NZ626252B2

NZ626252B2 - Expression cassette

Info

Publication number: NZ626252B2
Application number: NZ626252A
Authority: NZ
Inventors: Gumy Christel Aebischer; Martin Bertschinger; Daniel Luescher; Pierre Moretti
Original assignee: Ichnos Sciences SA
Priority date: 2011-12-07
Filing date: 2012-12-05
Publication date: 2016-09-27

Abstract

Disclosed is an expression cassette which comprises a promoter, a polynucleotide sequence encoding a polypeptide, and expression enhancing element wherein expression enhancing element comprises a non-translated genomic DNA sequence downstream of a mammalian Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non-translated genomic DNA sequence downstream of the mammalian GAPDH promoter starts within a region spanning from nucleotide position around + 1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence downstream of the mammalian GAPDH promoter is from 95 to around 15000 nucleotides. Also disclosed is an expression cassette which comprises a promoter, a polynucleotide sequence encoding a polypeptide, and a non-translated genomic DNA sequence upstream of a mammalian GAPDH promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter starts within a region spanning from around the 5' end of the mammalian GAPDH promoter to nucleotide position around -3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, wherein the length of the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter is from 100 to around 15000 nucleotides, with the proviso that the expression cassette does not comprise a mammalian GAPDH promoter or fragments thereof. GAPDH) promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non-translated genomic DNA sequence downstream of the mammalian GAPDH promoter starts within a region spanning from nucleotide position around + 1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence downstream of the mammalian GAPDH promoter is from 95 to around 15000 nucleotides. Also disclosed is an expression cassette which comprises a promoter, a polynucleotide sequence encoding a polypeptide, and a non-translated genomic DNA sequence upstream of a mammalian GAPDH promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter starts within a region spanning from around the 5' end of the mammalian GAPDH promoter to nucleotide position around -3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, wherein the length of the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter is from 100 to around 15000 nucleotides, with the proviso that the expression cassette does not comprise a mammalian GAPDH promoter or fragments thereof.

Description

Expression Cassette Related ation This application claims beneﬁt of US provisional application No. 61/567,675, ﬁled on December 07, 201 1; all of which are hereby incorporated by reference in their ty.

The ﬁeld of the invention The present invention relates to an expression cassette useful for the expression of a polynucleotide sequence encoding a polypeptide. The present invention is also directed to vectors and host cells which comprise the expression cassette and uses of the expression te for the production of a polypeptide from a host cell.

Background of the invention Reference to any prior art in the speciﬁcation is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general dge in New Zealand or any otherjurisdiction.

Expression systems for the production ofreeombinant polypeptides are well-known in the state of the art and are described by, e.g., Marine MH (1989) Biopharm, 2: 18—33; Goeddel DV el al., (1990) Methods l 185: 3—7; Wurm F & Bernard A (1999) Curr Opin Biotechnol 10: 156- 159. Polypeptides for use in pharmaceutical applications are preferably produced in mammalian cells such as CHO cells, NSO cells, SPZ/O cells, COS cells, HEK cells, BHK cells, or the like. The essential elements of an expression vector used for this purpose are normally selected from a prokaryotic plasmid propagation unit, for e E. coli, comprising a prokaryotic origin of ation and a prokaryotie selection marker, optionally a eukaryotic selection marker, and one or more expression cassettes for the expression ofthe structural gene(s) of interest each comprising a promoter, a polynucleotide ce encoding a polypeptide, and optionally a ription terminator including a polyadenylation signal. For ent expression in mammalian cells a mammalian origin of replication, such as the SV4O Ori or OriP, can be included. As promoter a constitutive or inducible promoter can be selected. For optimized transcription a Kozak sequence may be included in the 5’ untranslated region. For mRNA processing, in ular mRNA splicing and transcription termination, mRNA splicing signals, ing on the organization of the structural gene (exon/intron organization), may be included as well as a polyadenylation .

Expression of a gene is performed either in transient or using a stable cell line. The level of stable and high expression of a ptide in a tion cell line is crucial to the overall process of the production of recombinant polypeptides. The demand for biologic molecules such as proteins and speciﬁcally antibodies or antibody fragments has increased signiﬁcantly over the last few years.

High cost and poor yield have been limiting s in the availability of biologic molecules and it 1001476446 has been a major challenge to develop robust processes that increase the yield of desirable biological molecules on an industrial scale. Thus there is still a need for improving the efficiency of expression s to obtain high expression in recombinant polypeptide production.

Summary of the invention As used herein, the term "comprise" and ions ofthe term, such as "comprisingH H , comprises" and "comprised", are not intended to exclude other additives, ents, integers or steps.

The present invention relates generally to expression systems such as expression cassettes and expression vectors which can be used to obtain increased expression in recombinant polypeptide production. In one , the present disclosure provides an sion cassette which ses a promoter, a polynucleotide sequence encoding a polypeptide, and a non-translated genomic DNA ce downstream ol‘a eukaryotic Glyceraldehyde 3~phosphate dehydrogenase (GAPDH) promoter, wherein the polypeptide encoded by the polynucleotide ce is not GAPDH, and wherein the non—translated genomic DNA ce downstream ofthe eukaryotic GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length ofthe non—translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides.

In a further aspect, the t disclosure provides an expression cassette which comprises a promoter, a polynucleotide sequence encoding a polypeptide, and a non-translated genomic DNA sequence upstream ofa eukaryotic GAPDH promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non~translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter starts within a region spanning from around the 5’ end ofthe eukaryotic GAI’DH er to nucleotide position around —3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, wherein the length of the non—translated c DNA sequence upstream of the eukaryotic GAPDH promoter is from 100 to around 15000 nucleotides, with the proviso that the expression te does not comprise a eukaryotic GAPDH promoter or fragments thereof. In a further aspect, the present sure provides an sion vector sing an expression cassette and a host cell comprising an expression cassette or an expression vector comprising an expression cassette.

In still further aspects, the present disclosure provides an in vitro method for the expression of a polypeptide, comprising transfecting a host cell with an expression cassette or an expression vector and recovering the polypeptide and the use of an expression cassette or an expression vector for the expression of a heterologous polypeptide from a mammalian host cell.

Brief ption of the ﬁgures Figure 1 shows reporter expression construct (REP) consisting of mouse galovirus promoter (mCMV), 1g donor acceptor fragment (IgDA) containing the ﬁrst intron, IgGl antibody light chain (IgGl LC), Internal Ribosomal Entry Sites derived from Eneephalomyoearditis virus (IRES), IgGl antibody heavy chain (IgGl HC), green ﬂuorescent protein (GFP) and simian virus 40 polyadenylation signal (poly (A)).

Figure 2 shows transient sion of IgG1 antibody in CHO-S cells on day 5 post- transfection (Mean of IgG titers are plotted for two independent transfections). Cells were transfected using the GAPDH_A and GAPDH_B s (GAPDH_A and GAPDHwB), the same vectors without GAPDH upstream and downstream elements (A and B) and the l). The concentration of the accumulaed lgGi antibody in the supernatant was ined using the Octet instrument (Fortebio, Menlo, CA, USA).

Figure 3 shows expression of IgG1 antibody in HEK293 EBNA cells. Cells were transfected using the GAPDH_A and GAPDH_B vectors (GAPDH_A and GAPDH_B) and the pGLEX41 vector as a control (pGLEX41). The supernatant was ted and analysed on day 10 after transfection using the Octet instrument. The data ent N = 3 independent transfections in tubespins per vector.

Figure 4 shows an expression level study on a batch production using cellular pools. Cells were transfected and pools of stable cells were created using GAPDH_A and GAPDH_B vectors (GAPDH_A(1), A(2), GAPDH_B(1) and GAPDH_B(2)), the same s without the GAPDH upstream and downstream ts (A(1) and A(2)) and the pGLEX41 vector as a control (pGLEX41). After 7 days of culture the supernatant was analyzed using the Octet ment for accumulated antibody in the atant. Mean of IgG titers are given (pg/ml) for each pool. The data represent N: 2 batches per pool.

Figure 5 shows an expression level study on tions generated by stable ection and limiting dilution. Cells were transfected using the GAPDH_A and GAPDH_B vectors (GAPDH_A and GAPDH_B), the same vectors without the GAPDH upstream and downstream elements (A and B) and the pGLEX41 vector as a control (pGLEX4l). The mean value of GFP ﬂuorescence expressed by clones and minipools from stable transfections was read 14 days after transfection. Cells were cultivated under selection pressure in 96-well plates. The data represent N= 48 clones or minipools per vector.

Figure 6 shows the effect of medium additives insulin and PMA (phorbol lZ-myristate 13- acetate, a phorbol ester) on sion of IgG1 antibody in the supernatant. After ection with the GAPDH_A vector (GAPDH_A) and the pGLEX41 vector as a l (pGLEX4l) the cells were either diluted in PowerCH02 medium, 4mM Gln, +/- insulin and PowerCH02, 4mM Gln, PMA +/- insulin. No difference in expression could be ed compared to the standard medium for pGLEX4l (ﬁlled bars) or GAPDH_A (open bars).

Figure 7 shows an ew of the human GAPDH locus. The GAPDH gene is ﬂanked by the genes NCAPD2 and IFFOl.

Figure 8 shows details of the human GAPDH gene, the GAPDH up and downstream elements and the fragments created for the is of the GAPDH upstream fragmentation study. The NruI restriction site was introduced to facilitate cloning steps and is not part of the genomic 5’ GAPDH am sequence (it is therefore highlighted using an asterisk). The sizes of the nts are: Fragment 1 (SEQ ID NO: 9): 511 bps, Fragment 2 (SEQ ID NO: 10): 2653 bps, Fragment 3 (SEQ ID NO: 11): 1966 bps, Fragment 4 (SEQ ID NO: 12): 1198 bps, Fragment 8 (SEQ ID NO: 13): 259 bps, Fragment 9 (SEQ ID NO: 14): 1947 bps, Fragment 11 (SEQ ID NO: 15): 1436 bps, and Fragment l7 (SEQ ID NO: 16): 1177 bps.

Figure 9 shows expression results of fragmentation of the GAPDH upstream and downstream elements. Expression results were obtained in transient transfection in CHO cells on day 10 after transfection. The quantification was done using the Octet instrument. Vector pGLEX4l serves as negative control. pGLEX41-ampiA also is a negative control showing the basal expression of the vector t the GAPDH ﬂanking elements. pGLEX41-up/down contains the full length ﬂanking (upstream and downstream) regions and serves as positive control.

PCT/[B2012/056977 pGLEX4l—up contains only the upstream ﬂanking region and pGLEX4l-down contains only the downstream ﬂanking . All other constructs contain the fragments described in Figure 8. The fragments 2 and 3 were either cloned in the same direction as IgGl LC and IgGl HC or in opposite direction in relation to IgGl LC and IgG1 HC (AS).

Figure 10 shows transient expression of IgG1 dy in CHO-S cells on day 8 post- transfection (Mean of lgG titers are plotted for three independent ections; error bars: SD +/—). Cells were transfected using vectors with the Chinese r GAPDH upstream element in combination with the mouse CMV (A_GAPDHﬂUP) or the Chinese hamster GAPDH promoter (A_GAPDH_UP__PR). The plasmids having only the mouse CMV (A) or the Chinese r GAPDH promoter (A_PR) were transfeeted as a l. The tration of the accumulated IgGl antibody in the supernatant was determined using the Octet QK instrument (Fortebio, Menlo, CA, USA).

Detailed description of the invention The present disclosure relates to expression cassettes and expression vectors which comprise a promoter, a polynueleotide sequence encoding a polypeptide, and a non-translated genomic DNA sequence downstrvm cf a eukaryotic aldehyde 3rphesphate dehydrogenase (GAPDH) promoter, wherein the polypeptide encoded by the polynueleotide sequence is not GAPDH, and wherein the non-translated genomic DNA sequence downstream ofthe eukaryotic GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, wherein the nucleotide position is ve to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence downstream ofthe eukaryotic GAPDH promoter is from around 100 to around 15000 tides.

The present disclosure further relates to an expression te which comprises a promoter, a eleotide sequence encoding a ptide, and a non-translated genomic DNA encoded by ce upstream of a eukaryotic GAPDH promoter, wherein the polypeptide the polynueleotide sequence is not GAPDH, and wherein the non-translated genomic DNA from ce upstream of the eukaryotic GAPDH promoter starts within a region spanning around the 5’ end of the eukaryotic GAPDH promoter to nucleotide position around —3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, wherein the length of the non-translated genomic DNA sequence upstream of the eukaryotic 2012/056977 GAPDH promoter is from 100 to around 15000 nucleotides, with the proviso that the expression cassette does not comprise a eukaryotic GAPDH promoter or fragments thereof.

The term “expression cassette” as used herein includes a polynucleotide sequence encoding a polypeptide to be expressed and sequences controlling its expression such as a promoter and optionally an enhancer sequence, ing any combination of cis-acting transcriptional control elements. The sequences controlling the expression of the gene, i.e. its ription and the ation of the transcription product, are commonly referred to as regulatory unit.

Most parts of the regulatory unit are d upstream of coding sequence of the gene and are operably linked thereto. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site. The regulatory unit of the invention is either ly linked to the gene to be expressed, i.e. transcription unit, or is ted therefrom by intervening DNA such as for example by the 5 '—untranslated region of the heterologous gene. Preferably the expression cassette is ﬂanked by one or more suitable restriction sites in order to enable the insertion of the expression cassette into a vector and/or its on from a . Thus, the expression cassette according to the present invention can be used for the uction of an expression vector, in particular a mammalian expression vector. The expression cassette of the present invention may comprise one or more e.g. two, three or even more non-translated genomic DNA sequences downstream of a eukaryotic GAPDH promoter or fragments thereof, and/or one or more e.g. two, three or even more non- ated genomic DNA sequences am of a eukaryotic GAPDH promoter or fragments thereof. If the expression cassette of the present invention comprises more than one DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter or fragments thereof these DNA ces may be directly linked, i.e. may comprise linker sequences e.g. linker ’- and 3 ’- ends and that allow sequences containing restriction sites that are attached to the 5 comfortable sequential cloning of the sequences or fragments thereof. Alternatively, the DNA sequences downstream and/or upstream of a eukaryotic GAPDH promoter or fragments thereof may be not ly linked, i.e. may be cloned with intervening DNA sequences.

The term “polynucleotide sequence ng a polypeptide” as used herein includes DNA coding for a gene, preferably a heterologous gene expressing the polypeptide.

The terms “heterologous coding sequence”, “heterologous gene sequence”, “heterologous PCT/IBZOIZ/056977 gene”, “recombinant gene” or “gene” are used interchangeably. These terms refer to a DNA recombinant heterologous protein sequence that codes for a recombinant, in ular a product that is sought to be expressed in a host cell, preferably in a mammalian cell and harvested. The product of the gene can be a polypeptide. The heterologous gene sequence is naturally not present in the host cell and is derived from an sm of the same or a different species and may be genetically modiﬁed.

The terms “protein” and eptide” are used interchangeably to e a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

The term ranslated genomic DNA sequence” as used herein includes DNA that constitutes genetic information of an organism. The genome of almost all organisms is DNA, the only ions being some s that have a RNA . Genomic DNA molecules in most organisms are organized into DNA—protein xes called chromosomes. The size, number of chromosomes, and nature of genomic DNA varies between different organisms. Viral DNA genomes can be single— or double-stranded, linear or circular. All other organisms have double—strande DNA s. Bacteria have a single, circular chromosome. In eukaryotes, most genomic DNA is located within the nucleus (nuclear DNA) as multiple linear chromosomes of different sizes. Eukaryotic cells additionally contain genomic DNA in the mitochondria and, in plants and lower eukaryotes, the chloroplasts. This DNA is usually a circular molecule and is present as multiple copies within these organelles. A non-translated genomic DNA sequence is normally not ly linked to a promoter and thus is not translated. It may n gene(s) which are not translated, thus gene(s) that encode e.g. a protein which is not expressed.

The term “non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter” as used herein corresponds to non-translated eukaryotic genomic DNA 3’ of a eukaryotic GAPDH promoter. Non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter normally starts at nucleotide position around +1, preferably at nucleotide position +1, wherein the nucleotide position is relative to the ription start of the GAPDH mRNA i.e.‘ is relative to the origin of the transcription start of the eukaryotic ream of a gene coding for GAPDH. The non—translated genomic DNA sequence eukaryotic GAPDH promoter is usually of the same origin as the eukaryotic GAPDH promoter, e.g. if the GAPDH promoter is of human origin the non—translated genomic DNA sequence ream of the human GAPDH promoter is as well of human origin and corresponds to the naturally occurring human genomic DNA sequence downstream of the human GAPDH promoter.

The term “non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter” as used herein corresponds to non-translated eukaryotic c DNA 5’ of a eukaryotic GAPDH promoter. Non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter normally starts at a nucleotide position around the 5 ’ end of the eukaryotic GAPDH er, preferably at the nucleotide position immediately after the 5’ end of the eukaryotic GAPDH promoter. The non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter is usually of the same origin as the eukaryotic GAPDH promoter, e.g. if the GAPDH promoter is of human origin the non-translated genomic DNA sequence upstream ofthe human GAPDH er is as well of human origin and corresponds to the naturally occurring human genomic DNA sequence upstream of the human GAPDH promoter.

Positions of the eukaryotic GAPDH promoter, the non-translated genomic DNA sequence downstream or upstream of the eukaryotic GAPDH promoter and other DNA ces as indicated herein are relative to the transcription start of the GAPDH mRNA e.g. are relative to the origin of the transcription start of the eukaryotic GAPDH if not speciﬁcally otherwise indicated.

