A method for increasing protein expression in cells

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U.S. patent 6,762,038 suggests the use of mutant embryonic fibroblasts cell lines (MEFs) generated from mice having a homozygous disruption in their RNase L gene for enhanced expression of transfected genes. However, the effect of enhanced expression is restricted to these particular cell lines. Furthermore, the creation of new RNase L-null cell lines is a very laborious process and not readily applicable to all mammalian and/or eukaryotic expression systems.

Mammalian expression systems have become an important means for therapeutic protein and antibody production and possess several advantages over prokaryotic expression systems, e.g. with respect to proper protein folding and posttranslational modifications, such as glycolysation. However, in many systems, low protein expression yields represent a costly technical obstacle. This is particularly problematic, if the desired protein is inherently difficult to express, e.g. membrane proteins, such as G-protein linked receptors (GPCRs), large proteins, antibodies, fusion proteins, protein complexes, vaccines, and blood plasma proteins. Reasons for this difficulty could be an inherent instability of the protein itself, an inherent instability of the mRNA, as it is the case for AU-rich elements containing mRNAs, or weak promoter activity. Common solutions to these problems focused on increasing the expression of proteins by providing strong promoters, such as the CMV promoter, and enhancer elements upstream or downstream of the promoter including specific types of introns, such as intron A of CMV. Other solutions involved chromatin Matrix Attachment R.egion (MAR) elements which are 300-3000 base pairs long DNA elements that are important in nuclear and chromosomal architecture. It was proposed that these elements prevent the neighbouring chromatin from affecting transgene expression, which leads to an increased probability of isolating a clone exhibiting the desired regulated expression (U.S. patent 7,129,062). This particular approach is potentially problematic as it may involve several different vector constructs to achieve the effect. Despite the availability of these approaches, there is still a need to further increase the protein expression of proteins that are difficult to express, such as those mentioned above.

Codon optimization is another method known in the art to boost protein production. It is based on the observation that, if a nucleic acid sequence encoding the protein to be expressed contains codons (triplets) that are rarely used by the host, its expression level will not be maximal. Codon optimization basically involves altering the rare codons in the target nucleic acid sequence, so that they more closely reflect the codon usage of the host. The information usually used for the optimization process is therefore the DNA or protein sequence to be optimized and a codon usage table of the respective host. The codon usage table lists the relative frequency of each possible codon for a particular amino acid in a given organism.. Several web-based programs are also available to optimize codons based on codon usage bias for a given host organism. Codon optimization may be successful in some situations where genes of non-human or non-mammalian origin are expressed in human or other mammalian host cells, and vice versa. However, codon usage is just one of many factors influencing the expression level of a protein, and the effect of codon optimization is often limited.

It was an object of the present invention to provide for a method to significantly improve the yield of endogenous and exogenous (recombinant) proteins expressed in cells of any organism, preferably in eukaryotic cells, including proteins that are inherently difficult to express. This method should be time and cost efficient and should allow for the large-scale production of proteins in cells of any type of organism including prokaryotic and eukaryotic cells. It was another object of the present invention to achieve this improvement in expression yields without changing regulatory approved features for the production of recombinant proteins, such as the cell lines used, recombinant protein characteristics, or the use of exogenous materials, in order to allow for the production of therapeutically used proteins.

The objects of the present invention are solved by a method for increasing the expression of a protein in cells, preferably eukaryotic cells, said method comprising the step of reducing the number of RNase L cleavage sites in the nucleic acid sequence of said protein. In one embodiment, said cells are prokaryotic cells; in another embodiment, said cells are eukaryotic cells.

The nucleic acid sequence of a protein comprises both coding and non-coding regions, i.e. regions that are translated into a sequence of amino acids (also referred to as exons) and regions that are not translated into a sequence of amino acids. Non-coding regions of the nucleic acid sequence are for example the 5' untranslated region (5'UTR), the 3' untranslated region (3'UTR), and introns. All of these elements (5'UTR, 3'UTR, introns, and the coding region) can control gene and protein expression, and are, thus, targets for the above method. According to the invention, said step of reducing the number of RNase L cleavage sites reduces said number either in the coding region or non-coding region, or in both.

In one embodiment said number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50% (compared to the number of RNase L cleavage sites in the wild type nucleic acid sequence).