FIELD OF THE INVENTION
The present invention relates to identification of novel MAR sequences and characterization of those sequences in increasing therapeutic protein production in CHO cell lines.
BACKGROUND AND PRIOR ART OF THE INVENTION
Prokaryotic expression systems were part of the early repertoire of research tools in molecular biology (Yin et. al. 2007). 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 (Geisse, S. et. al. 1996).
However, the integration of foreign DNA into the genome of a host cell is typically a 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 (Henikoff, S.1992). 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

(Kleinjan, DJ. et. al. 1998). Two common approaches can be used to protect DNA from negative position effects or integration-dependent repression. One approach is being 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).
Insulator is a class of DNA sequence that possesses a common ability to protect genes from inappropriate signals emanating from their surrounding environment. Insulator protects an expressing gene from its surroundings in two different ways (i) by blocking the action of a distal enhancer on a promoter. It happens when insulator is placed between the enhancer and promoter and it can prevent an enhancer from activating expression of an adjacent gene, (ii) by acting as a barrier which prevents the advance of a nearby condensed chromatin which might otherwise silence the expression.
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 (Allen, GC. et. al. 2000). S/MARs are sequences that anchor the chromatin loops to the nuclear matrix. Length of S/MAR vary considerably (300-3000bps). 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 (Girod, PA. and Mermod, N. 2003). 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 (Girod, PA. and Mermod, N. 2003).

As such, they may define boundaries of independent chromatin domains, 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 (Girod, PA. and Mermod, N. 2003).
Analyses of experimentally identified S/MARs have revealed a typical element to be as short as 300 base pairs and up to several kbs long. These S/MARs may contain several sequence motifs, including AT-rich nucleotide motifs ((> 70% A-T) (Girod, PA. and Mermod, N. 2003). Most MARs appear to contain a MAR-specific sequence called "MAR recognition signature," which is a bipartite sequence that consists of two individual sequences AATAAYAA and AWWRTAANNWWGNNNC from one another. Other sequences, proposed to be indicative of MAR sequences, are the DNA-unwinding motif (AATATATTAATATT), replication initiator protein sites (ATTA and, ATTTA), homo-oligonucleotide repeats (e.g., the A-box AATAAAYAAA and the T-box TTWTWTTWTT), DNase I-hypersensitive sites, potential nucleosome-free stretches, polypurinejpolypyrimidine tracks, and sequences that may adopt non-B-DNA or triple-helical conformations under conditions of negative supercoiling (Boulikas,T. 1995).
It was speculated that S/MAR could form genetic boundaries between chromosomal domains that independently organize into structures permissive or non-permissive for gene expression, referred to as euchromatin and heterochromatin domains, respectively. A transgene flanked by S/MAR elements may therefore constitute an autonomous chromatin domain whose expression would remain independent of the adjacent chromosomal environment. Recently, S/ MARs have been shown to increase the expression of adjacent transgenes when co-inserted into a chromosomal environment confirming the hypothesis (Kalos, M and Foumier, REK 1995 and Phi-Val, L. et. al 1990). Alternatively, the MAR may actively reconfigure chromatin

around its chromosomal integration site and thereby prevent transgene silencing, for instance by mediating histone modifications or changes in subnuclear localization.
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 (Zahn-Zabal, M. et. al. 2001). 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 (Girod, PA. et. al. 2005).
The major objective of the invention is to to identify novel MAR sequences and also demonstrate improved efficiency in enhancing the production of recombinant proteins from eukaryotic cells lines by using these newly identified MAR sequences.
OBJECTIVES OF THE PRESENT INVENTION
The principal object of the present invention is to provide novel Matrix Attachment
Region [MAR] sequences.
Another object of the present invention is to make use of novel MAR sequences to
provide increased protein production.
Yet another object of the present invention is to provide process for obtaining MAR
sequences.
Still another object of the present invention is to provide MAR sequences, its
complementary sequences, variants and fragments thereof
STATEMENT OF THE INVENTION
Accordingly, the present invention provides a matrix attachment region sequence [s] or its complementary sequence[s], variant[s] and fragment[s] thereof; a process to obtain a matrix attachment region sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof; a method for increasing protein production in eukaryotic cells, said method comprising steps of: introducing a matrix attachment region sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof in eukaryotic cell followed by its expression to obtain the proteins; an expression vector carrying a matrix attachment region sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof; and a eukaryotic cell