The term “non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extends to” or “non-translated genomic DNA sequence ream of a eukaryotic GAPDH promoter extends to” is used to deﬁne extension of the length of non-translated c DNA sequence upstream and/or downstream of a eukaryotic GAPDH promoter from the start to a ular c element e.g. extension to an intron. This extension includes the full length of the DNA ce encoding the genetic element e.g. the intron or a part thereof.

The eukaryotic GAPDH promoter and the eukaryotic genomic DNA upstream and/or downstream of the GAPDH er can be found for human, rat and mouse in the NCBI public databank (Entries for human, mouse, rat and Chinese hamster GAPDH gene are Gene IDs 2597 (mRNA: NMHOO2046.3), 14433 (mRMA: NM_008084.2), 24383 (mRNA: NM_017008.3) and 100736557 (mRNA: NM_001244854.2), respectively; National Center for Biotechnology ation (NCBI): http://www.ncbi.nlm.nih.gov/) and are exemplarily shown in Figure 7 and 8 for the human GAPDH gene.

The eukaryotic GAPDH promoter is usually considered to stretch from around bps —500 to around +50 relative to the ription start of the GAPDH mRNA. The human GAPDH considered by promoter is located on chromosome 12. The human GAPDH promoter is Graven et al. (Graven etal., (1999) Biochimica et Biophysics Acta, 147: 203-218) to stretch from bps -488 to +20 relative to the transcription start of the GAPDH mRNA based on a fragmentation study. According to the NCBI public databank the human GAPDH promoter stretches from bps -462 to +46 relative to the transcription start of the GAPDH mRNA as deﬁned by the NCBI public databank. If not speciﬁcally otherwise indicated, the human GAPDH er as referred to herein stretches from «462 to position +46 relative to the transcription start of the GAPDH mRNA which correspond to the sequence stretching from bps 4071 to 4578 of SEQ ID NO: 17.

The ing used for the DNA of the GAPDH gene, the lFFOl gene and the NCAPD2 « fl.“ “,1 Wu AW U 9} used for gene 01 uuman, mouse 11ui L UL lgin as You,““Wed he 0'" corresponds tn the numberinu Llwrwlll u “Av A .l. ‘1 AD these genes in the NCBI public databank (http:/'/www.ncbi.nlm.nih.gov0.

The term "promoter" as used herein deﬁnes a regulatory DNA sequence generally located upstream of a gene that mediates the initiation of transcription by directing RNA polymerase to bind to DNA and initiating RNA synthesis.

The term cer" as used herein deﬁnes a nucleotide sequence that acts to potentiate the transcription of genes independent of the ty of the gene, the position of the sequence in relation to the gene, or the ation of the sequence. The vectors of the present ion optionally e enhancers.

The terms "functionally linked" and "operably " are used interchangeably and refer to a functional relationship between two or more DNA segments, in particular gene sequences to be expressed and those sequences controlling their expression. For example, a promoter and/or enhancer ce, including any combination of cis-acting transcriptional control elements is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding ce in an appropriate host cell or other sion system. Promoter regulatory sequences that are operably linked to the transcribed gene sequence are physically contiguous to the transcribed ce.

"Orientation" refers to the order of nucleotides in a given DNA sequence. For example, an orientation of a DNA sequence in opposite direction in relation to another DNA sequence is one in which the 5' to 3' order of the sequence in relation to another sequence is reversed when compared to a point of reference in the DNA from which the sequence was obtained.

Such reference points can include the direction of ription of other speciﬁed DNA sequences in the source DNA and/or the origin of replication of replicable s containing the sequence.

The term "expression vector" as used herein includes an isolated and puriﬁed DNA le which upon transfection into an appropriate host cell provides for a high-level sion of a recombinant gene product within the host cell. In addition to the DNA sequence coding for the recombinant or gene t the expression vector comprises regulatory DNA sequences that are required for an efﬁcient transcription of the DNA coding sequence into mRNA and for an efﬁcient translation of the mRNAs into ns in the host cell line.

The terms “host cell” or “host cell line” as used herein include any cells, in particular ian cells, which are capable of growing in culture and expressing a desired recombinant product protein.

The term “fragment” as used herein includes a portion of the respective nucleotide sequence e.g. a portion of the non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter or a portion ofthe nucleotide sequence encoding a ular genetic element such as a er. Fragments of a non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter may retain biological activity and hence alter e.g. increase the expression patterns of coding sequences operably linked to a promoter. Fragments of a non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter may range from at least about 100 to about 3000 bp, preferably from about 200 to about 2800 bp, more preferably from about 300 to about 2000 bp nucleotides, in particular from about 500 to about 1500 bp nucleotides. In order to clone the fragments of the non—translated c DNA sequence downstream and/or of the present upstream of a eukaryotic GAPDH promoter in the expression cassette ion, usually linker sequences containing restriction sites that allow comfortable cloning are ed to the 5’- and 3’- ends of the fragments. herein The term “nucleotide sequence identity” or “identical nucleotide sequence” as used include the percentage of nucleotides in the candidate sequence that are identical with and/or nucleotide sequence of e.g. the non-translated genomic DNA sequence downstream and ucing upstream of a otic GAPDH promoter, aﬁer aligning the sequences Thus sequence ty gaps, if necessary, to achieve the maximum percent sequence identity. to e the similarity in can be determined by standard s that are ly used position of the nucleotides of two nucleotide sequences. Usually the nucleotide sequence identity of the candidate sequence to the non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter is at least 80%, preferably at least 85%, in particular 96%, more more preferably at least 90%, and most preferably at least 95%, particular 97%, even more ular 98%, most particular 99%, ing for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

The term “CpG site” as used herein include regions ofDNA where a cytosine nucleotide its length. "CpG" is occurs next to a guanine nucleotide in the linear sequence of bases along shorthand for "——C—phosphate—G~—", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA. The "CpG" notation is used to distinguish this linear sequence from the CG base-pairing of cytosine and guanine.

The term “alternative codon usage” as used herein includes usage of alternative codons coding for the same amino acid in order to avoid the CpG sequence motif. This includes using preferably codons not having an internal CpG site (for example GCG coding for Alanine and containing a CpG site, might be replaced by either GCT, GCC or GCA) as well as ng joining oftwo codons that leads to a new CpG site.

The term “around” as used herein in relation to the length of a DNA ce and in relation to a nucleotide position which is relative to the transcription start of the GAPDH mRNA e.g. is ve to the origin of the transcription start of the eukaryotic GAPDH includes values with deviations of a maximum oft 50 % of a maximum of i 10 % of the stated , usually PCT/182012/056977 values e.g. “around 3000 nucleotides” includes values of 2700 to 3300 nucleotides, preferably 2900 to 3100 nucleotides, more preferably 2995 to 3005 tides, “around 100 nucleotides” includes values of 50 to 150 nucleotides, preferably 90 to 110 nucleotides, more preferably 95 to 105 nucleotides, “around 15000 nucleotides” includes values of 13500 to 16500 nucleotides, preferably 14500 to 15500 nucleotides, more preferably 14990 to 15010 nucleotides, most preferably 14995 to 15005 nucleotides, “around 200 nucleotides” es values of 150 to 250 tides, ably 190 to 210 nucleotides, more preferably 195 to 205 tides, “around 8000 nucleotides” includes values of 7200 to 8800, preferably 7500 to 8500 tides, more preferably 7990 to 8010 nucleotides, most preferably 7995 to 8005 nucleotides, “around 500 nucleotides” includes values of 450 to 550 nucleotides, preferably 475 to 525, more ably 490 to 510, most preferably 495 to 505 nucleotides, “around 5000 nucleotides” includes values of 4500 to 5500 nucleotides, preferably 4750 to 5250, more preferably 4990 to 5010, most ably 4995 to 5005 nucleotides, “around 1000 nucleotides” includes values of 900 to 1100 tides, preferably 950 to 1050, more ably 990 to 1010, most preferably 995 to 1005 nucleotides, “around 4500 nucleotides” includes values of 4050 to 4950 nucleotides, preferably 4250 to 4750, more preferably 4490 to 4510, most preferably 4495 to 4505 nucleotides, “around 1500 nucleotides” includes values of 1350 to 1650 nucleotides, preferably 1450 to 1550, more preferably 1490 to 1510, most preferably 1495 to 1505 nucleotides, “around 4000 nucleotides” includes values of 3600 to 4400 nucleotides, preferably 3800 to 4200, more preferably 3990 to 4010, more ably 3995 to 4005 nucleotides, d 2000 nucleotides” includes values of 1800 to 2200 nucleotides, preferably 1900 to 2100, more preferably 1990 to 2010, most preferably 1995 to 2005 nucleotides, “around 3500 nucleotides” es values of 3 150 to 3850 nucleotides, preferably 3300 to 3700, more preferably 3490 to 3510, most preferably 3495 to 3505 nucleotides, “around 2700 nucleotides” includes values of 2430 to 2970 nucleotides, preferably 2600 to 2800, more preferably 2690 to 2710, most preferably 2695 to 2705 nucleotides, “around 3300 nucleotides” includes values of 2970 to 3630 nucleotides preferably 3100 to 3500, more preferably 3290 to 3310, most preferably 3295 to 3305 nucleotides, “around 3200 nucleotides” includes values of 2880 to 3520 nucleotides, preferably 3000 to 3400, more preferably 3190 to 3210, most preferably 3195 to 3205 nucleotides, around +7000 or around position +7000 includes positions +6300 to +7700, preferably positions +6700 to +7300, more preferably positions +6990 to +7010, most ably positions +6995 to +7005, around +1 or around position +1 includes positions -10 to +10, preferably positions —5 to +5, more preferably positions —1 to +2, around 3500 or around position -3500 includes positions -3150 to -3850, preferably positions -3300 to -3700, more preferably positions -3490 to -5010, most preferably positions -3495 to -3505.

The term “around” as used herein in relation to the numbering used for the DNA of the GAPDH gene, the IFFOl gene and the NCAPD2 gene of human, mouse and rat origin as referred herein or used herein in relation to a position in a sequence of a SEQ ID number es values with deviations of a maximum ofi 500 bps, preferably 1: 100 bps, more preferably d: 10 bps, most preferably i 5 bps.

In one embodiment, the present disclosure provides an expression cassette which comprises a promoter, a polynucleotide sequence encoding a polypeptide, and a anslated genomic DNA ce downstream of a eukaryotic GAPDH promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non4translated genomic DNA sequence downstream ofthe otic GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the anslated genomic DNA sequence downstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides.

In one embediment, the length of the nonetranslated genomic DNA sequence denstream of a eukaryotic GAPDH promoter is at least around 100 nucleotides and extends at its maximum to the second last intron of the IFF01 gene or to a part thereof. In one embodiment, the length of the non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter is at least around 100 nucleotides and extends at its maximum to the last intron of the IFF01 gene.

The human IFFOl gene is located in human DNA around bps 6665249 to 6648694 of chromosome 12 (NCBI gene ID: 25900). In one embodiment, the length of the non—translated genomic DNA sequence downstream of a otic GAPDH er extending at its maximum to the last intron of the IFFOl gene in human stretches at its maximum to around bps 6650677 of chromosome 12 coding for the IFFOl gene in human (position +7021). In one embodiment, the length of the non-translated genomic DNA ce downstream of a eukaryotic GAPDH promoter extending at its maximum to the second last intron of the IFFOl gene in human stretches at its maximum to around bps 6657230 of some 12 coding the IFFOl gene in human (position + 13574). The non-translated genomic DNA ces ream of a eukaryotic GAPDH promoter extending at its maximum to the last intron of PCT/182012/056977 the IFF01 gene in human and to the second last intron of the IFFOI gene in human, respectively, are included in SEQ ID NO: 17 which shows bps 6657230 to 6639125 of chromosome 12 (NCBI gene ID: . The non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the last intron stretches to around bps 11553 of the nucleotide sequence as shown by SEQ ID NO: 17 and the non-translated genomic DNA sequence downstream of a otic GAPDH promoter extending to the second last intron stretches to around bps 18106 of the nucleotide sequence as shown by SEQ ID NO: 17.

The mouse IFFOI gene (NCBI gene ID: 320678) is located in mouse DNA around bps 125095259 to 125111800 of chromosome 6. In one embodiment, the length of the non- translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending at its maximum to the last intron of the IFF01 gene in mouse stretches at its maximum to around bps 125109211 of chromosome 6 coding for the IFFOI gene in mouse (position + 6391). In one embodiment, the length of the non—translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending at its maximum to the second last intron of the IFFOI gene in mouse stretches at its maximum to around bps 125103521 of chromosome 6 coding for the IFFOI gene in mouse (position ). The non-translated c DNA sequences downstream of a eukaryotic GAPDH promoter extending at its maximum to the last intron and to the second last intron of the IFFOI gene in mouse, respectively are included in SEQ ID NO: 18 which shows bps 125103521 to 125119832 of chromosome 6 (NCBI gene ID: 320678). The non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the last intron of the IFFOl gene in mouse stretches to around bps 10622 of the tide sequence as shown by SEQ ID NO: 18 and the non-translated genomic DNA sequence downstream of a otic GAPDH promoter extending to the second last intron of the IFFOI gene in mouse stretches to around bps 16312 of the nucleotide sequence as shown by SEQ ID NO: 18.

The rat IFFOI gene (NCBI gene ID: 362437) is located in rat DNA around bps 161264966 to 161282150 of some 4. In one embodiment, the length of the non-translated c DNA sequence downstream of a eukaryotic GAPDH er extending at its maximum to the last intron of the IFFOI gene in rat stretches at its maximum to around bps 161280937 of the chromosome 4 coding for IFF01 gene in rat (position + 5154). In one ment, the length of the non—translated genomic DNA sequence ream of a eukaryotic GAPDH PCTHB2012/056977 er extending at its maximum to the second last intron of the IFFOI gene in rat stretches at its maximum to around bps 161279451 of chromosome 4 coding for the 1FF01 gene in rat (position +6640).

The anslated c DNA sequences downstream of a eukaryotic GAPDH promoter ing at its maximum to the last intron and to the second last intron of the IFF01 gene in rat, respectively are included in SEQ ID NO: 19 which shows bps 161279451 to 161290508 of chromosome 4 (NCBI gene ID: 362437). The non—translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the last intron of the IFF01 gene stretches to around bps 9572 of the nucleotide sequence as shown by SEQ ID NO: 19 and the non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the second last intron of the IFFOl gene stretches to around bps 11058 bps of the nucleotide sequence as shown by SEQ ID NO: 19.

The Chinese hamster IFFOl gene (NCBI gene ID: 100753382) is located in Chinese hamster DNA around bps 3577293 to 3593683. In one embodiment, the length of the non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending at its re last intren of the IFFQI gene in hinese hamster stretches at its m to around bps 3579014 coding for IFFOI gene in Chinese hamster (position +6883). In one ment, the length of the non-tramlated genomic DNA sequence downstream of a eukaryotic GAPDH er extending at its maximum to the second last intron of the IFFOl gene in Chinese hamster stretches at its maximum to around bps 3585061 coding for IFFOl gene in Chinese hamster (position ). The chromosomal location is not yet annotated in the NCBI databank and the t sequence ation contains many unknown bases. Therefore the precise annotation of the limits may change with the availability of more accurate sequence information.

The non-translated genomic DNA sequences downstream of a eukaryotic GAPDH promoter extending at its maximum to the last intron and to the second last intron of the lFFOl gene in Chinese hamster, respectively are included in SEQ ID NO: 29 which shows bps 3567932 to 3585061. The non-translated genomic DNA ce downstream of a eukaryotic GAPDH promoter extending to the last intron of the IFFOl gene stretches to around bps 11083 of the nucleotide sequence as shown by SEQ ID NO: 29 and the non-translated genomic DNA last intron of sequence downstream of a eukaryotic GAPDH er extending to the second PCT/I82012/056977 the IFFOl gene stretches to around bps 17130 bps of the nucleotide sequence as shown by SEQ ID NO: 29.

In a further embodiment, the non-translated genomic DNA sequence downstream of a otic GAPDH promoter starts at the eukaryotic GAPDH polyadenylation site e.g. starts at the ﬁrst nucleotide encoding the eukaryotic GAPDH polyadenylation site. Preferably the non-translated genomic DNA ce downstream of the eukaryotic GAPDH er starts downstream of the eukaryotic GAPDH polyadenylation site e.g. starts immediately after the last nucleotide encoding the eukaryotic GAPDH polyadenylation site. Even more preferred the non-translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter starts downstream of the eukaryotic GAPDH polyadenylation site and the length of the non-translated c DNA sequence downstream of the eukaryotic GAPDH er is at least around 100 nucleotides and extends at its maximum to the second last intron of the IFF01 gene.

In one embodiment, the non-translated genomic DNA sequence downstream of the eukaryotic GAPDH er starts within a region spanning from tide position around +3881 to nucleotide position around +5000, preferably within a region spanning from nucleotide position around +3 93 1 to nucleotide position around +5000, more preferably within a region spanning from nucleotide position around +4070 to nucleotide position around +5000, wherein the nucleotide position is ve to the transcription start of the GAPDH mRNA.

A non—translated genomic DNA sequence downstream of the otic GAPDH er which starts e.g. downstream of the eukaryotic GAPDH polyadenylation site used in the present invention usually starts at a tide position around position +3 931, preferably at a nucleotide position around +4070, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA.

In human the non-translated genomic DNA sequence downstream of the human GAPDH polyadenylation site starts at around nucleotide position +3931 (relative to the transcription start of the GAPDH mRNA which corresponds to hp 8463 as shown in SEQ ID NO: 17).

