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 natoe 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.
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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 tiie 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 vsith 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/MARl/ 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 fimctional eukaryotic protein. Therefore, Mammalian expression system is generally preferred for manuftujturing 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 domams, such that only the encompassing cis-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 iysozyme 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 fi-equently 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.

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 ScaffoldAlatrix 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.