with a matrix attachment region sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The present invention relates to a matrix attachment region sequence[s] or its
complementary sequence[s], variant[s] and fragment[s] thereof.
In another embodiment of the present invention said sequence increases protein
production by modulating transcription efficiency.
In yet another embodiment of the present invention said sequence promotes transient
and stable transfection to enhance expression of recombinant proteins.
The present invention is in relation to a process to obtain a matrix attachment region
sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof.
The present invention is in relation to a method for increasing protein production in
eukaryotic cells, said method comprising steps of: introducing a matrix attachment
region sequence[s] or its complementary sequence[s], variant[s] and fragment[s]
thereof in eukaryotic cell followed by its expression to obtain the proteins.
The present invention is in relation to an expression vector carrying a matrix
attachment region sequence[s] or its complementary sequence[s], variant[s] and
fragment[s] thereof.
In another embodiment of the present invention, said expression vector is mammalian
expression vector.
The present invention is in relation to a eukaryotic cell with a matrix attachment
region sequence [s] or its complementary sequence [s], variant[s] and fragment[s]
thereof
The following definitions are used in order to help in understanding the
invention:
"Chromosome" is organized structure of DNA and proteins found inside the cell.
"Chromatin" is the complex of DNA and protein, found inside the nuclei of
eukaryotic cells, which makes up the chromosome.
"DNA" or Deoxyribonucleic Acid, contain genetic informations. It is made up of
different nucleotides A, G, T or C.
A "gene" is a deoxyribonucleotide (DNA) sequence coding for a given mature
protein, "gene" shall not include untranslated flanking regions such as RNA
transcription initiation signals, polyadenylation addition sites, promoters or enhancers.

"Promoter" is a nucleic acid sequence that controls expression of a coding sequence or functional RNA. Promoters may be derived from a native gene, or be composed of different elements derived from different promoters found in nature. "Enhancer" refers to the sequence of gene that acts to initiate the transcription of the gene independent of the position or orientation of the gene.
"Repressor" refers to the sequence of the gene that acts to inhibit the transcription of the gene independent of the position or orientation of the gene. The term "signal peptide" refers to an amino terminal polypeptide precedign the secreted mature protein. In mature protein it is not present as it is cleaved. The definition of "vector" referes herein is a nucleic acid molecule capable of transporitgn another nucleic acid to which it has been linked. Vectors, usually derived from plasmids, functions like a "molecular carrier", which will carry fragments of DNA into a host cell.
"Plasmid" are small circular double stranded polynucleotide structures of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome.
"Replication" refers to the synthesis of DNA from its template DNA strand. "Transcription" refers the synthesis of RNA from a DNA template. "Translation" means the synthesis of a polypeptide from messenger RNA. "Cis" refers to the placement of two or more DNA elements linked on the same plasmid.
"Trans" refers to the placement of two or more elements on two or more different plasmids.
"Orientation" refers to the order of nucleotides in the DNA sequence. As used herein, an "isolated nucleic acid fragment" is a polymer of DNA or RNA that is single or double stranded. AN isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
"Gene amplification" refers to the selective, repeated replication of a certain gene or genes without proportional increase in the copy number of other genes. It is an important widespread developmental and evolutionary process in many organism. Gene amplification can be classified in two categories (i) developmentally regulated gene expression as seen in Xenopus oocytes and (ii) spontaneously occuring gene