Preferably, if the non-translated genomic DNA sequence downstream of the GAPDH polyadenylation site is from human, the non-translated genomic DNA sequence downstream of the GAPDH polyadenylation site starts at around +3931 (relative to the transcription start of the GAPDH mRNA; which corresponds to bp 8463 as shown in SEQ ID NO: 17) and its length is around 3357 bps corresponding to the sequence from around bps 8463 to around 11819 as shown in SEQ ID NO: 17, more preferably it starts at around +4070 (relative to the transcription start of the GAPDH mRNA which corresponds to bp 8602 as shown in SEQ ID NO: 17) and its length is around 3218 bps corresponding to the sequence from around bps 8602 to around 11819 as shown in SEQ ID NO: 17 .

In a further embodiment, the non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter ses the nucleotide sequence selected from the group consisting of SEQ ID NOs: 8 and 21 or ﬁagments f.

In a further embodiment, the non-translated genomic DNA ce downstream of the eukaryotic GAPDH promoter comprises a nucleotide ce complementary to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 8 and 21 or fragments thereof.

In a further embodiment, the non—translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter comprises a nucleotide sequence at least 80 /0 identical to the nucleotide sequence selected from the group ting of SEQ ID NOs: 8 and 21 or nts thereof.

In some embodiments, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 8 and 21 or fragments thereof, ses ﬁve or less, preferably four or less, more preferably three or less, most preferred two or less, in particular one nucleic acid modiﬁcation, wherein the nucleic acid modiﬁcation(s) are ably a nucleic acid substitution.

In a further ment, the length of the non—translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter is preferably from around 200 to around 8000 nucleotides, more preferably from around 500 to around 5000 nucleotides, even more preferably from around 1000 to around 4500 nucleotides, most preferably from around 1500 to around 4000 nucleotides, in particular from around 2000 to around 3500 nucleotides, more particular from around 2700 to around 3300, even more particular around 3200, most particular 3218 nucleotides. The length of the non—translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter as deﬁned herein does not include any linker sequences added to the non-translated genomic DNA sequence.

In a further embodiment, the anslated genomic DNA sequence downstream of the eukaryotic GAPDH promoter is orientated in the same direction as the polynucleotide sequence encoding a polypeptide.

In a further embodiment, the non-translated genomic DNA ce downstream of the eukaryotic GAPDH promoter is orientated in opposite direction in on to the polynucleotide sequence encoding a polypeptide.

In some embodiments, the expression cassette which comprises a promoter, a polynucleotide sequence encoding a ptide, and a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter ﬁlrther comprises a non—translated c DNA sequence am of a eukaryotic GAPDH promoter, wherein the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter starts within a region spanning from around the 5’ end of the eukaryotic GAPDH promoter to nucleotide position around -3500, wherein the nucleotide on is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides.

In a another embodiment, the expression te comprises a promoter, a cleotide sequence encoding a ptide, and a non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter starts within a region spanning from around the 5’ end of the eukaryotic GAPDH promoter to nucleotide position around -3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, wherein the length of the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter is from 100 to around 15000 tides, with the proviso that the sion cassette does not comprise a eukaryotic GAPDH promoter or fragments thereof.

In some embodiments, the expression cassette further ses a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter, wherein the non—translated within a genomic DNA sequence downstream ofthe eukaryotic GAPDH promoter starts region spanning from nucleotide position around +1 to nucleotide on around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, of the and wherein the length of the non-translated genomic DNA sequence downstream eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides. In these ments the non-translated c DNA sequence downstream of a eukaryotic GAPDH promoter used is e.g. as described supra.

In some embodiments, the length of the non-translated genomic DNA ce upstream the eukaryotic GAPDH promoter is preferably from around 200 to around 8000 nucleotides, even more preferably from more preferably from around 500 to around 5000 tides, around 1000 to around 4500 nucleotides, most preferably from around 1500 to around 4000 nucleotides, in particular from around 2000 to around 3500 nucleotides, more particular from around 2700 to around 3300, even more particular around 3200, most particular 3158 nucleotides in length. The length of the non-translated genomic DNA sequence am of the eukaryotic GAPDH promoter as deﬁned herein does not e any linker sequences added to the non-translated genomic DNA sequence.

In a further embodiment, the length of the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter is at least around 100 nucleotides and extends at its maximum to the start codon of the NCAPD2 gene. In a further embodiment, the length of the nonutranslated genomic DNA sequence upstream of the eukaryotic GAPDH promoter is at least around 100 nucleotides and extends at its maximum to the third last intron of the NCAPD2 gene. In a further embodiment, the length of the non-translated genomic DNA is at least around 100 nucleotides and sequence upstream of the eukaryotic GAPDH promoter extends at its maximum to the second last intron of the NCAPD2 gene. In a further embodiment, the length of the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter is at least around 100 nucleotides and extends at its maximum to the last intron of the NCAPD2 gene.

The human NCAPD2 gene (NCBI gene 1D: 9918) is d in human DNA around bps 6603298 to 6641132 of chromosome 12. In one ment, the length of the non-translated genomic DNA sequence upstream of a otic GAPDH promoter extending at its maximum to the last intron of the NCAPD2 gene in human stretches at its maximum to around 6640243 bps of chromosome 12 coding for the NCAPD2 gene in human (position -3414 relative to the transcription start of the GAPDH gene which corresponds to bp 1119 in SEQ ID NO: 17).

In one embodiment, the length of the non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its maximum to the second last intron of the NCAPD2 gene in human stretches at its maximum to around 6639984 bps of chromosome 12 coding for the NCAPD2 gene in human (position -3673 relative to the ription start of the GAPDH gene which corresponds to bp 860 in SEQ ID NO: 17).

In one embodiment, the length of the non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its maximum to the third last intron of the NCAPD2 gene in human stretches at its maximum to around 6639125 bps of chromosome 12 coding for the NCAPD2 gene in human (position —4532 relative to the transcription start of the GAPDH gene; which corresponds to bp 1 in SEQ ID NO: 17).

The non-translated genomic DNA sequence am of a eukaryotic GAPDH promoter extending at its maximum to the last intron, to the second last intron and to the third last intron of the NCAPD2 gene in human, respectively are included in SEQ ID NO: 17, which shows bps 0 to 6639125 of chromosome 12 (NCBI gene ID: 9918).

The mouse NCAPD2 gene (Gene ID: 68298) is located in mouse DNA around on 125118025 to 125141604 of some 6. In one embodiment, the length of the non- translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter (estimated to have a a length of 500 bps upstream of the transcription start) ing at its maximum to the last intron of the NCAPD2 gene in mouse stretches at its maximum to around bps 125118607 of chromosome 6 coding for the NCAPD2 gene in mouse.

In one embodiment, the length of the non-translated genomic DNA sequence am of a otic GAPDH promoter extending at its maximum to the second last intron of the NCAPD2 gene in mouse stretches at its maximum to around 125118880 bps of chromosome 6 coding for the NCAPD2 gene in mouse. of a In one embodiment, the length of the non-translated genomic DNA sequence upstream eukaryotic GAPDH er extending at its maximum to the third last intron of the NCAPD2 gene in mouse stretches at its maximum to around 125119832 bps of chromosome 6 coding for the NCAPD2 gene in mouse.

The non—translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its maximum to the last , to the second last intron and to the third last intron of the NCAPD2 gene in mouse, respectively are included in SEQ ID NO: 18, which shows bps 125103521 to 832 of chromosome 6 (NCBI gene ID: 68298). The non- ated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending to the last intron stretches to around bps 1226 of the nucleotide ce as shown by SEQ ID NO: 18 (-3006 relative to the transcription start of the mouse GAPDH mRNA). The non- translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending shown by to the second last intron stretches to around bps 953 of the nucleotide sequence as SEQ ID NO: 18 (—3279 ve to the transcription start of the mouse GAPDH mRNA). non—translated c DNA sequence downstream of a eukaryotic GAPDH promoter extending to the third last intron stretches to around bp 1 of the nucleotide sequence as shown by SEQ ID NO: 18 (—4231 relative to the transcription start of the mouse GAPDH mRNA).

The rat NCAPD2 gene (Gene ID: 362438) is located in eukaryotic DNA around position 161288671 to 417 of chromosome 4. In one embodiment, the length of the non- translated genomic DNA ce upstream of a eukaryotic GAPDH promoter extending at its m to the last intron of the NCAPD2 gene in rat stretches at its maximum to around 161289191 bps of chromosome 4 coding for the NCAPD2 gene in rat. In one embodiment, the length of the non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its m to the second last intron of the NCAPD2 gene in rat stretches at its maximum to around 161289446 bps of chromosome 4 coding for the NCAPD2 DNA sequence gene in rat. In one embodiment, the length of the non-translated genomic upstream of a eukaryotic GAPDH promoter extending at its maximum to the third last intron of the NCAPD2 gene in rat stretches at its maximum to around 161290508 bps of chromosome 4 coding for the NCAPD2 gene in rat.

The non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its maximum to the last intron, to the second last intron and to the third last intron of the NCAPD2 gene in rat, respectively are included in SEQ ID NO: 19, which shows bps 161279451 to 161290508 of chromosome 4 (NCBI gene ID: 362438). The anslated genomic DNA ce upstream of a eukaryotic GAPDH promoter extending to the last intron stretches to around bps 1318 of the tide sequence as shown by SEQ ID NO: 19 (—3 101 relative to the transcription start of rat GAPDH mRNA). The non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the second last intron stretches to around bps 1063 of the nucleotide ce as shown by SEQ ID NO: 19 (position -3356 relative to the transcription start of rat GAPDH mRNA). The non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the third last intron stretches to around bp 1 of the nucleotide sequence as shown by SEQ ID NO: 19 (position -4418 relative to the transcription start of rat GAPDH mRNA).

The Chinese hamster NCAPD2 gene (Gene ID: 100753087) is located in eukaryotic DNA around position 3544184 to 3569879. The chromosomal location is not ble on the NCBI database. In one embodiment, the length of the non—translated genomic DNA sequence upstream of a eukaryotic GAPDH er extending at its maximum to the last intron of the NCAPD2 gene in Chinese hamster stretches at its maximum to around 0 bps in Chinese hamster. In one embodiment, the length of the non—translated c DNA sequence upstream of a eukaryotic GAPDH promoter ing at its maximum to the second last intron of the NCAPD2 gene in Chinese hamster stretches at its maximum to around 3569131 bps in Chinese hamster. In one embodiment, the length of the non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its maximum to the third last intron of the NCAPD2 gene in Chinese hamster hes at its maximum to around 2 bps in Chinese hamster.

The non—translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending at its maximum to the last intron, to the second last intron and to the third last intron of the NCAPD2 gene in Chinese hamster, respectively are included in SEQ ID NO: 29, which shows bps 3567932 to 3585061. The non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter extending to the last intron stretches to around bps 1449 ofthe nucleotide sequence as shown by SEQ ID NO: 29 (-2752 relative to the transcription start of Chinese hamster GAPDH mRNA). The non-translated genomic DNA sequence downstream of a otic GAPDH promoter ing to the second last intron hes to around bps 1200 of the nucleotide sequence as shown by SEQ ID NO: 29 (position -3001 relative to the transcription start of Chinese hamster GAPDH mRNA). The 2012/056977 non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter extending to the third last intron stretches to around bp 1 of the nucleotide sequence as shown by SEQ ID NO: 29 (position -4200 relative to the transcription start of Chinese r GAPDH mRNA).

In some embodiments, the non-translated genomic DNA ce upstream of a otic GAPDH er starts usually within a region spanning from nucleotide position around -5 00 to a nucleotide position around -3500, preferably within a region spanning from nucleotide on around —576 to nucleotide position around -3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA.

In some embodiments, the non—translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter starts usually at a nucleotide position around position —5 00, preferably at a nucleotide on around -576, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA.

In human the non-translated genomic DNA sequence upstream of the human GAPDH promoter strrts "t around nucleotide position —463 (relative to the transcription start of the GAPDH mRNA which ponds to hp 4533 as shown in SEQ ID NO: 17). Preferably, if the non—translated genomic DNA sequence upstream of the GAPDH promoter is from human, the anslated genomic DNA sequence upstream of the GAPDH promoter starts at around —5 00 (relative to the transcription start of the GAPDH mRNA; which corresponds to bp 4533 as shown in SEQ ID NO: 17). More ably, if the non-translated genomic DNA sequence upstream of the GAPDH promoter is from human, the non-translated genomic DNA sequence upstream of the GAPDH promoter starts at around -576 (relative to the transcription start of the GAPDH mRNA; which corresponds to hp 4533 as shown in SEQ ID NO: 17) and its length is around 3158 bps corresponding to the sequence from around bps 800 to around 3957 as shown in SEQ ID NO: 17.

In a further embodiment, the non—translated genomic DNA sequence upstream of the otic GAPDH promoter comprises a nucleotide sequence selected from the group ting of SEQ ID N03: 7, 9, 10, 11, 12, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or fragments thereof, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 9, 10, ll, 12, 13, 14, 15 and 16 or fragments thereof, or a nucleotide PCT/182012/056977 sequence selected from the group consisting of SEQ ID N03: 20, 22, 23, 24, 25, 26, 27, 28 and 16 or fragments f. More red is a nucleotide sequence selected from the group consisting of SEQ ID N05: 10, 12, 15 and 16 or fragments thereof, more preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 12, 15 and 16 or fragments thereof, wherein nucleotide sequences comprising SEQ ID NOs: 10 and/or 16 are orientated in opposite direction in relation to the cleotide sequence encoding a polypeptide, and nucleotide sequences sing SEQ ID NOs: 12 and/or 15 are orientated in the same direction as the polynucleotide sequence encoding a polypeptide. Equally more preferred is a nucleotide sequence selected from the group consisting of SEQ ID NOS: 23, 25, 28 and 16 or fragments thereof, more preferably a nucleotide ce selected from the group consisting of SEQ ID N05: 23, 25, 28 and 16 or nts thereof, wherein nucleotide sequences comprising SEQ ID N03: 23 and/or 16 are orientated in opposite direction in on to the polynucleotide sequence encoding a polypeptide, and tide sequences sing SEQ ID NOs: 25 and/or 28 are orientated in the same direction as the polynucleotide sequence encoding a ptide.

In a further embodiment, the non-translated genomic DNA sequence upstream of the cukaryotic GAPDH promoter comprises a nucleotide ce complementary to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 9, 10, ll, l2, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or fragments thereof, preferably a nucleotide sequence complementary to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 9, 10, 11, 12, 13, 14, 15 and 16 or fragments thereof, or a nucleotide sequence complementary to the nucleotide ce selected from the group consisting of SEQ ID NOs: 20, 22, 23, 24, 25, 26, 27, 28 and 16 or fragments thereof. More preferred is a nucleotide sequence complementary to the nucleotide ce selected from the group consisting of SEQ ID NOs: 10, 12, 15 and 16 or fragments thereof. Equally more preferred is a nucleotide sequence complementary to the nucleotide sequence selected from the group consisting of SEQ ID N03: 23, 25, 28 and 16 or fragments thereof.

In a r embodiment, the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter comprises a nucleotide sequence at least 80% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 9, 10, 11, 12, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or nts f, preferably a nucleotide sequence at least 80% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOS: 7, 9, 10, 11, 12, 13, 14, 15 and 16 or fragments thereof, or a tide from the group ting sequence at least 80% identical to the nucleotide sequence selected of SEQ ID NOS: 20, 22, 23, 24, 25, 26, 27, 28 and 16 or fragments thereof. More preferred is a nucleotide sequence at least 80% identical to the nucleotide sequence ed from the group consisting of SEQ ID NOS: 10, 12, 15 and 16 or fragments thereof, more ably a nucleotide sequence at least 80% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOS: 10, 12, 15 and 16 or nts thereof, n nucleotide direction in sequences comprising SEQ ID NOS: 10 and/or 16 are orientated in opposite on to the polynucleotide sequence ng a polypeptide, and nucleotide sequences comprising SEQ ID NOS: 12 and/or 15 are orientated in the same direction as the polynucleotide sequence encoding a polypeptide. Equally more preferred is a nucleotide sequence at least 80% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOS: 23, 25, 28 and 16 or fragments thereof, more ably a nucleotide the group consisting sequence at least 80% identical to the nucleotide sequence selected from of SEQ ID NOS: 23, 25, 28 and 16 or fragments thereof, wherein nucleotide sequences comprising SEQ ID NOS: 23 and/or 16 are orientated in opposite direction in relation to the polynucleotide sequence encoding a polypeptide, and nucleotide sequences comprising SEQ ID NOS: 25 and/or 8 are orient"th in the same direction as the polynucleotide sequence encoding a ptide.

In some embodiments, the nucleotide sequence selected from the group consisting of SEQ ID NOS: 7, 9, 10, 11, 12, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or fragments thereof, comprises ﬁve or leSS, preferably four or less, more preferably three or less, most preferred two or less, in particular one nucleic acid modiﬁcation, wherein the nucleic acid modiﬁcation(s) are preferably a nucleic acid substitution.

In some embodiments, the tide sequence selected from the group consisting of SEQ ID NOS: 7, 9, ll, 14, 20, 22, 24 and 27 or fragments thereof, comprises five or less, preferably four or less, more ably three or less, most red two or less, in particular one nucleic acid modiﬁcation, wherein the nucleic acid modiﬁcation(s) are preferably a nucleic acid substitution.

In some embodiments, the tide sequence selected from the group consisting of SEQ ID NOS: 7, 9, 11, 14, or fragments thereof, comprises one nucleic acid substitution at position 16 2012/056977 relative to the start of the nucleotide sequence of SEQ ID NOs: 7, 9, 11, 14. Preferably G at position 16 relative to the start of the nucleotide sequence is replaced with T.

