protein (NESP)", BRITISH JOURNAL OF CANCER, vol. 84, no. SUPP.1, 2001, pages 3 - 10, XP001059714, DOI: doi:10.1054/bjoc.2001.1746 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2819866C2 (en) * | 2022-04-21 | 2024-05-28 | Общество с ограниченной ответственностью "Протеиновый контур" | Expression vector, eukaryotic producer strain and method of producing darbepoetin alpha |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2625277B1 (en) | Expression vector for high level expression of recombinant proteins | |
| JP5225107B2 (en) | Selection of host cells that express proteins at high levels | |
| US20110097798A1 (en) | Mammalian cell expression vectors and utilization | |
| AU2018200686B2 (en) | DNA element having the activity of enhancing foreign gene expression | |
| KR101385223B1 (en) | Expression vector for Animal Cell | |
| Gupta et al. | Vector-related stratagems for enhanced monoclonal antibody production in mammalian cells | |
| WO2018066492A1 (en) | PROMOTER OF Hspa5 GENE | |
| US9862969B2 (en) | Promoter derived from human gene | |
| WO2020032153A1 (en) | PROMOTOR FOR H spa 8 GENE | |
| WO2011070405A1 (en) | An expression vector carrying scaffold/matrix attachment region(s) | |
| WO2023210607A1 (en) | PROMOTER OF Eno1 GENE | |
| Kayser et al. | Cell line engineering methods for improving productivity | |
| WO2011015916A2 (en) | Vectors and compounds for expression of recombinant infliximab | |
| WO2011015917A2 (en) | Vectors and compounds for expression of recombinant tnk-tpa (tenecteplase) | |
| CN118581155A (en) | CRISPR/Cas12 a-based multi-target-point glycosylase gene editing method | |
| WO2011015924A2 (en) | Vectors and compounds for expression of recombinant trastuzumab |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10835556 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 10835556 Country of ref document: EP Kind code of ref document: A1 |