Mammalian expression vector comprising the mcmv promoter

Mammalian protein expression systems are the best choice for the production of eukaryotic proteins, especially when correct folding and post-translational modification is required. They produce eukaryotic recombinant proteins in the most natural state, with native tertiary structure, physiochemical characteristics and bioactivities. They have been successfully applied in the biopharmaceutical production of cytokines, monoclonal antibodies, growth factors and so on. Email:marketing@medicilon.com.cn web:www.medicilon.com

The invention relates to a mammalian expression vector comprising a murine CMV promoter and the first intron of the major immediate early gene of the human cytomegalovirus, mammalian host cells containing the expression vector and a process for the production of a recombinant protein by using the expression vector.

Mammalian expression vector comprising the mCMV promoter and first intron of hCMV major immediate early gene

The present invention relates to a mammalian expression vector comprising a murine CMV promoter and the first intron of the major immediate early gene of the human cytomegalovirus, CHO cells and CHO cell line containing this expression vector and a process for the production of a recombinant protein by using this expression vector.

The Chinese Hamster ovary cell (CHO) mammalian expression system is widely used in production of recombinant protein. Apart from lymphoid cell lines such as NS-O, it is one of the few cell types allowing for simple and efficient high-density suspension batch culture of animal cells. Furthermore, CHO cells allow for very high product yields and are comparatively robust to metabolic stresses whereas lymphoid cells are more difficult to culture at an industrial scale. In view of considerable costs of production, it is of utmost importance to maximize the yield of recombinant protein per bioreactor run. Choice of culture medium composition and bioreactor design and operation are parameters that impact yield and may be quite complex to optimize. Other factors which significantly affect the amount of polypeptide produced by a cell line are the gene copy number, the efficiency with which the gene is transcribed and the mRNA is translated, stability of the mRNA and the efficiency of secretion of the protein. Therefore, increases in the strength or transcriptional activity of the promoter controlling expression of product protein enhance yield. Incremental increases at the single cell level will translate into considerable improvements of product yield in high-density batch or fed-batch culture showing stationary phase gene expression at cell densities in the range of 106 to 107 cells/ml.

For transduction of mammalian cells, the majority of gene transfer experiments to date have used viral vectors encoding transgenes under the control of promoter elements derived from viruses. One of the most frequently used promoters in these expression cassettes is that of the human cytomegalovirus immediate early gene. The enhancer/promoter of this gene directs high levels of transgene expression in a wide variety of cell types. The activity of this promoter depends on a series of 17, 18, 19 and 21 bp imperfect repeats, some of which bind transcription factors of the NF-κB cAMP responsive binding protein (CREB) and the nuclear factor- 1 families. A disadvantage, however, of the hCMV IE promoter is its pronounced species preference.

US 5,866,359 describes a method of enhancing expression from an already strong hCMV promoter in CHO and NSO cells by co-expressing adenoviral ElA protein from a weak promoter. ElA is a multifunctional transcription factor which may act on cell cycle regulation and has both independent transcriptional activating and repressing functional domains. Fine tuning of El A expression is crucial to achieve the ideal balance between gene transactivation and any negative impact on cell cycle progression. However, unwanted overexpression of ElA expression could reduce the capacity of the cell to synthesise the recombinant protein of interest.

US 5,591,639 describes vectors containing the promoter, enhancer and complete 5'- untranslated region of the major immediate early gene of the human cytomegalovirus (hCMV-MIE) including intron A upstream of a heterologous gene. This approx. 2100 bp DNA sequence results in high level expression of several heterologous gene products. However, Chapman et al., Nucleic Acids Research, 19 (1991), 3979-3986, report that when the first 400 bp of this human sequence were present in expression plasmids, poor expression of glycoproteins was observed in both monkey kidney cells (COS7) and Chinese hamster ovary cells (DXBI l). Deletion of these upstream modulatory sequences led to higher levels of expression for several mammalian glycoproteins in these cell types. Furthermore, a comparison of the SV40 early and hCMV immediate-early promoters/enhancers showed that the activity of the hCMV promoter can be increased by inserting intron A of the major immediate early gene of the human cytomegalovirus.

It is known that the transcriptional activity of the promoter of the major immediate early gene of the mouse cytomegalovirus in CHO cells is much higher than that of the hCMV promoter. The mCMV IE promoter is able to drive high levels of expression without the pronounced species preference observed for the hCMV IE promoter (Addison, et al. Journal of General Virology (78 (1997), 1653-1661). However, attempts to enhance the activity of the mCMV promoter analogously to the hCMV promoter by inserting the natural first intron of the murine major immediate early gene downstream of the mCMV promoter failed. In contrast to the situation with hCMV promoter, such natural first intron of mCMV was found to decrease significantly the expression of a recombinant gene from the mCMV promoter (cf. WO 2004/009823 Al)

In the art there is still a need to enhance the activity of the mCMV promoter. Therefore, the technical problem underlying the present invention is to provide a mCMV promoter based expression system which allows for enhanced protein expression from the mCMV promoter in mammalian host cells, in particular CHO cells.

According to the present invention, this technical problem is solved by providing a mammalian expression vector comprising a murine CMV promoter and the first intron of the major immediate early gene of the human cytomegalovirus (first hCMV intron) operably linked to a heterologous gene sequence encoding a desired recombinant protein. The mCMV promoter plus the first hCMV intron located downstream of the mCMV promoter form a regulatory unit which drives the expression of the downstream coding sequence. The mammalian expression vector of the present invention is a particular useful expression vector construct for high level expression of recombinant gene products in CHO cells. Surprisingly, the present vector comprising within the expression cassette the mCMV promoter in combination with the first hCMV intron drives the expression of heterologous protein products at levels higher than those seen for vectors only containing the mCMV promoter. The levels of heterologous proteins expressed by the mCMV promoter plus the first hCMV intron are at least equivalent to the protein levels expressed by the hCMV promoter plus the first hCMV intron. Not wishing to be bound by any particular theory, it is believed that the presence of the first hCMV intron obviously promotes efficient protein synthesis from the corresponding mRNA. These finding are unexpected and surprising in view of the fact that the activity of the mCMV promoter could not be increased by combining with the first mCMV intron.

In the context of the present invention a "mammalian expression vector" is a, preferably isolated and purified, DNA molecule which upon transfection into an appropriate mammalian host cell provides for a high level expression of a recombinant gene product within the host cell. In addition to the DNA sequence coding for the recombinant or heterologous gene product the expression vector comprises regulatory DNA sequences that are required for an efficient transcription of mRNAs from the coding sequence and an efficient translation of the mRNAs in the host cell line. In particular the expression vector according to the invention contains at least one regulatory unit comprising at least one mCMV promoter sequence in combination with the first intron (intron A) of the major immediate early gene of the human cytomegalovirus which is operably linked to the sequence coding for the recombinant protein and drives the expression of the encoded protein. The regulatory unit comprising the raCMV promoter plus the first hCMV intron is either directly linked to the coding sequence of the heterologous gene or is separated therefrom by intervening DNA such as for example by the 5 '-untranslated region of the heterologous gene or a part therefrom.

According to the present invention the promoter of the mammalian expression vector is that of the major immediate early gene of the murine cytomegalovirus (mCMV IE or mCMV promoter). The murine CMV (mCMV) IE promoter was originally described by Dorsch-Hasler et al., Proc. Natl. Acad. Sci. USA, 82 (1985), 8325-8329, the entire disclosure of which is incorporated by reference into the present text.