In some embodiments, the tide sequence selected from the group consisting of SEQ ID NOS: 20, 22, 24 and 27 or fragments thereof, comprises one nucleic acid substitution at position 13 relative to the start of the nucleotide sequence of SEQ ID NOs: 20, 22, 24 and 27.

Preferably G at position 13 relative to the start of the tide sequence is replaced with T.

In a further embodiment, the anslated c DNA ce upstream of the otic GAPDH er is orientated in the same direction as the polynucleotide sequence encoding a polypeptide.

In a further embodiment, the non-translated genomic DNA sequence upstream of the otic GAPDH promoter is orientated in opposite direction in relation to the cleotide sequence encoding a polypeptide.

In a preferred embodiment, the sion cassette comprises a promoter, a polynucleotide sequence encoding a polypeptide, and a non—translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter and a non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter as described supra. Preferably the origin of the non—translated genomic DNA sequence ream of a eukaryotic GAPDH promoter and the non- translated genomic DNA ce upstream of a eukaryotic GAPDH promoter is the same i.e. is of the same species. More preferably the origin of the non—translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter, the non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter and the host cell is the same i.e. is of the same species, e.g. the origin of the non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter, the non—translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter and the host cell is from the same mammal e.g. from human.

In some embodiments, if the non-translated genomic DNA sequence downstream and/or upstream of the eukaryotic GAPDH promoter is non-translated genomic DNA sequence from one species, the promoter of the expression cassette is not a GAPDH promoter from the same species.

PCT/1B2012/056977 In some embodiments, if the non-translated genomic DNA sequence downstream and/or upstream of the eukaryotic GAPDH promoter is non—translated genomic DNA sequence downstream and/or am of human , the promoter of the expression te is not a human GAPDH er.

In some embodiments, the er of the sion cassette is not a GAPDH er.

In one embodiment, if the expression cassette comprises a promoter, a polynucleotide DNA ce downstream sequence encoding a polypeptide, and a non—translated genomic of a eukaryotic Glyceraldehyde 3—phosphate ogenase (GAPDH) promoter, wherein the polypeptide encoded by the polynucleotide ce is not GAPDH, and wherein the non- translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, wherein the nucleotide position is ve to the transcription start of the GAPDH mRNA, and wherein the length of the non—translated genomic DNA ce downstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides and wherein the expression cassette further comprises a non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promot r, wherein the non—translated genomic DNA from sequence upstream of the eukaryotic GAPDH promoter starts within a region spanning around the 5’ end of the eukaryotic GAPDH promoter to nucleotide position around —3 500, wherein the tide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides, the promoter of the expression cassette may be a eukaryotic GAPDH promoter, preferably a mammalian GAPDH promoter, more preferably a rodent or human GAPDH promoter. In this embodiment the non-translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter starting within a region spanning from around the 5’ end of the eukaryotic GAPDH promoter to nucleotide position around -3500 is preferably located directly upstream of the eukaryotic GAPDH er, more preferably in this embodiment the expression cassette comprises the naturally occurring genomic DNA sequence comprising the eukaryotic GAPDH promoter and extending to nucleotide position around -3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA. 28 ‘ In some embodiments, the non—translated genomic DNA sequence downstream and/or upstream of the eukaryotic GAPDH promoter is of mammalian origin, e.g. the eukaryotic GAPDH promoter is a mammalian GAPDH promoter and non-translated genomic DNA sequence downstream and/or upstream of the mammalian GAPDH promoter is used as described herein.

In some embodiments, the non-translated genomic DNA sequence downstream and/or upstream of the eukaryotic GAPDH promoter is of rodent or human , e.g. the eukaryotic GAPDH promoter is a rodent or human GAPDH er and non-translated genomic DNA sequence downstream and/or upstream of the rodent or the human GAPDH promoter is used as described herein.

Preferably the anslated genomic DNA sequence downstream and/or upstream of the eukaryotic GAPDH promoter is selected from human, rat or mouse origin, more preferably from human or mouse origin, most preferably from human origin.

In some embodiments, the non-translated genomic DNA sequence downstream and/or upstream of the eukaryotic GAPDH promoter is not operably linked to the cleotide sequence encoding the polypeptide.

In some embodiments, the expression cassette ses a polyadenylation site. ably the polyadenylation site is selected from the group consisting of SV40 poly(A) and BGH (Bovine Growth Hormone) poly(A).

In some embodiments, the er and the polynucleotide sequence encoding a polypeptide of the expression cassette are operably linked.

In some ments, the promoter of the expression te is selected from the group consisting of SV40 promoter, human tk promoter, MPSV promoter, mouse CMV, human CMV, rat CMV, human EFlalpha, Chinese hamster EFlalpha, human GAPDH, hybrid promoters ing MYC, HYK and CX promoter. ‘ 29 In some embodiments, the polypeptide encoded by the expression cassette can be a non- glycosylated and glycosylated polypeptide. Glycosylated polypeptides refer to polypeptides having at least one oligosaccharide chain.

Examples for non-glycosylated proteins are e. g. non-glycosylated hormones; non- glycosylated enzymes; non-glycosylated growth factors of the nerve growth factor (NGF) family, of the epithelial growth factor (EGF) and of the ﬁbroblast growth factor (FGF) family and non-glycosylated receptors for hormones and growth factors.

Examples for glycosylated proteins are hormones and hormone releasing factors, clotting factors, anti-clotting factors, receptors for hormones or growth factors, neurotrophic factors cytokines and their receptors, T-cel] receptors, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, antibodies, chimeric proteins, such as immunoadhesins, and fragments of any of the ylated proteins. Preferably the polypeptide is selected from the group consisting of antibodies, antibody fragments or antibody dcrivates (e.g. Fc fusion proteins and particular antibody formats like bispecific antibodies). Antibody fragment as used herein includes, but is not limited to, (i) a domain, (ii) the Fab fragment consisting of ‘ L, VH, CL or CK and CH1 domains, including Fab' and Fab’-SH, (iii) the Fd nt ting of the VH and CH1 s, (iv) the dAb nt (Ward ES er al., (1989) Nature, 341(6242): 544-6) which ts of a single le domain (v) F(ab‘)2 nts, a bivalent fragment comprising two linked Fab fragments (vi) single chain FV molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird RE et al., (1988) Science, 242(4877): 423-6; Huston JS et al., (1988) Proc Natl Acad Sci U S A, 85(16): 5879-83), (vii) "diabodies" or "triabodies", multivalent or multispeciﬁc fragments constructed by gene fusion (Holliger P et al., (1993) Proc Natl Acad Sci U S A, 90(14): 6444— 8; Holliger P et al,, (2000) Methods Enzymol, 326: 461-79), (viii) scFv, diabody or domain dy fused to an Fc region and (ix) scFV fused to the same or a different antibody.

In some embodiments the expression cassette r comprises a genetic element selected from the group consisting of an additional promoter, an enhancer, transcriptional control ts, and a selectable marker, preferably a selectable marker which is expressed in animal cells. Transcriptional control elements are e.g. Kozak sequences or ription terminator elements.

In one embodiment, the genetic t is a selectable marker wherein the content of CpG sites contained in the polynucleotide sequence encoding the selectable marker is 45 or less, ably 40 or less, more preferably 20 or less, in particular 10 or less, more particular 5 or less, most particular 0 (CpG sites have been completely removed).

In a further aspect, the present disclosure provides an expression vector, preferably a mammalian expression vector comprising an sion cassette as bed supra.

In some embodiments, the expression vector comprises at least two separate transcription units. An expression vector with two separate transcription units is also referred to as a -gene vector. An example thereof is a vector, in which the first ription unit encodes the heavy chain of an antibody or a fragment f and the second transcription unit encodes the light chain of an antibody. r example is a double-gene vector, in which the two ription units encode two different subunits of a protein such as an enzyme.

However, it is also possible that the expression vector of the present invention comprises more than two separate transcription units, for example three, four or even more separate transcription units each of which comprises a different nucleotide sequence encoding a different polypeptide chain. An example therefore is a vector with. four separate transcription units, each of which contains a different nucleotide sequence encoding one subunit of an enzyme consisting of four different subunits.

In some embodiments, the expression vector further comprises a genetic element selected from the group consisting of an additional promoter, an enhancer, transcriptional control elements, an origin of replication and a selectable .

In some ments, the sion vector further comprises an origin of replication and a selectable marker wherein the content of the CpG sites contained in the polynucleotide sequence of the expression vector encoding the origin of replication and the selectable marker is 200 or less, preferably 150 or less, in particular 100 or less, more particular 50 or less, most particular 30 or less.

Any selection marker commonly employed such as thymidine kinase (tk), dihydrofolate reductase (DHFR), puromycin, neomycin or glutamine synthetase (GS) may be used for the expression cassette or the expression vector of the present invention. Preferably, the 2012/056977 expression vectors of the invention also comprise a limited number of useful restriction sites for insertion of the expression cassette for the secretion of a heterologous protein of the present ion. Where used in particular for transient/episomal expression only, the expression vectors of the invention may further comprise an origin of replication such as the oriP origin of Epstein Barr Virus (EBV) or SV40 virus for autonomous replication/episomal maintenance in eukaryotic host cells but may be devoid of a selectable marker. Transient expression in cell lacking relevant factors to facilitate replication of the vector is also possible.

The expression vector ring the expression cassette may ﬁirther comprise an expression cassette coding for a ﬂuorescent , an expression cassette coding for an ncRNA, an expression cassette coding for an antiapoptotic protein, or an expression cassette coding for a n increasing the capacity of the secretory pathway.

In a further aspect, the present disclosure provides an expression , which comprises in order: a) a non-translated genomic DNA sequence upstream and/or downstream of a otic GAPDH promoter b) a promoter c) a polynucleotid“ sequence encoding a polypeptide d) a polyadenylation site 0) an enhancer f) a non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter, or a) a non-translated genomic DNA ce am and/or downstream of a eukaryotic GAPDH promoter b) an enhancer c) a promoter (1) a cleotide sequence encoding a polypeptide e) a polyadenylation site f) a non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH promoter, or a) an enhancer b) a non-translated genomic DNA sequence upstream and/or downstream of a otic GAPDH c) a promoter d) a polynucleotide sequence ng a polypeptide e) a polyadenylation site f) non-translated genomic DNA sequence downstream and/or upstream of a eukaryotic GAPDH, wherein inclusion of the enhancer is optional, and wherein the polypeptide d by the polynucleotide ce is not GAPDH, and wherein the non-translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence downstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 nucleotides and wherein the non- translated genomic DNA sequence upstream of the eukaryotic GAPDH promoter starts within ’ end of the eukaryotic GAPDH promoter to nucleotide a region spanning from around the 5 position around -3500, n the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the anslated c DNA sequence upstream of the eukaryotic GAPDH promoter is from around 100 to around 15000 tides, with the proviso that if a) or b) is a non-translated genomic DNA sequence upstream of a eukaryotic GAPDH f) is a non-translated genomic DNA sequence downstream of a otic GAPDH and if a) or b) is a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH f) is a anslated genomic DNA sequence upstream of a eukaryotie GAPDH.

In some embodiments, the present disclosure provides an sion vector, which comprises in order: a) a non-translated genomic DNA sequence upstream of a otic GAPDH promoter b) a promoter c) a polynucleotide sequence encoding a polypeptide d) a polyadenlyation site e) an er f) a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter, wherein inclusion of the enhancer is al.

In a further aspect, the present disclosure provides an expression vector, which comprises in order: WO 84157 PCTﬂ82012/056977 a) a non-translated genomic DNA sequence upstream of a eukaryotic GAPDH promoter b) an enhancer c) a promoter (1) a polynucleotide ce ng a polypeptide e) a polyadenlyation site f) a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH, wherein inclusion of the enhancer is optional.

In a further aspect, the present sure provides an expression , which comprises in order: a) an enhancer b) a non-translated genomic DNA ce upstream of a eukaryotic GAPDH c) a promoter (1) a polynucleotide sequence encoding a polypeptide e) a polyadenlyation site i) non-translated genomic DNA ce downstream of a eukaryotic GAPDH, wherein inclusion of the enhancer is optional. in a further aspect, the present disclosure provides an expression vector, which ses in order: a) a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter b) a promoter c) a polynucleotide sequence encoding a polypeptide d) a polyadenlyation site e) an enhancer f) a non-translated genomic DNA sequence am of a eukaryotic GAPDH er, wherein inclusion of the enhancer is optional.

In a further aspect, the present disclosure provides an expression vector, which comprises in order: a) a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter b) an enhancer c) a promoter (1) a polynucleotide sequence encoding a polypeptide PCT/132012/056977 e) a polyadenlyation site t) a non-translated genomic DNA sequence am of a eukaryotic GAPDH, wherein inclusion of the enhancer is optional.

In a further aspect, the present disclosure provides an expression vector, which comprises in order: a) an enhancer b) a non-translated genomic DNA sequence downstream of a eukaryotic GAPDH c) a er (1) a polynucleotide sequence encoding a ptide e) a polyadenlyation site t) non—translated genomic DNA sequence am of a eukaryotic GAPDH, n ion of the enhancer is optional.

Non—translated genomic DNA sequence upstream of a eukaryotic GAPDH, enhancer, promoter, polynucleotide sequence encoding a polypeptide, polyadenlyation site and non- translated genomic DNA sequence downstream of a eukaryotic GAPDH promoter of the expression vectors are e. g. as described supra.

In a further , the present disclosure provides a host cell comprising an sion cassette or an expression vector as described supra. The host cell can be a human or non— human cell. Preferred host cells are mammalian cells. Preferred examples alian host cells include, without being restricted to, Human embryonic kidney cells (Graham FL et al., J.

Gen. Virol. 36: 59—74), MRCS human ﬁbroblasts, 983M human melanoma cells, MDCK canine kidney cells, RF cultured rat lung ﬁbroblasts isolated from Sprague—Dawley rats, B16BL6 murine melanoma cells, P815 murine mastocytoma cells, MTl A2 murine mammary adenocarcinoma cells, PER:C6 cells (Leiden, Netherlands) and Chinese hamster ovary (CHO) cells or cell lines (Puck et al., 1958, J. Exp. Med. 108: 5).

In a particular preferred embodiment the host cell is a Chinese hamster ovary (CHO) cell or cell line. Suitable CHO cell lines include e.g. CHO-S (Invitrogen, Carlsbad, CA, USA), CHO Kl (ATCC ), CHO pr03-, CHO DG44, CHO P12 or the dhfr- CHO cell line DUK-BII (Chasin eta1., PNAS 77, 1980, 4216—4220), DUXBI 1(Simonsen et al., PNAS 80, 1983, 2495-2499), or CHO-KlSV (Lonza, Basel, Switzerland).

In a further aspect, the t disclosure provides an in vitro method for the expression of a polypeptide, comprising transfecting a host cell with the expression cassette or an expression vector as described supra and recovering the polypeptide. The polypeptide is preferably a heterologous, more preferably a human polypeptide.

For transfecting the expression cassette or the expression vector into a host cell according to the present invention any transfection technique such as those well-known in the art, e.g. poration, m phosphate cipitation, extran transfection, lipofection, can be employed if appropriate for a given host cell type. It is to be noted that the host cell transfected with the expression cassette or the expression vector of the present invention is to be ued as being a transiently or stably transfected cell line. Thus, according to the present invention the present expression cassette or the expression vector can be maintained episomally i.e. transiently transfected or can be stably integrated in the genome of the host cell i.e. stably transfected.

A transient transfection is characterised by non—appliance of any selection pressure for a vector borne selection . In transient expression experiments which commonly last 2 to vector are up to 10 days post transfection, the transfected expression cassette or expression maintained as episomal elements and are not yet integrated into the . That is the transfected DNA does not usually integrate into the host cell genome. The host cells tend to lose the transfected DNA and overgrow transfected cells in the population upon culture of the ently transfected cell pool. Therefore sion is strongest in the period immediately following transfection and decreases with time. Preferably, a transient transfectant according to the present invention is understood as a cell that is maintained in cell culture in the absence of selection pressure up to a time of 2 to 10 days post transfection.

In a red embodiment of the ion the host cell e.g. the CH0 host cell is stably transfected with the expression cassette or the expression vector of the present invention.

Stable transfection means that newly introduced foreign DNA such as vector DNA is becoming incorporated into genomic DNA, y by random, mologous recombination events. The copy number of the vector DNA and concomitantly the amount of the gene product can be increased by selecting cell lines in which the vector sequences have been ampliﬁed after integration into the DNA of the host cell. Therefore, it is possible that such stable ation gives rise, upon exposure to further increases in selection pressure for gene ampliﬁcation, to double minute chromosomes in CHO cells. rmore, a stable transfection may result in loss of vector sequence parts not directly related to expression of the recombinant gene product, such as e.g. ial copy number control regions rendered superﬂuous upon genomic integration. Therefore, a transfected host cell has integrated at least part or different parts of the sion cassette or the expression vector into the genome.

In a further aspect, the present disclosure provides the use of the expression cassette or an expression vector as described supra for the expression of a heterologous polypeptide from a mammalian host cell, in particular the use of the expression cassette or an expression vector as described supra for the in vitro expression of a heterologous polypeptide from a mammalian host cell.

Expression and recovering of the protein can be carried out according to methods known to the person d in the art.

For the expression of a polypeptide, the anslated genomic DNA sequence ream and/or upstream of a eukaryotic GAPDH promoter of the expression cassette or of the expression vector as described supra and the host cell as described supra are used and are usually of the same origin. Surprisingly it has been found that an se of sion is obtained if the non-translated genomic DNA sequence downstream and/0r upstream of a eukaryotic GAPDH promoter of the expression cassette or of the expression vector and the host cell are of different origin e.g. if human DNA sequences downstream and/or upstream of a eukaryotic GAPDH er are used in CHO cells.