expression as amplification of the lac region reported in Escherichia coli. The best known gene amplification in mammalian cells is dihydrofolate reductase (DHFR). "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritence. Host organisms containingthe transformed nucelic acid fragments are referred to as "transformed" organisms.
"Eukaryotic cell" refers to any cell from a eukaryotic organism whose cells are organized into complex structures by internal membrane and cytoskeleton. Any eukaryotic cell that can be used for gene/protein manipulation and also can be maintained under cell culture conditions and subsequently transfected would be included in this invention. Especially preferable cell types include, e. g., stem cells, embryonic stem cells, Chinese hamster ovary cells (CHO), COS, BHK21, NIH3T3, HeLa, C2C12, HEK, MDCK, cancer cells, and primary differentiated or undifferentiated cells.
"Transfection" means the introduction of a foreign material like DNA into eukaryotic cells by any means of trasnfer. Different method of transfection includes Calcium phosphate, electroporation, lipofectamine and DEAE-Dextran transfection etc. "Transfected cell" refers to the eukaryotic cell in which the foreign DNA has been introduced into the eukaryotic cells. This DNA can be part of the host chromosome or replicate as an extra chromosomal element. "Cotransfection" is the method of simultaneous transfection.
"Transient gene expression" refers to a convenient method for the rapid production of small quantities of protein. Generally COS cells are mostly used for transient expression characterization.
"Stable gene expression" means preparation of stable cell line that permanently express the gene of interest depending on the stable integration of plasmid into the host chromosome.
The major objective of the invention is to to identify novel MAR sequences and also demonstrate improved efficiency in enhancing the production of recombinant proteins from eukaryotic cells lines by using these newly identified MAR sequences. This object has been achieved by providing novel MAR nucleotide sequences, and the use of these characterized active MAR sequences in both transient and stable transfection method to enhance the expression of recombinant proteins.

This invention relates to a purified and isolated DNA sequence having characteristics of protein production increasing activity by modulating the transcription efficiency. This invention similarly provides the purified and isolated DNA sequence usually consists of a S/MAR nucleotide sequence or a fragment thereof Included in the present invention are complementary sequences of the above-mentioned sequences or fragment, which can be produced by any means. Encompassed by this present invention variants of the above mentioned sequences, that is nucleotide sequences that vary from the reference sequence by conservative nucleotide substitutions, whereby one or more nucleotides are substituted by another with same characteristics.
Additionally, in a specific embodiment the invention refers to cloning of the above mentioned sequences in suitable mammalian expression vector. According to the present invention, the above mentioned nucleotide sequences could be located at both the 5' and the 3' ends of the sequence containing the promoter and the gene of interest in the expression vector.
In a preferred embodiment, this invention also provides a method for the use of above mentioned sequence in increasing the protein production activity. Included in the present invention is the use of above mentioned sequences in increasing the activity of protein production. "Protein production increasing activity" means that after introduction of DNA sequence under suitable conditions into a eukaryotic host cell, the sequence is capable of enhancing protein production levels in cell culture as compared to control cell culture where cells are transfected without said DNA sequence.
References:
1. Yin, J. et. al. Journal of Biotech. Ill, 335-347 (2007)
2. Geisse, S. et. al. Protein Express. Purif. 8, 271-282 (1996)
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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)
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9. Phi-Val, L. et. al. Mol. Cell. Biol. 10, 2302-2307 (1990)
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We Claim:
1) A matrix attachment region sequence[s] or its complementary sequence[s],
variant[s] and fragment[s] thereof.
2) The sequence as claimed in claim 1, wherein said sequence increases protein
production by modulating transcription efficiency.
3) The sequence as claimed in claim 1, wherein said sequence promotes transient and
stable transfection to enhance expression of recombinant proteins.
4) A process to obtain a matrix attachment region sequence[s] or its complementary
sequence[s], variant[s] and fragment[s] thereof
5) A method for increasing protein production in eukaryotic cells, said method
comprising steps of: introducing a matrix attachment region sequence[s] or its
complementary sequence[s], variant[s] and fragment[s] thereof in eukaryotic cell
followed by its expression to obtain the proteins.
6) An expression vector carrying a matrix attachment region sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof.
7) The expression vector as claimed in claim 6 wherein said expression vector is mammalian expression vector.
8) A eukaryotic cell with a matrix attachment region sequence[s] or its
complementary sequence[s], variant[s] and fragment[s] thereof.
9) The matrix attachment region sequence, a process, eukaryotic cell and a method
for increasing protein production are substantially as herein described along with
accompanying examples.