WO2011070405A1 - Vecteur d'expression portant une/des région(s) s/mar - Google Patents

Vecteur d'expression portant une/des région(s) s/mar Download PDF

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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
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WIPO (PCT)
Prior art keywords
expression vector
expression
vector
darbepoetin alfa
recombinant
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Application number
PCT/IB2010/000484
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English (en)
Inventor
Villoo Morawala Patell
Rajesh Ullanat
Sunit Maity
Sudhir Joshi
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Avesthagen Limited
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Publication date
Application filed by Avesthagen Limited filed Critical Avesthagen Limited
Publication of WO2011070405A1 publication Critical patent/WO2011070405A1/fr

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    • 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

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  • 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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un vecteur d'expression eucaryote portant une/des région(s) S/MAR (Scaffold/Matrix Attachment Region(s)). Elle concerne l'utilisation de la production de la derbépoïétine alpha recombinante en grande quantité.
PCT/IB2010/000484 2009-12-07 2010-03-05 Vecteur d'expression portant une/des région(s) s/mar WO2011070405A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN3008/CHE/2009 2009-12-07
IN3008CH2009 2009-12-07

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WO2011070405A1 true WO2011070405A1 (fr) 2011-06-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2819866C2 (ru) * 2022-04-21 2024-05-28 Общество с ограниченной ответственностью "Протеиновый контур" Вектор экспрессии, эукариотический штамм продуцент и способ получения дарбэпоэтина альфа

Citations (7)

* Cited by examiner, † Cited by third party
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 (fr) * 2000-12-15 2002-06-20 Pangen Biotech Inc. Vecteur d'expression pour cellule animale contenant une region de liaison matricielle nucleique de l'interferon $g(b)
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 (fr) * 2003-07-11 2005-01-20 Cytos Biotechnology Ag Systeme d'expression genetique
WO2005040377A2 (fr) * 2003-10-24 2005-05-06 Selexis S.A. Transfert et expression genique a efficacite elevee dans des cellules de mammiferes au moyen d'une procedure de transfection multiple de sequences mar
US20070124829A1 (en) * 2001-03-30 2007-05-31 Rapp Jeffrey C Avians containing a lysozyme promoter transgene

Patent Citations (7)

* Cited by examiner, † Cited by third party
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 (fr) * 2000-12-15 2002-06-20 Pangen Biotech Inc. Vecteur d'expression pour cellule animale contenant une region de liaison matricielle nucleique de l'interferon $g(b)
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 (fr) * 2003-07-11 2005-01-20 Cytos Biotechnology Ag Systeme d'expression genetique
WO2005040377A2 (fr) * 2003-10-24 2005-05-06 Selexis S.A. Transfert et expression genique a efficacite elevee dans des cellules de mammiferes au moyen d'une procedure de transfection multiple de sequences mar

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2819866C2 (ru) * 2022-04-21 2024-05-28 Общество с ограниченной ответственностью "Протеиновый контур" Вектор экспрессии, эукариотический штамм продуцент и способ получения дарбэпоэтина альфа

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