In a further aspect, the present disclosure provides the use of the expression cassette or the expression vector as described supra for the preparation of a medicament for the treatment of a disorder.

In a further aspect, the present disclosure es the expression cassette or the sion vector as described supra for use as a medicament for the treatment of a disorder. 2012/056977 In a further aspect, the present disclosure provides the expression cassette or the expression vector as described supra for use in gene therapy.

PCT/132012/056977 Examples Example 1: Cloning of expression vectors: 1. Materials and Methods 1.1 Plasmids constructs 1.1.1. LB culture plates 500 ml of water were mixed and boiled with 16 g of LB Agar (Invitrogen, Carlsbad, CA, USA) (1 litre of LB contains 10 g tryptone, 5 g yeast extract and 10 g NaCl). After cooling down, the respective antibiotic was added to the solution which is then plated (ampicillin plates at 100 pg/ml and kanamycin plates at 50 . 1.1.2. Polymerase Chain on (PCR) All PCR were performed using 1 p1 of dNTPs (10 mM for each dNTP; Invitrogen, Carlsbad, CA, USA), 2 units of Phusion® DNA Polymerase (Finnzymes Oy, Espoo, Finland), 25 nmol of Primer A (Mycrosynth, Balgach, Switzerland), 25 nmol of Primer B (Mycrosynth, Balgach, Switzerland), 10 pl of 5X HF buffer (7.5 mM MgC12, Finnzymes, Espoo, Finland), 1.5 pl of Dimethyl sulfoxide (DMSO, Finnzymes, Espoo, Finland) and 1-3 pl of the template (1-2 pg) in a 50 pl ﬁnal volume. All primers used are listed in Table l.

The PCR were d by an initial denaturation at 98°C for 3 minutes, followed by 35 cycles of 30 sec denaturation at 98°C, 30 sec annealing at a ~specific temperature (according to CG content) and 2 min tion at 72°C. A ﬁnal elongation at 72°C for 10 min was med before cooling and keeping at 4°C.

Table 1: Summary of primers used in PCRs. GAPDH: Glyceraldehyde phate dehydrogenase “T” (underlined) in primer G1nPr1172 sequence, 5’: upstream sequence, 3: downstream ce. The was introduced in order to avoid the formation ofprimer dimers.

Sequence Primer sequence ampliﬁed ATTATTCGCGATGGCTCCTGGCA SEQ ID GGACCGAGGC ATCGTCGCGAAGCTTGAGATTGI SEQ ID CCAAGCAGGTAGCCAG AGCAAGTACTTCTGAGCCTTCA SEQ ID GTAATGGCTGCCTG 3’GAPDH TGGCAGTACTAAGCTGGCACCA SEQ ID CTACTTCAGAGAACAAG 1.1.3. Restriction digest For all restriction digests around 1 pg of plasmid DNA (quantified with NanoDrop, ND—lOOO Spectrophotometer (Thenno Scientiﬁc, Wilmington, DE, USA)) was mixed to 10—20 units of each enzyme, 4 pl of ponding 10X NEBuffer (NEB, Ipswich, MA, USA), and the volume was completed to 40 HI with sterile H20. Without further indication, digestions were incubated 1 h at 37°C.

After each preparative digestion of backbone, 1 unit of Calf Intestinal ne Phosphatase (CIP; NEB, Ipswich, MA, USA) was added and the mix was incubated 30 min at 37°C.

If the digest was done in NEBuffer 3 (NEB, Ipswich, MA, USA), the buffer was changed to NEB buffer 4 before adding the CIP because this enzyme has a strong activity in this buffer and may also digest some of the nucleotides at the external ends. 1.1.4. PCR puriﬁcation and agarose gel electrophoresis 1.1.4.1. PCR clean up To allow digestion all PCR fragments were cleaned prior to ction digests using the Macherey Nagel Extract II kit (Macherey Nagel, Oensingen, Switzerland) ing the manual ofthe cturer using 40 p1 of elution buffer. This protocol was also used for changing buffers of DNA samples.

WO 84157 PCT/IBZOI2/056977 1.1.4.2. DNA extraction For gel electrophoresis, 1% gels were prepared using UltraPureTM Agarose (Invitrogen, Carlsbad, CA, USA) and 50X Tris Acetic Acid EDTA buffer (TAE, pH 8.3; Bio RAD, Munich, Germany). For staining ofDNA 1 ul of Gel Red Dye (Biotum, Hayward, CA, USA) was added to 100 m1 of agarose gel. As a size marker 2 ug of the 1 kb DNA ladder (NEB, Ipswich, MA, USA) was used. The electrophoresis was run for around 1 hour at 125 Volts.

The bands of interests were cut out from the agarose gel and puriﬁed using the kit Extract II (Macherey-Nagel, Oensingen, Switzerland), following the manual of the manufacturer using 40 ul of elution . 1.1.5. Ligation For each ligation, 4 ul of insert were mixed to 1 ul of vector, 400 units of ligase (T4 DNA ligase, NEB, Ipswich, MA, USA), 1 ul of 10X ligase buffer (T4 DNA ligase buffer; NEB, Ipswich, MA, USA) in a 10 ul volume. The mix was incubated for l—2 h at RT. 1.1.6. Transformation of ligation products into competent bacteria For the cloning ofpGLEX4 l -[REP] and for constructs made with the pCR—Blunt vector which contain a standard origin of replication, TOP 10 (One Shot® TOP 10 Competent E. coli; Invitrogen, ad, CA, USA) were used.

For replication initiation of plasmid containing the R6K origin of replication, the expression of the 1: protein, coded by the pir sequence, is required. The 7: protein is expressed by One Shot® PIRl competent E. coli (Invitrogen, Carlsbad, CA, USA). These bacteria were used for all s containing the R6K sequence.

To transform ent bacteria with the ligation product, 25-50 ul of bacteria were thawed on ice for 5 s. Then, 3-5 ul of ligation product were added to competent bacteria and ted for 20-30 min on ice before the thermic shock for 1 minute at 42°C. Then, 500 ul of S.O.C medium rogen, Carlsbad, CA, USA) were added per tube and incubated for 1 hour at 37°C under agitation. Finally, the bacteria are put on a LB plate with ampicillin (Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 37°C. For the cloning in pCR-Blunt vectors, plates with kanamycin (Sigma-Aldrich, St. Louis, MO, USA) were used.

W0 2013/084157 1.1.7. Plasmid preparation in small (mini) and medium scale (midi) 1. Minipreparation For minipreparation, es of transformed bacteria were grown for 6-16 hours in 2.5 ml of LB and ampicillin or cin at 37°C, 200 rpm. The DNA was extracted with a plasmid ation kit for Ecoli (QuickPure, Macherey Nagel, Oensingen, Switzerland), following the provided manual.

Plasmid DNA from minipreparations was quantiﬁed once with the op O Spectrophotometer (Thermo Scientiﬁc, Wilmington, DE, USA) by measuring the absorbance at 260 nm and assessing the ratio of the OD260 nm/OD280 nm that had to be between 1.8 and 2. A control ion was performed before sending the sample to Fasteris SA (Geneva, Switzerland) for sequence conﬁrmation.

For BAC extraction, the QuickPure kit (Macherey Nagel, Oensingen, Switzerland) was used with the following modiﬁcation of the protocol: 10 ml of LB and chloramphenicol (12.5 ug/ml) (Sigma—Aldrich, St. Louis, MO, USA) were seeded with bacteria containing pBACe3.6 vector. After incubation on a shaking platform at 37°C over night, the culture was centrifuged for 5 min at 13 300 rpm before being resuspended in 500 ul of Al Buffer. 500 ul ef A2 Lysis Buffer were added and the solution was ted 5 min at RT. Then, it was neutralized with 600 ul of A3 buffer and centrifuged 10 min at 13 300 rpm. The supernatant was loaded on a column and from this step onwards the rd protocol of QuickPure miniprep kit was used. 2. Midipreparation For midipreparation, transformed bacteria were grown at 37°C overnight in 200 to 400 ml of LB and ampicillin (or kanamycin). Then, the culture was centrifuged 20 min at 725 g and the plasmid was puriﬁed using a commercial kit (NucleoBond Xtra Midi; Macherey Nagel, Oensingen, Switzerland) following the low plasmid protocol provided in the manual of the manufacturer.

Plasmid-DNA from midipreparation was quantiﬁed three times with the NanoDrop O Spectrophotometer, conﬁrmed by restriction digest and ﬁnally sent for sequencing (Fasteris SA, Geneva, Switzerland).

PCT/132012/056977 II. Results and Discussion II.1. Cloning of DNA regions upstream and downstream of the GAPDH expression cassette (5’ and 3’GAPDH) The BAC clone RPCIB753F11841Q was ordered at Imagene (Berlin, Germany). This clone contains the human GAPDH sequence in a pBACe3.6 vector backbone, containing a chloramphenieol resistance gene. After DNA extraction by minipreparation, the vector concentration was determined by Nanodrop to 27 ng/ul.

DNA sequences immediately surrounding the GAPDH sion cassette upstream of the promoter and downstream of the denylation site were ampliﬁed by PCR using 27 ng of the puriﬁed clone RPCIB753F11841Q as template. The 3 kb fragment upstream of the promoter was ampliﬁed with s GlnPr1171 (SEQ ID NO: 1) and GlnPr1172 (SEQ ID NO: 2) leading to the amplicon with SEQ ID No. 5. As primer GlnPrl 172 (SEQ ID NO: 2) carries a base change (G to T) relative to the template sequence, all sequences derived from this PCR reaction will carry this base change, too. The Change is located in position —3721 ve to the transcription start of the GAPDH gene (bp 812 of SEQ ID NO: 17, position 23 relative to the start of SEQ ID NO: 5). The 3kb fragment downstream of the polyadenlyation site was ampliﬁed with primers GlnPr1173 (SEQ ID NO: 3) and GlnPrl 174 (SEQ ID NO: 4) leading to the amplicon with SEQ ID NO: 6 (Table 1). The annealing temperature used for these PCRs was 72°C.

The 5’and 3’GAPDH fragments (SEQ ID NOS: 5 and 6) were cloned in unt, a commercially available oduct cloning vector (pCR-Blunt, PCR Zero Blunt cloning kit, Invitrogen). The on products were transformed into TOPIO competent bacteria and plated on kanamycin LB-agar plates. Colonies were ampliﬁed and plasmids were isolated by minipreps. Control digests were performed to identify positives clones yielding unt- H and pCR—Blunt-3 ’GAPDH constructs.

II.2. Preparation of the DNA fragment coding for the reporter proteins GFP and a recombinant IgGl monoclonal dy (LC-IRES-HC-IRES-GFP) The reporter construct (REP) used in the present work consisted in a polycistronic gene: IgGl monoclonal antibody light chain RES— IgGl onal antibody heavy chainaiC} IRES- green fluorescent protein (GFP). The presence of Internal Ribosomal Entry Sites (IRES) derived from Encephalomyocarditis virus (Gurtu er al., Biochem Biophys Res PCTle2012/056977 Commun; 229(1): 295~298, 1996)) allows the translation of the 3 peptides IgGl monoclonal antibody light chain (LC), IgGl monoclonal antibody heavy chain (HC) and GFP (Fig. 1).

Transfected cells will therefore secrete the IgGl monoclonal dy and accumulate intracellular GFP in a dependent manner. However, polycistronic mRNAs are not common in eukaryotic cells and their translation is not very efﬁcient, leading to relative low titers of IgGl and GFP expression.

A vector containing the REP construct was digested using the restriction enzymes NheI and BstBI (BstBI is used at 65°C). The REP fragment containing the sion construct was cut out, puriﬁed and used for further cloning steps. 11.3. Cloning of expression vectors The vector pGLEX4l, an sion vector derived from pcDNA3.l (+) (Invitrogen, ad, CA) was used for stable cell line production. It was used as l backbone that had been modiﬁed to generate the second generations of vectors A and B with and t the GAPDH sequences. For all vectors the same promoter-intron combination (mCMV and a donor~acceptor nt coding for the ﬁrst intron (IgDA)) was used (Gorman et al., (1990) Proc Natl Acad Sci USA, 87: 5459-5463).

Cloning of intermediate vector 1-HM-MCS-ampiA: The development of the new vector generation was started from pGLEX41. This vector was cut using the restriction s Nru] and Bsle in order to release the ampicillin resistance cassette. The backbone fragment was CIPed and puriﬁed by gel electrophoresis. The DNA fragment coding for a codon optimized (for expression in E. coli) version of the ampicillin resistance gene (including the bla promoter) has been ordered from GeneArt. The insert was cut out of the GeneArt cloning vector #1013237 using the restriction enzymes NruI and Bsle (the same enzymes as used for the backbone), puriﬁed and cloned into the ne.

Minipreps were analyzed by restriction digest. The clone pGLEX4l-HM-MCS-ampiA#2 had the expected restriction proﬁle and the integration of the correct fragment was conﬁrmed by sequencing. g of intermediate vector pGLEX41-MCS-R6K-ampiA In order to exchange the pUC origin of replication of the vector pGLEX41-HM-MCS- ampiA#2 the vector was digested using PvuI and Bsle. The ne fragment was CIPed PCT/182012/056977 and puriﬁed. The new insert fragment contains the R6K origin of replication and a d SV40 poly(A) sequence as part of the expression cassette. Unnecessary bacterial or viral backbone sequences around the SV40 poly(A) had been eliminated (see Table 2 below). The insert fragment has been ordered from GeneArt; it was cut out of the GeneArt cloning vector #1013238 using the enzymes PvuI and Bsle (the same as used for the backbone), puriﬁed and cloned into the backbone fragment. Minipreps were prepared and were conﬁrmed by sequence analysis. The clone pGLEX41-MCS—R6K—ampiA#1 had the correct sequence.

Table 2: Content of CpG in the different vectors — CpG content in expression vectors ors:“A”pGLEX41 Codon optimized CpG reduced Vectors “B” Cloning of intermediate vector 1—MCS-R6K-ampiB The vector 1— MCS—R6K-ampiA#1 was opened using the ction enzyme BspHI and CIPed in order to e the ampicillin resistance. The new insert fragment contains the ampicillin resistance codon optimized for expression in E. coli, but all the CpG sequences that could be eliminated by alternative codon usage had been replaced (see Table 2 . This fragment was ordered at GeneArt. In order to release the insert nt, the GeneArt cloning vector #1016138 was digested using BspHI. After puriﬁcation of both insert and backbone fragments by gel ophoresis, they were ligated and transformed into PIRl bacteria. The minipreps were directly sent for sequencing. pGLEX41-R6K-MCS-ampiB#l has the correct sequence and was used for further cloning steps.

Cloning of the reporter construct in pGLEX41-derived expression vectors In order to clone the er construct REP in the expression vectors pGLEX41- MCS-R6K- ampiA and pGLEX41-MCS-R6K-ampiB, the vectors were cut using the restriction enzymes NheI and ClaI. The expression vector pGLEX41-HM-MCS was opened using the restriction treated with CIP after enzymes NheI and BstBI (at 65°C). All vector backbones were ion and the backbones d by gel electrophoresis. The backbones were ligated with the NheI/BstBI (BstBl is compatible with ClaI) fragment coding for the reporter construct REP. The ligation products were transformed into PIRl or TOP 10 competent bacteria and plated on ampicillin LB-agar plates. Colonies were ampliﬁed and plasmids were isolated by minipreps. Positive clones could be identiﬁed by ction digest of minipreps and subsequent sequence conﬁrmation by Fasteris SA.

Addition of ﬂanking GAPDH sequences in pGLEX41 derived expression vectors All restriction digest of this paragraph were performed in a 80 pl ﬁnal volume and incubated over night at 37°C.

S’GAPDH ce (SEQ ID NO: 7) was excised from pCR—blunt—S‘GAPDH using the restriction enzyme Nrul and ligated in the expression vectors pGLEX4l—R6K-ampiA—[REP] and pGLEX4l-R6K—ampiB—[REP] which were ized using Nrul and d with ClP in order to avoid re-circularization. After ampliﬁcation of PlRl es (obtained by transformation of ligation products) minipreps were analyzed by restriction digest. Clones pGLEX4l—R6K-ampiA-5’GAPDH—[REP] #2 and l-R6K—ampiB—5’GAPDH-[REP] #1 showed bands of the expected size in the restriction analysis, were subsequently ed by sequencing and used for ﬁlI‘thCI‘ g steps. These new vectors were then opened with Seal and treated with CIP. The 3’GAPDH fragment (SEQ ID NO: 8) was excised from pCR— Blunt—3’GAPDH using the same enzyme and ligated into the two backbones in order to generate pGLEX41-R6K-ampiA-GAPDH-[REP] and pGLEX4 l —R6K-ampiB-GAPDH-[REP] expression vectors.

The control digest of clones pGLEX4l-R6K-ampiA-GAPDH-[REP] #2 and pGLEX4l—R6K- ampiB—GAPDH-[REP] #8 showed bands ofthe expected size in the restriction analysis .The insertion of the 3 ’GAPDH fragment in the correct orientation was subsequently conﬁrmed by sequencing (Fasteris). 11.4. Cloning of ance vectors Starting point for the cloning of the resistance vectors was the vector pGLEX-MCS-R6K- ampiA#l. As for expression of resistance genes a weak promoter is sufﬁcient, the mCMV promoter was replaced by the SV40 promoter. The genes coding for the resistance genes were PCT/182012/056977 ordered from GeneArt SA (Regensburg, Germany) and either optimized for sion in Chinese r (puromycin: puroA and neomycin: neoA) or reduced in CpG t by selective codon usage (puromycin: puroB and neomycin: neoB). g of pGLEX—R6K-AmpiA-PuroA/PuroB: In order to clone the puromycin resistance in the expression cassette, the vector pGLEX41- MCS-R6K-ampiA#l was opened using the restriction enzymes NruI and XbaI followed by treatment with CIP. The insert fragment was ordered from GeneArt and was provided as insert in GeneArt cloning vector #1013239. It contains the SV40 promoter and the codon optimized gene for the puromycin resistance (for codon usage of CHO . The insert was cut out of the GeneArt cloning vector using the enzymes NruI and XbaI (the same as used for the backbone), puriﬁed and cloned into the backbone fragment. Minipreps were prepared and analyzed by restriction digest. The clone pGLEX—MCS-R6K-ampiA—puroA#1 showed the correct proﬁle and could be conﬁrmed by sequencing.

This vector was used for the cloning of the vector pGLEX—MCS—R6K-ampiA-puroB by exchange of the coding region for the puromycin resistance gene, while leaving the SV40 promoter. The new insert fragment contains a optimized version of the puromycin gene, where all the CpG sequences that could be eliminated due to ative codon usage had been replaced. The fragmcnt has been ordered by GeneArt and was delivered in the cloning vector # 1016139. In order to release the insert fragment, the GeneArt vector was ed using the restriction enzymes XbaI and NotI. The insert fragment was d by gel electrophoresis and cloned into the backbone of pGLEX-MCS~R6K~ampiA-puroA, after release of the puromycin open reading frame by restriction digest using XbaI and NotI, followed by CIP treatment. The resulting vector pGLEX-MCS-R6K-ampiA-puroB#1 was conﬁrmed directly by ce analysis.

Cloning of the vectors pGLEX-R6K-ampiA—NeoA and pGLEX-R6K-ampiA-NeoB In order to clone the neomycin resistance in the expression cassette, the vector pGLEX-R6K- puroA#1 was opened using the restriction enzymes XbaI and NotI, followed by ent with CIP. The insert fragments were ordered from GeneArt and were provided as inserts in GeneArt cloning vectors #1013242 (neoA) and #1026894 (neoB). They contain the codon optimized gene for the neomycin resistance for codon usage of CHO cells and the CpG reduced version of the in resistance, respectively. The inserts were cut out of the WO 84157 GeneArt cloning vectors using the enzymes XbaI and NotI (the same as used for the backbone), puriﬁed and cloned into the backbone fragment. Minipreps were prepared and the clones were conﬁrmed by sequencing. g of vectors pGLEX-R6K-ampiB—NeoB and pGLEX41-R6K-ampiB-puroB: The vector pGLEX4l-R6K-puroB#1 was opened using the ction enzyme BspHI and subsequently CIPed. The insert fragment contains the llin resistance gene that was codon optimized for expression in E. coli, while all CpG sequences that could be eliminated due to alternative codon usage had been replaced. This fragment has been ordered at GeneArt and arrived in the cloning vector #1016138. In order to release the insert fragment the GeneArt cloning vector was digested using BspHI. Aﬂer ation of both insert and backbone fragment by gel electrophoresis, they were ligated and transformed into PIRl bacteria. The minipreps were directly sent for sequencing and could be confirmed (pGLEX4l~ ampiB—R6K-puroB#l).

The g leading to vector pGLEX—R6K-ncoB-ampiB was done by opening pGLEX—R6K— ncoB-ampiA using the restriction enzymes BspHI in order to create the backbone fragment.

Digestion of pGLEX-RéK-ampiB-hygroB using the same restriction enzyme combination yielded the insert fragment coding for ampiB. The ampiB insert was cloned into the pGLEX- R6K-neoB—ampiA backbone. 11.5 Addition of sequences upstream and downstream of the human GAPDH gene into resistance vectors The vector pCR-blunt-S ’GAPDH was digested with NruI in order to obtain the 5 ’GAPDH insert (3164 bps). The vectors coding for resistance genes were digested with Nrul, subsequently d with CIP (Calf intestinal phosphatase, NEB, Ipswich, MA) in order to e the backbone fragments. The 4 different ne fragments -R6K—neoA- ampiA, pGLEX-R6K-neoB-ampiB, pGLEX-R6K—puroA-amp'LA and pGLEX-puroB—ampiB) were ligated with the 3164 bps 5 ’GAPDH insert and transformed into PIRl competent bacteria. Restriction digest of minipreps using ApalI allowed the identiﬁcation of clones pGLEX-R6K-neoB-ampiB-5’GAPDH#5, R6K-neoA-ampiA—5’GAPDH #6, pGLEX- R6K—puroA-ampiA-5 ’GAPDH #16 and pGLEX-puroB-ampiB-S’GAPDH #5.

PCT/[82012/056977 These intermediate vectors were then cut with the restriction enzyme Seal and treated with CIP in order to prepare the backbones for ligation. The vector carrying the second insert fragment, pCR-Blunt-3 ’GAPDH, was cut using Seal in order to release the insert fragment (3224 bps) the GAPDH downstream ﬂanking region. The four different ne molecules were ligated with the puriﬁed 3224 bps insert nt and transformed into PIRl competent cells. Minipreps were analyzed by restriction digest. Clones showing restriction nts of the expected size were pGLEX-R6K—neoB-ampiB-GAPDH #8, pGLEX-R6K-neoA-ampiA- GAPDH #1, pGLEX-R6K-puroA—ampiA—GAPDH #1 and puroB-ampiB-GAPDH #4.

The clones were subsequently conﬁrmed by cing analysis ris, Geneva, Switzerland). 11.1.5. Midipreparations of ds cloned for transfection In order to have sufﬁcient quantities of plasmids, midipreps were prepared using the Macherey Nagel kit (NucleoBond Xtra Midi; Macherey Nagel, Oensingen, Switzerland).

After conﬁrmation by restriction digest and sequencing, the plasmids were linearized and used for transfection in CHO-S cells. Table 3 summarizes the trations of plasmid DNA batches obtained in mi dipreparations, linearized DNA preps that had been prepared for transfection, the enzymes used for linearization and the sequence ﬁles from Fasteris SA conﬁrming the identity and the sequence information of the respective plasmid. All midipreps were conﬁrmed by sequencing before being used for transfections.

Table 3: Summary of plasmids cloned. Concentration of DNA midipreparation and linearized midipreparation (with the ponding enzyme). The GSC number codes for the respective plasmid and allows to identify relevant sequencing ﬁles.

Cone. of Midi— Cone. of Glenmark Plasmlds, E me for preparation l'niflbrization linearized plasmid (pg/ml) plasmids (pg/ml) code pGLEX4l-R6K-AmpiA- 1538 EcoRV 1019 GSC 2774 [REP]-GAPDH pGLEX41-R6K-AmpiB- 1243 EcoRV 1233 GSC 2775 GAPDH pGLEX-RéK-AmpiA-neoA- GSC 2776 GAPDH pGLEX-R6K-AmpiB-neoB- GAPDH pGLEX-R6K- AmpiA- 917 GSC 2778 puroA- GAPDH pGLEX-AmpiB—puroB- GAPDH pGLEX4 l— [REP] 21 19 88 GSC 2239 pGLEX4 1 —R6K-AmpiA— BspHI GSC 2240 [REP] pGLEX4 l -R6K-AmpiB- 1751 Bsle GSC 2249 [REP] pGLEX—R6K-AmpiA-neoA— BspHI GSC 2214 pGLEX-RéK—AmpiB-neoB Bsle GSC 2244 R6K-AmpiA—puroA GSC 2220 pGLEX—R6K—AmpiB-puroB BspHI GSC 2213 PCTH82012/056977 Example 2: Transfection of cells with expression vectors: 1. Materials and Methods CHO-S cells and HEK293 cells Mammalian cells are the preferred host to express proteins e they are capable of correct folding, assembly and post-transcriptional modiﬁcation of recombinant proteins. The CHO cell line was used because they are well characterized and do not serve as a host for most human pathogenic viruses, making them a relatively safe host for stable therapeutic protein tion. Chinese Hamster Ovary cells (CHO-S, Invitrogen, Carlsbad, CA, USA) were cultured in suspension in PowerCHO-2 CD medium (Lonza, rs, Belgium), supplemented with 4 mM L-glutamine (Applichem, Germany) and incubated in a g incubator (200 rpm with a circular stroke of 2.5 cm) at 37°C, 5% C02 and 80% humidity.

HEK293 cells are used because they are easy to transfect and allow rapid production of recombinant proteins up to lower gram amounts. The cells used are HEK293-EBNA cells (ATCC, Manassas, VA) and are routinely cultured in suspension in Ex—cell 293 medium -Aldrich, St. Louis, MI).

Subcultures of CHO—S and HEK293 EBNA cells were routinely carried out every 3—4 days using a seeding density of 0.5x106 Viable cells/ml in fresh . The cells were cultivated using 10 ml of medium in 50 ml ctor tubes (Tubespin Bioreactor 50; TPP, Trasadingen, Switzerland) containing a permeable ﬁlter allowing gas exchange. The cell Viability and concentration were determined with the ss automated cell counter (Invitrogen, Carlsbad, CA, USA) using the trypan blue cell exclusion method. Cell tration was conﬁrmed by determination of the packed cell volume (PCV) method using PCV tubes (TPP, Trasadingen, Switzerland) for CHO-S cells.

Packed cell volume (PCV) The PCV method is based on the centrifugation of a speciﬁc volume of culture liquid in a mini-PCV tube (PCV Packed Cell Volume Tube; TPP, Trasadingen, Switzerland) for 1 min at 5000 rpm. During centrifugation, the cells are pelleted in the graduated capillary at the base of the tube. The tage of packed cell volume is then ined by assessing the volume of the pellet in relationship to the amount of cell culture ﬂuid centrifuged. For example, 1% PCV indicated that 10 ul of cell pellet was present in 1 ml of culture ﬂuid.

PCT/182012/056977 For routine cell counting of cells, 200 pl of each sample was pipetted in a PCV tube and the volume of the corresponding pellet (in ul) was read with a ruler (“easy read” measuring ; TPP, Trasadingen, Switzerland). This volume was multiplied by 5 to have the value for 1 m1 and then it was multiplied using a cell speciﬁc correlation factor to obtain an estimation of the tration of viable cells (in millions of cells/ml).

“Automatized” cell counting Cell tration and viability was determined with the Countess® Automated Cell Counter rogen, Carlsbad, CA, USA) in mixing the sample with the same amount of trypan blue.

The solution is then pipetted into the Countess® chamber slide before being read by the instrument. This instrument allows an automatic read-out of the Neubauer chamber which, after calibration, determines cell viability and the tration of dead and living cells.

Flow Cytometry analysis Flow try is a technique for the analysis of multiple parameters of individual cells. This technique allows the quantitative and qualitative analysis of cells that are phenotypically different from each other, for instance dead from Viable cells (according to the size and the granularity of cells). It also allows the quantiﬁcation of cells which express a protein of interest, such as GFP. Cells were ted from the culture by sterile ing 300 iii of samples and were analyzed with a scence-Associated Cell Sorting (FACS) Calibur ﬂow cytometer (Becton, son and Company, Franklin Lakes, NJ, USA) equipped with an air- cooled argon laser emitting at 488 nm. The analyses were made with the CellQuest software.

GFP emission was detected with the FL-l, using a 530/30-nm band pass ﬁlter.

In the ﬁrst gate, cell debris as well as dead cells were ed from the analysis in a SSC/FSC dotplot on linear scale. Then, the GFP ﬂuorescence of living cells was displayed in a histogram on logarithmic scale. The median value of the ﬂuorescence distribution was used to assess the GFP expression level of the analyzed cell populations.

IgG quantiﬁcation method: OCTET QK The Octet QK system (FortéBio, Menlo Park, CA, USA) performs label-free quantitation of antibodies, proteins, peptides, DNA and other biomolecules and provides kinetic characterization of biomolecular binding interactions. A correlation between the binding rate 2012/056977 (nm) and the accumulated IgGl concentration (pg/ml) of the sample allows quantiﬁcation of the lgG titer with a calibration curve.

Cell s were centrifuged 5 min at 300 g. The supernatant was then d (1/5 for IgGl antibody) with the Octet Buffer in a 96 well plate before being analyzed with the Octet using Protein A biosensors (Protein A DIP and READTM Biosensor, Forte Bio, USA) to obtain the antibody concentration per well.

Transient transfection using JetPEI ent and stable ection of CHO-S and HEK293 EBNA cells was performed using polyethyleneimine (PEI; JetPEI, Polyplus—transfection, Illkirch, France). PEI is a cationic polymer which can complex with vely charged molecules such as DNA. The positive charged DNA-PEI complex binds to the vely charged cell surface and is internalized by endoeytosis. It reaches the lysosome compartment from where it is ed by lysis to the nucleus. The high transfection efﬁciency with DNA-PEI complexes is due to the ability of PEI to protect DNA from lysosomal degradation. The cells were ected according to the manual provided by the manufacturer.

All plasmids were linearized before stable transfection (100 ug of DNA re-suspended in 100 pl Tris-EDTA, pH 7.5). For transient transfection circular plasmids were directly used from midipreparation DNA. In this study, transient ections were kept in 50 ml bioreactor tubes and no antibiotics were added.

Stable CHO—S clones expressing IgG1 and GFP were obtained by co-transfeeting one expression vector and two resistance vectors (coding for puromycin or neomycin resistance, respectively).

Selection of stable pools and minipools Transfeetion efﬁciency was determined 24h after transfection by Flow Cytometry (BD FACS Calibur cytometer, #1293) by analysing the intracellular GFP expression. If the percentage of GFP positive cells was higher than 20 %, the transfeeted cells were diluted with selective medium and distributed into 96 well plates (for limiting dilution to generate isolated stable minipools) or in T-Flasks (to generate stable pools). The selective medium used was PCT/IBZOIZ/056977 PowerCHO-Z, 4 mM glutamine, supplemented with different concentrations of genetiein and puromycin.

Seven days after transfection, the selection stringency was renewed by adding selection medium to the cells. As soon as colonies in 96 well plates were confluent, the plates were read using a ﬂuorescence reader.

The pools in T-Flasks were expanded to in scale using antibiotic-free PowerCHO-Z, 4 mM L-glutamine. Their viability and concentration were evaluated with the Countess ted cell counter (lnvitrogen, Carlsbad, CA, USA). As soon as the cell density allowed it, a seed train was d for every pool by seeding cells at a density of 0.5x106 cells/ml in 10 ml medium in 50 ml bioreactor tubes (incubated in a shaker (200 rpm) at 5% C02, 37°C and 80% ty). Each seed train was passaged twice a week by seeding the cells at 0.5x106 ml in growth medium (cell concentration was determined by PCV analysis). The seed train was used for the inoculum of all productions runs (batches).

For the next 4-5 weeks productions runs were seeded once a week in duplicates. The pool stability was ted by FACS and IgG expression as described above for clonal populations.

Production runs (batch fermentation) The batch runs of cell pools were seeded at a concentration of 0.5x106 cells/m1 using the seed train for ation and cells were then cultured for 7 days in Feed media. On day 4 and 8, 200 pl of cells were centrifuged for 5 min at 300 g and the supernatant was analyzed for accumulated IgG using the Octet. In addition, the GFP expression of each batch was analyzed by FACS. 2. Results 2.1 Expression in transient in CHO cells: The vectors compared in this study differ mainly in their backbone. The entire expression cassette (Promoter, ﬁrst intron, expression construct, poly (A)) is y the same for all vectors. The vectors are derived from the vector pGLEX4l as bed in Example 1. In one vector, the ampicillin resistance gene was codon optimized for expression in E. coli and the bacterial backbone was reduced to a minimum: pGLEX41-R6K-AmpiA-[REP] (in short A).

In a second vector, the ampicillin resistance gene was codon optimized for expression in E. coli, but all CpG sequences were avoided, by using alternate codons (when le): This vector is called pGLEX4l-R6K-AmpiB-[REP] (in short B). The third modiﬁcation included the use of the GAPDH ﬂanking ces that were cloned upstream and downstream of the expression cassette of the s A and B giving the vectors pGLEX4l-R6K-AmpiA-[REP]- GAPDH (in short GAPDH_A) and pGLEX4l-R6K-AmpiB-[REP]—GAPDH (in short GAPDH_B).

Transient transfections of CHO-S cells rogen) were done in order to compare the expression level of the reporter proteins expressed in the t of the different plasmid backbones. The transfections (in duplicate) were performed in 50 ml bioreactor tubes (TPP, Trasadingen, Switzerland) using 10 ml of ﬁnal medium volume and analyzed on day 5 after transfection by Octet (Fig. 2).

All s (A and B) with corrected backbone show a slightly higher expression level than the control vectors pGLEX4 1. There is only a minor difference between the vectors A and B.

This is expected, because the only difference in the backbone is the ampicillin resistance which should not have an impact on transient sion.

The most striking observation is the positive effect of the GAPDH sequences on expression.

A 2—fold higher expression level is ed with the plasmid harbouring the GAPDH ﬂanking sequences compared to the ones without the GAPDH sequences. This is true for both A and B constructs. Compared to the pGLEX41 vector, a 3-fold higher expression can be observed. This is even more surprising if the size of the plasmids is taken into t. The vector A (7048 bps) is almost half the size compared to the vector GAPDH-A (13436 bps).

Therefore, assumed that the amount of delivered DNA during the process of transient transfection is the same for all plasmids, only half the molar amount of GAPDH-A is delivered to the nucleus. 2.2 Expression in transient in HEK293 cells Transient transfections of HEK293 EBNA cells were done in order to compare the expression level of the er proteins sed in the context of the different plasmid backbones. The transfections (in duplicate) were performed in 50 ml bioreactor tubes (TPP, Trasadingen, Switzerland) using 10 ml of ﬁnal medium volume and were analyzed on day 10 after transfection by Octet (Fig. 3).

The results shown in ﬁgure 3 show a signiﬁcant increase in sion that can be obtained using the GAPDH ﬂanking regions in HEK293 EBNA cells. The GAPDH-B vector is showing a threefold increase in expression, whereas the GAPDH-A vector shows an even higher increase in expression of 5-fold. These vectors do not contain the oriP element and might therefore have a potential for even higher . 2.3 Expression in stable CHO cell lines Establishment of stable transfected cells Stable populations were generated by co—transfecting an expression vector and vectors coding for resistance genes, followed by selection pressure mediated by antibiotics. The selection the generation of stable pressure was removed 14 days after transfection. These steps allowed minipools and stable pools which were cultured in r intervals in production runs in order to compare the sion levels of the reporter proteins (IgGl antibody and GFP) of the different constructs and the stability of expression.

Reporter protein expression study on production runs performed with cell pools Pools were ted by stable transfection. During the selection procedure (the ﬁrst 14 days after transfection) the pools were analyzed by FACS. An increase of the GFP positive cell on together with the viability ofthe e could be observed over the time. The selection pressure mediated by the antibiotics was removed from the pools after 14 days.

Using this approach no cell pools transfected with the “B” plasmids could be ed. The expression level of the generated pools was assayed as soon as the cells could be cultured in 50 ml bioreactor tubes. Batches were done in duplicates. The cells were analyzed by FACS for GFP expression and the accumulation of IgG in the supernatant was assayed by Octet after 8 days of expression.

A tional relationship could be observed between the IgG titers and the GFP expression ofthe pools. Therefore, only the IgG data are shown in ﬁgure 4. All pools ected with vectors containing GAPDH sequence show higher expression compared to the vector pGLEX41 or with the same vector t GAPDH sequence (factor of 2.8 between A and A- GAPDH. No conclusion could be drawn between B and B—GAPDH as no B pools survived).

PCT/182012/056977 Transfections performed with A—GAPDH and B-GADPH induced a higher expression of IgG (2.7 and 3.5 folds more respectively) than pGLEX4l transfection (for batch-2). Therefore in pools, the GAPDH g sequences seem to be favourable for the tion of proteins.

Finally, transfections performed with B-GADPH vectors induced a higher expression of IgG than the transfection performed with A-GAPDH (factor of 1.25). Therefore, the CpG reduction in resistance genes seems to be able for the stable production of proteins, too.

Expression level study on clonal populations Cells were transfected and buted in 96 well plates in selective medium in order to obtain clonal or oligoelonal tions. After 7 days the selection pressure was refreshed by addition of selective medium to the cells. The expression of GFP was assessed 14 days after transfection by using an ELISA-plate reader. The results are shown in ﬁgure 5.

Conﬁrming the results obtained in cellular pools, cells transfected with vectors containing GAPDH ﬂanking sequences expressed signiﬁcantly more GFP than the same backbone t GAPDH up-and downstream sequences (factors from 1.7 to 2 fold) or the other s used as control (pGLEX41: 2.5 fold) (Fig. 5). In addition, populations with vectors containing resistance sequences which had been CpG reduced (B) d a higher expression than the corresponding vectors which had only been codon optimized (A) (15 fold between A and B; 1.2 fold between B and H).

From the expression study several conclusions could be drawn. First, the GAPDH up- and downstream sequence allows higher expression than the standard vector that was used as a benchmark (pGLEX41). Also a lower expression level is obtained when cells are transfected with the same vector backbone without the GAPDH sequences conﬁrming that the beneﬁcial effect on the expression is related to the inserted GAPDH g sequences. In addition, the reduction of CpG number in the expression and selection plasmids seems to be slightly favourable for expression, too.

Example 3: ent expression level of CHO-S GMP cells transfected with new ed vectors It has been described in the literature that the 5’ region of the GAPDH promoter harbours a potential insulin as well as a phorbol ester se element (Alexander-Bridges et al., (1992) Advan Enzyme Regul, 32: 149-159). The phorbol ester response element (—1040 —1010 bps) is situated upstream of what is usually referred to as the GAPDH promoter (-488 - +20). In a deletion study performed in stable H35 Hepatoma cell lines, the s were not able to demonstrate a signiﬁcant effect of the deletion of irs -1200 to -488 (relative to the transcription ng point). ore the phorbol ester response element might not be functionally linked to the expression driven from the GAPDH promoter. Nevertheless a transient transfection experiment was med in order to evaluate the contribution of insulin and PMA (phorbol-l2-myristateacetate, the most common phorbol ester) in the increase in transient and stable expression that was observed using the plasmids containing the GAPDH ﬂanking elements.

In order to obtain insulin free growth medium, PowerCH02 was prepared from powder medium and no insulin was added. PMA was purchased from Sigma (St. Louis, MO), and was dosed at a ﬁnal concentration of 1.6 uM (corresponding to the concentration used by Alexander—Bridges on H35 Hepatoma cell lines) in PowerCH02 (+/— Insulin). ections were performed in 50 m1 bioreactor tubes (Tubespins, TPP, Trasadingen, Switzerland) as described previously. In order to avoid the presence of insulin provided by M (Life technologies, Carlsbad, CA), the transfection medium was changed to RPMII640 (PAA, Pasching, Austria) supplemented with 4 mM Gln and 25 mM HEPES.

After transfection, the cells were distributed in 12 well plates and 1 ml of the four different media was added (PowerCH02, 4mM Gln, ulin; PowerCH02, 4mM Gln, 1.6 uM PMA, +/— insulin). Again, the reporter construct expressing IgG1 and GFP using two IRES was used (described in example 2). This vector allowed veriﬁcation of the transfection ncy. The percentage and the viability of transfected cells were found similar in all four different media preparations.

As shown in ﬁgure 6, no signiﬁcant effect of insulin depletion and/or PMA on could be observed during this experiment. Similar titers were obtained in all media used for expression.

This suggests that the potential phorbol ester and the insulin response elements present in the upstream ﬂanking sequence of the GAPDH gene do not affect transient transgene expression.

WO 84157 Example 4: Fragmentation analysis of DNA ﬂanking the GAPDH expression cassette upstream of the promoter and downstream of the polyA site in order to study the effect on reporter gene expression The human GAPDH locus is located on chromosome 12 of the human genome. GAPDH is described to be tutively active in all cells of mammalian origin, as the enzyme is a key player in the metabolism of e. Upstream of the er, the GAPDH gene is ﬂanked by NCAPDZ, a gene that hes over more than 30000 bps. Downstream of the polyadenylation site, the GAPDH gene is ﬂanked by IFFOl (see ﬁgure 7 for details).

Not only GAPDH and the promoter, but also the ﬂanking regions are well conserved between ent species (see Table 4).

Table 4: Stretches of high homologies between human, rat and mouse GAPDH ﬂanking regions.

Analysis was done using clone r 9 (ScieED, Cary, NC, USA). The numbering is relative to the ﬁrst base of the am or the downstream ﬂanking element, respectively (Sequence ID NO: 7 and Sequence ID NO: 8, respectively). Sequences used for alignment were for mouse bases 532-3731 (upstream) and 8164-1 1364 (downstream) of Sequence ID No 18 and for rat bases 719-3918 (upstream) and 8495-1 1058 (downstream) of Sequence ID No 19.

Upstream region Downstream Sequences of Sequences of Sequences of Sequences of homolog homolo homolo ' homolo_ [mouse] [rat] [mouse] >80 % >80 0/0 V\DO =\° VasG g V\DG g 161—249 279—331 15—69 278— 764 1614-1671 1904-2061 256—338 554—623 159—249 546— 1894-2067 1888-2072 2927-3071 515—659 273-342 __— 2918—3082 2296- 5 15 -647 2349 2381- 1143- 2513 1223 - 2736- 1957- 2818 2009 - A comparison of the DNA homology between rodent and human shows a minimum ofDNA conservation of 38%. The presence of a conserved stretch of DNA outside of a promoter region or a region coding for a gene indicates that there might be a selection pressure on the cell to maintain the DNA sequence or to allow only certain/minor changes. In our speciﬁc maintain a case, the GAPDH ﬂanking regions might be important for the cells because they high expression level of the GAPDH genes. Changes in the DNA ce leading to decrease of expression would be selected against.

In order to evaluate the contribution of the upstream and the ream GAPDH element to the observed increase in expression, constructs were made containing only the upstream GAPDH ﬂanking region (SEQ ID NO: 7), fragments of the upstream GAPDH ﬂanking region or the ream GAPDH ﬂanking region (SEQ ID NO: 8). The reporter IgGl type antibody was expressed by an IRES construct (Light chain-IRES-heavy chain), therefore avoiding co-transfection of multiple plasmids. Details on the fragmentation of the GAPDH upstream fragment are shown in ﬁgure 8. The following fragments of the upstream GAPDH ﬂanking region were used: nt 1 (SEQ ID NO: 9), fragment 2 (SEQ ID NO: 10), fragment 3 (SEQ ID NO: 11), fragment 4 (SEQ ID NO: 12), fragment 8 (SEQ ID NO: 13), fragment 9 (SEQ ID NO: 14), fragment 11 (SEQ ID NO: 15), fragment 17 (SEQ ID NO: 16).

The upstream GAPDH ﬂanking region (SEQ ID NO: 7) used does n 2 times 3 (in total 6) nucleotides of the NruI restriction site of which three are linked to the genomic DNA at its ’ and three are linked to the genomic DNA its 3’ end. The downstream GAPDH ﬂanking region (SEQ ID NO: 8) used does contain two times 3 (in total 6) nucleotides of the ScaI restriction site of which three are linked to the genomic DNA at its 5’ and three are linked to the c DNA its 3’ end. The upstream GAPDH ﬂanking region and the downstream GAPDH ﬂanking region without the tides of the respective restriction site are shown in SEQ ID NO: 20 (upstream GAPDH ﬂanking region without ction sites) and SEQ ID NO: 21 (downstream GAPDH ﬂanking region without ction sites). The fragments of the upstream GAPDH ﬂanking region used does each contain 3 nucleotides of the tive restriction site at its 5’ and/or its 3’ end linked to the genomic DNA (Fragment 1 contains 3 nucleotides of the NruI restriction site at its 5’end; Fragment 2 contains 3 nucleotides of the NruI restriction site at its 3 ’end; Fragment 3 contains 3 nucleotides of the NruI restriction site at its 5’end: Fragment 4 contains 3 tides of the NruI ction site at its 3’end; Fragment 8 ns 3 nucleotides of the NruI restriction site at its 3 ’end; Fragment 9 contains 3 nucleotides of the NruI restriction site at its 5’end and 3 nucleotides of the NruI restriction site at its 3’end; Fragment 11 contains 3 nucleotides of the NruI restriction site at its 3’end).

Fragment 17 does not contain tides of a restriction site. The fragments of the upstream GAPDH ﬂanking region without the nucleotides of the respective restriction site are shown in SEQ ID NO: 22 (fragment 1 without restriction site), SEQ ID NO: 23 (fragment 2 Without restriction site) SEQ ID NO: 24 (fragment 3 without restriction site), SEQ ID NO: 25 (fragment 4 without restriction site), SEQ ID NO: 26 (fragment 8 without restriction site), SEQ ID NO: 27 ent 9 without restriction sites), SEQ ID NO: 28 (fragment 11 without restriction site).

The effect of the upstream and the downstream GAPDH elements on expression was assessed on day 10 after transfection using the Octet (Fortebio, Menlo, CA, USA) in order to quantify the amount of secreted IgGl in the supernatant (see ﬁgure 9). pGLEX41, the original vector is giving lower expression results (80%) compared to the ed new vector design used in the pGLEX41-ampiA backbones. Compared to the original pGLEX41 backbone the new design includes codon optimization of the ampiA gene necessary for ampicillin resistance in E. coli, a different origin of replication (R6K instead ofpUC origin of replication) and elimination of unnecessary linker (or spacer) ces of bacterial origin. Both vectors have approximately the same size.

Surprisingly, l —ampiA including the upstream (SEQ ID NO: 7) and ream element (SEQ ID NO: 8), (named 1-up/down in ﬁgure 9 showing the expression results) is giving higher expression (factor 1.5) compared to the same vector without the upstream and ream sequences. If one considers the difference in size (up/down fragments increase the size of the plasmid by approximately 6000 bps) and ore the differences in delivered plasmid copies during transfection, the effect might even more important on a per plasmid basis.

The vector containing only the upstream fragment (up) is showing an expression level similar to the original expression construct 1-ampiA. The vector ning only the downstream fragment (down) is g a signiﬁcant increase (factor 1.2) in expression compared to the original expression uct pGLEX41-ampiA. A further increase in expression can be ed if both, the up- and the downstream fragment are present. This is conﬁrmed by the fragmentation of the upstream fragments. Fragment 9 and the promoter proximal fragment 8 do not show any difference in expression compared to pGLEX41- ampiA. Fragment 1, 11 and 17 show an increase in expression. The highest increase was observed for fragment 4. It should be ghted that the promoter al fragment 8 is not 012/056977 showing any effect. ore the increase in expression cannot be explained by previously published sequences (Alexander-Bridges et al., (1992) Advan Enzyme Regul, 32: 149-159), Graven et al., (1999) Biochimica et Biophysics Acta 147: 8).

Interestingly, fragments 2 and 3 lead to a signiﬁcant decrease in expression. This is unexpected, especially in View of the fact that these fragments cloned in the opposite direction (antisense (AS) in figure 9) do not cause this effect. For the fragments l, 8, 9, 11 and 17 no difference in expression was observed for fragments that were integrated in sense or antisense orientation (data not shown). Fragment 11, gh a part of nt 2, does not show this effect. Therefore the sequence element that seems to be detrimental to expression should be at least partially on the BstBI-BstBI fragment that was deleted in fragment 2 in order to obtain fragment 11.

In addition, the hypothesis that a ve element is located (at least partially) on the BstBl— BstBI fragment is supported by the increase in expression observed between fragment 3 (which includes the BstBI-BstBI fragment) and fragment 1.

While it seems easy to localize the fragment having a negative effect (BstBI-BstBI), from this study it is less obvious how this negative effect observed for fragment 2 and 3 is compensated by sequence elements present in the complete upstream nt. It could be that this negative effect is balanced out by the small positive effect that was observed by nt 1 and fragment 4 (but the increase in expression for fragment 1 is less than for fragment 4).

Nevertheless the positive effect for fragment 4 (factor 1.25) observed seems less important compared to the negative effect r 0.4). Furthermore fragment 9, which is the entire upstream region without the BstBI fragment does not show sed expression ed to the entire GAPDH upstream ﬂanking region (nevertheless, fragment 9 includes the EcoRV-BstBI fragment which is part of fragment 2 and 3 and might have a ve effect on expression).

It can only be speculated about the mechanism behind the observed effects. The orientation dependency of the negative effect on expression observed with fragments 2 and 3 excludes the expression of non—identified open reading frames (for example expression of an ncRNA), because there are no surrounding promoters that could trigger the expression of only one orientation. The fact that the expression is reduced below the basal level shows not only the absence of a positive effect (for example an enhancer activity), but rather the presence of an orientation ent negative .

In summary, a surprising increase of expression in transient in CHO cells is observed if both ﬂanking regions, the upstream and the downstream region, are present in the expression plasmid. Although fragment 4 seems to have a signiﬁcant positive effect on expression, no single fragment could be identiﬁed that is responsible for the entire increase of expression that was observed. The increase of expression of the expression vector pGLEX41-ampiA (up/down) seems to be the summary effect of both, up- and downstream flanking . e 5: Cloning of the non-translated genomic DNA sequence upstream of the Chinese hamster GAPDH gene and the Chinese hamster promoter 1.1 Cloning of the non-translated genomic DNA ce upstream of the Chinese hamster GAPDH gene into an expression vector The non-translated genomic DNA sequence upstream of the Chinese hamster GAPDH gene was ampliﬁed from genomic DNA of CHO—S (Life Technologies) cells by PCR. Genomic DNA was extracted as described in e 1. Constructs were prepared using the mouse CMV promoter or the Chinese r GAPDH promoter for the sion of the reporter gene uct [REP] described in Example 1.

For cloning of the genomic DNA sequence upstream of the Chinese hamster GAPDH gene in combination with the mouse CMV promoter, primers GlnPr1896 and GlnPr1897 were used for ampliﬁcation of the 3 kbs fragment (bps 672 to 3671 of SEQ ID No 29) using the PCR protocol described in Example 1 and leading to the on with the SEQ ID No 30. The amplicon contains the genomic DNA sequence am of the Chinese hamster GAPDH gene and 5’ and 3’ restriction sites that were introduced by the primers.

For cloning of the genomic DNA sequence upstream of the Chinese hamster GAPDH gene in combination with the Chinese hamster GAPDH er, primers GlnPr1902 and GlnPr1905 were used in order to amplify the 3508 bps fragment containing the c DNA sequence ing the genomic DNA sequence upstream of the Chinese hamster GAPDH gene and the GAPDH promoter (bps 672 to 4179 of SEQ ID No 29) leading to the amplicon with the SEQ ID No 31. In a second PCR, GlnPr1901 and GlnPr1902 were used for ampliﬁcation of the 508 bps fragment ning only the promoter region (bps 3672 to 4179 of SEQ ID No 29), leading to the SEQ ID No 32. The intron used in the vector “A” (described in Example 1) was amplified using primers GlnPrl 903 and GlnPr1904.

A ﬁrst fusion PCR was performed with primers GlnPr1904 and GlnPrl90l using the amplicon with SEQ ID NO: 32 and the amplicon with the intron sequence as tes. The amplicon contains the Chinese hamster GAPDH promoter, an intron and 5’ and 3’ restriction sites that were introduced by the primers. All primers are shown in Table 5.

A second fusion PCR was performed with primers G1nPrl905 and G1nPrl904 using the amplicon with SEQ ID No. 31 and the amplicon with the intron sequence as templates. The amplicon ns the genomic DNA sequence upstream of the Chinese r GAPDH intron and 5’ and 3’ restriction sites that gene, the Chinese hamster GAPDH promoter, an were introduced by the primers.

After puriﬁcation on a 1% agarose gel, the bands of interest were cut out and puriﬁed using the kit “NucleoSpin Gel and PCR up” (Macherey Nagel, Oensingen, Switzerland). The d fragments were cloned into the plasmid pCR_Blunt using the Zero Blunt PCR cloning Kit (Invitrogen, Carlsbad, CA, USA). on products were transformed into competent Ecoli TOPlO (One Shot® TOP 10 Competent E. coli; Invitrogen, Carlsbad, CA, USA) and analyzed by restriction analysis of minipreps. This led to the plasmids pCR_blunt[CHO—upstreamGAPDH], ning the genomic DNA sequence upstream of the Chinese hamster GAPDH gene, pCR__Blunt[CHO—upstreamGAPDH_GAPDHpromoter] containing the genomic DNA sequence upstream of the Chinese r GAPDH gene and the GAPDH promoter and intron from vector “A” and pCR_Blunt[CHO-GAPDHpromoter] containing the GAPDH promoter and the intron from vector “A”.

For evaluation of the amplicons on their effect on expression of a secreted gene, the vector “A” (described in Example 1) was used. As described previously, the sion te used in this vector contains a polycistronic gene coding for a secreted IgGl and GFP (see Example 1). Transfected cells will therefore secrete the IgGl monoclonal antibody and accumulate intracellular GFP in a dependent manner.

In order to e the 3 kb insert fragment containing the genomic DNA sequence upstream of the Chinese hamster GAPDH gene, the plasmid pCR_Blunt[CHO-upstreamGAPDH] was digested using the restriction enzyme NaeI. This insert was cloned in the ne of “A”, digested using the restriction enzyme NruI and CIPed (CIP; NEB, Ipswich, MA, USA).

Backbone and insert were ligated together using T4 DNA ligase (T4 DNA ligase, NEB, Ipswich, MA, USA) and subsequently transformed into competent Ecoli PIRl. Clones were picked for ep preparation and subsequent restriction analysis. The resulting plasmid was called “A_GAPDH_UP”, conﬁrmed by sequencing analysis and produced in midiprep scale using the NucleoBond Xtra Midi kit (Macherey Nagel, Oensingen, Switzerland).

For the cloning of expression ucts using the Chinese hamster GAPDH promoter, the insert fragments were released from plasmids pCR_Blunt[CHO-upstreamGAPDH_GAPDH promoter] and unt[CHO—GAPDHpromoter] by digestion using the restriction s NheI and NruI. The resulting fragments were cloned in the backbone of vector “A”, opened using the same enzymes and CIPed. After ligation with T4 DNA ligase and transformation into competent Ecoli PIRI, clones were picked for miniprep restriction analysis. The resulting plasmids were called “A_GAPDH_UP_Prom” (plasmid with non— translated c DNA sequence am of the Chinese r GAPDH and the promoter) and “A_PR” (plasmid with only the promoter) conﬁrmed by sequencing analysis and produced in midiprep scale using the kit NucleoBond Xtra Midi (Macherey Nagel, Oensingen, Switzerland). 2. Assessment of the effect of the non-translated genomic DNA sequence upstream of the Chinese hamster GAPDH gene on the expression of the reporter gene construct CHO-S cells were transfected in tubespins ctors using 10 ml of medium volume (as bed in Example 2). The ected cells were incubated in a shaking incubator with 200 rpm agitation at 37°C, 5 % C02 and 80 % humidity. The supematants of the cells were analyzed for IgGl expression using the Octet QK system with Protein A biosensors, (FortéBio, Menlo Park, CA, USA). The results are shown in Figure 10.

The expression level of the plasmid containing the GAPDH promoter (“A_PR”) compared to the mouse CMV promoter (A) is reduced by 50 %, indicating that the Chinese hamster GAPDH promoter is not as strong as the viral er. The plasmid containing the non— translated genomic DNA sequence upstream of the Chinese hamster GAPDH gene in combination with the Chinese hamster GAPDH promoter (“A_GAPDHﬁUP~Prom”) shows a two fold increase in expression compared to the construct having only the GAPDH promoter (“A~PR”). The plasmid containing the non-translated genomic DNA sequence upstream of the Chinese r GAPDH gene and the mouse CMV promoter “(A_GAPDH_UP”) shows the highest expression and an increase of more than 40% over the plasmid containing only the mouse CMV promoter (“A”). This conﬁrms that the non-translated genomic DNA sequence upstream of the Chinese hamster GAPDH gene has an enhancer effect on the expression of the er protein.

Table 5: Primers used for cloning in Example 5 Primer SEQ ce Orien— ID No tation TACGGCCGGCTTCACTGTACAGTGGCACAT forward TCAGGCCGGCCGTGGTTCTTCGGTAGTGAC reverse TACTCGCGAAGAAGATCCTCAACTTTTCCACAGCC GTTCACTAAACGAGCTCTGCTATTTATAGGAACTGGGGTG I./ AGTTCCTATAAATAGCAGAGCTCGTTTAGTGAAC I./ CGCTAGCACCGGTCGATCGA TACTCGCGATTCACTGTACAGTGGCACATAC

Claims

Claims An expression cassette which ses a promoter, a polynucleotide sequence encoding a polypeptide, and expression enhancing element wherein expression enhancing element comprises a non-translated genomic DNA sequence downstream of a mammalian Glyceraldehyde 3-phosphate dehydrogenase ) promoter, wherein the ptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non-translated c DNA ce downstream ofthe mammalian GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, 10 wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated c DNA sequence downstream ofthe mammalian GAPDH promoter is from 95 to around 15000 nucleotides. The sion cassette of claim 1, wherein the sion cassette further comprises a 15 non-translated genomic DNA sequence upstream of a mammalian GAPDH promoter, wherein the non—translated genomic DNA sequence am of the mammalian GAPDH promoter starts within a region spanning from around the 5’ end of the mammalian GAPDH promoter to nucleotide position around —3 500, n the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the 20 non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter is from around 100 to around 15000 nucleotides. An expression cassette which comprises a promoter, a polynucleotide ce encoding a polypeptide, and a non-translated genomic DNA sequence upstream of a ian 25 GAPDH promoter, wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the anslated genomic DNA sequence upstream ofthe mammalian GAPDH promoter starts within a region spanning from around the 5’ end of the mammalian GAPDH promoter to nucleotide position around -3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, n the 30 length of the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter is from 100 to around 15000 nucleotides, with the proviso that the expression cassette does not comprise a mammalian GAPDH promoter or fragments thereof. 1000660930 The expression cassette of claim 3, n the expression cassette further comprises a non-translated genomic DNA sequence downstream of a mammalian GAPDH promoter, n the non-translated genomic DNA sequence downstream of the ian GAPDH promoter starts within a region spanning from tide position around +1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non-translated genomic DNA sequence downstream ofthe mammalian GAPDH promoter is from 95 to around 15000 nucleotides. 10 The expression cassette of any one of claims 1 to 4, wherein the non-translated genomic DNA sequence downstream and/or upstream of the mammalian GAPDH promoter is not operably linked to the polynucleotide sequence encoding the ptide. The expression cassette of any one of claims 1 to 4, wherein the expression cassette 15 further comprises a polyadenylation site. The expression cassette of claim 1 or 4, wherein the length of the non—translated genomic DNA sequence downstream of the mammalian GAPDH promoter is around 10 nucleotides and extends at its maximum to the second last intron of the IFF01 gene or to a 20 part thereof. The expression cassette of claim 1 or 4, wherein the non-translated genomic DNA sequence downstream of the mammalian GAPDH er starts downstream ofthe mammalian GAPDH polyadenylation site and wherein the length of the non-translated 25 genomic DNA sequence ream ofthe mammalian GAPDH promoter is at least 95 nucleotides and extends at its maximum to the second last intron of the IFF01 gene. The expression cassette of claim 2, wherein the length of the anslated genomic DNA sequence upstream of the ian GAPDH promoter is at least around 100 nucleotides 30 and extends at its maximum to the start codon of the NCAPD2 gene. 10. The expression cassette of claim 2, wherein the length of the non-translated genomic DNA sequence am of the mammalian GAPDH promoter is at least around 100 nucleotides 1 000660930 and extends at its m to the third last intron of the NCAPD2 gene. 11. The sion cassette of claim 3, wherein the length of the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter is at least 100 nucleotides and extends at its maximum to the start codon of the NCAPD2 gene. 12. The expression cassette of claim 3, wherein the length of the non-translated genomic DNA sequence upstream ofthe mammalian GAPDH promoter is at least 100 nucleotides and extends at its maximum to the third last intron of the NCAPD2 gene. 13. The expression te of any one of claims 1 to 4, wherein the non-translated genomic DNA sequence downstream and/or upstream of the mammalian GAPDH promoter is of rodent or human origin. 15 14. The expression cassette of claim 1 or 4, wherein the non-translated genomic DNA ce downstream of the mammalian GAPDH promoter ses the nucleotide sequence selected from the group consisting of SEQ ID NOS: 8 and 21 or fragments thereof. 20 15. The expression cassette of claim 1 or 4, wherein the non—translated genomic DNA sequence ream of the mammalian GAPDH promoter ses a nucleotide sequence complementary to the nucleotide sequence selected from the group consisting of SEQ ID NOS: 8 and 21 or fragments thereof. 25 16. The expression cassette of claim 1 or 4, wherein the non-translated genomic DNA sequence downstream of the mammalian GAPDH promoter comprises a nucleotide sequence at least 80% identical to the tide sequence selected from the group consisting of SEQ ID NOS: 8 and 21 or fragments thereof. 30 17. The expression cassette of claim 2 or 3, wherein the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter comprises a tide sequence selected from the group consisting of SEQ ID NO: 7, 9, 10, 11, 12, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or nts thereof. 1 000660930 18. The expression cassette of claim 17, wherein the nucleotide sequence selected from the group consisting of SEQ ID NOS: 7, 9, 10, 11, 12, l3, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or nts thereof comprises five or less c acid modiﬁcations. 5 19. The expression cassette of claim 2 or 3, wherein the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter comprises a nucleotide sequence complementary to the nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 9, 10, ll, 12, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or fragments thereof. 10 20. The expression cassette of claim 2 or 3, wherein the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter comprises a nucleotide sequence at least 80% identical to the nucleotide sequence selected from the group ting of SEQ ID NO: 7, 9, 10, ll, 12, 13, 14, 15, 16, 20, 22, 23, 24, 25, 26, 27 and 28 or fragments 21. The sion cassette of any one of claims 1 to 4, wherein the er and the polynucleotide sequence encoding a polypeptide are operatively linked. 22. The expression cassette of claim 1 or 4, wherein the non-translated c DNA 20 sequence downstream of the mammalian GAPDH promoter is orientated in the same direction as the polynucleotide sequence encoding a polypeptide. 23. The sion cassette of claim 1 or 4, wherein the non-translated c DNA sequence downstream of the mammalian GAPDH promoter is orientated in opposite 25 direction in relation to the polynucleotide ce encoding a polypeptide. 24. The expression cassette of claim 2 or 3, wherein the non-translated genomic DNA sequence upstream of the mammalian GAPDH promoter is orientated in the same direction as the polynucleotide sequence encoding a polypeptide. 25. The sion cassette of claim 2 or 3, wherein the non-translated genomic DNA sequence upstream of the ian GAPDH promoter is orientated in opposite direction in relation to the polynucleotide sequence encoding a polypeptide. 1000660930 1001476446 26. The expression cassette of any one of claims 1 to 4, wherein the er is selected from the group consisting of SV40 promoter, MPSV promoter, mouse CMV, human tk, human CMV, rat CMV, human EFl alpha, Chinese hamster EFl alpha, human GAPDH, hybrid promoters including MYC, HYK and CX promoter. 27. The expression cassette of any one of claims 1 to 4, n the polypeptide is selected from the group consisting of dies, antibody fragments or antibody derivates. 28. The expression cassette of claim 6, wherein the poiyadenylation site is selected from the 10 group ting of BGH poly(A) and SV4O poly(A). 29. The expression cassette of any one of claims 1 to 4, further comprising a genetic element selected from the group consisting of an additional promoter, an enhancer, transcriptional control elements, and a selectable marker. 30. The expression cassette of claim 29, wherein the c element is a selectable marker wherein the content of CpG sites contained in the cleotide sequence encoding the selectable marker is 45 or less. 2O 31. An expression vector comprising an expression cassette of any one of claims 1 to 30. 32. An sion vector, which comprises in order: a) a nonetranslated genomic DNA sequence upstream and/or ream of a mammalian GAPDH promoter 25 b) a promoter 0) a polynucleotide sequence ng a polypeptide d) a polyadenylation site e) a non—translated genomic DNA sequence downstream and/or upstream of a mammalian GAPDH promoter, or 30 a) a non-translated genomic DNA sequence upstream and/or downstream of a mammalian GAPDH promoter b) a promoter 0) a polynucleotide sequence encoding a polypeptide d) a polyadenylation site 1001476446 e) a non—translated genomic DNA sequence downstream and/or upstream of a mammalian GAPDH promoter, or a) a non—translated genomic DNA sequence upstream and/or downstream of a mammalian GAPDH b) a promoter c) a polynucleotide sequence encoding a ptide d) a polyadenylation site e) non-translated genomic DNA sequence downstream and/or upstream of a mammalian GAPDH, 10 wherein the polypeptide encoded by the polynucleotide sequence is not GAPDH, and wherein the non—translated genomic DNA sequence downstream of the mammalian GAPDH promoter starts within a region spanning from nucleotide on around +1 to nucleotide on around +7000, wherein the nucleotide position is relative to the ription start of the GAPDH mRNA, and wherein the length of the anslated 15 genomic DNA sequence downstream of the mammalian GAPDH promoter is from 95 to around 15000 tides and wherein the non—translated genomic DNA sequence upstream of the ian GAPDH er starts within a region spanning from around the 5’ end of the mammalian GAPDH promoter to nucleotide position around — 3500, wherein the nucleotide position is relative to the transcription start of the GAPDH 20 mRNA, and wherein the length of the non—translated genomic DNA ce upstream of the mammalian GAPDH promoter is from around 100 to around 15000 nucleotides, with the proviso that if a) or b) is a anslated genomic DNA sequence upstream of a mammalian GAPDH e) is a non—translated genomic DNA sequence downstream ofa mammalian GAPDH and if a) or b) is a non—translated genomic DNA sequence 25 ream of a mammalian GAPDH e) is a non—translated genomic DNA sequence upstream of a mammalian GAPDH. 33. An expression vector, which comprises in order: a) a non—translated genomic DNA ce upstream and/or downstream of a mammalian GAPDH promoter 30 b) a promoter c) a polynucleotide sequence encoding a polypeptide d) a polyadenylation site e) an enhancer f) a non—translated genomic DNA sequence downstream and/or upstream of a mammalian 1001476446 GAPDH promoter, or a) a non-translated genomic DNA sequence upstream and/or downstream of a mammalian GAPDH promoter b) an enhancer c) a er d) a eleotide sequence encoding a polypeptide e) a polyadenylation site 0 a non—translated genomic DNA sequence ream and/or upstream of a mammalian GAPDH promoter, or 10 a) an enhancer b) a non-translated genomic DNA sequence upstream and/or downstream of a mammalian GAPDH e) a promoter d) a polynueleotide sequence encoding a polypeptide 15 e) a polyadenylation site i) non—translated genomic DNA sequence downstream and/or upstream ol’a mammalian GAPDH, wherein the polypeptide d by the polynueleotide sequence is not GAPDH, and wherein the non—translated genomic DNA sequence downstream of the mammalian 20 GAPDH promoter starts within a region spanning from nucleotide position around +1 to nucleotide position around +7000, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length 01‘ the non~translated genomic DNA sequence downstream of the mammalian GAPDH promoter is from 95 to around 15000 tides and wherein the non-translated genomic DNA sequence 25 upstream of the mammalian GAPDH promoter starts within a region spanning from around the 5’ end of the ian GAPDH promoter to nucleotide on around — 3500, wherein the nucleotide position is relative to the transcription start of the GAPDH mRNA, and wherein the length of the non—translated genomic DNA ce upstream of the mammalian GAPDH promoter is from around 100 to around 15000 nucleotides, with 30 the proviso that if a) orb) is a non-translated genomic DNA ce upstream of a mammalian GAPDH t) is a non—translated c DNA sequence downstream of a mammalian GAPDH and if a) or b) is a non—translated genomic DNA sequence downstream of a mammalian GAPDH t) is a non—translated genomic DNA sequence upstream of a mammalian GAPDH. 1001476446 34. The expression vector of any one of claims 31 to 33, wherein the expression vector further comprises a genetic element selected from the group consisting of an additional promoter, an enhancer, transcriptional l elements, an origin of replication and a selectable marker. 35. The expression vector of any one of claims 31 to 33, wherein the sion vector further comprises an origin of replication and a selectable marker wherein the t of CpG sites contained in the polynucleotide sequence of the expression vector encoding the origin 10 of replication and the selectable marker is 200 or less. 36. An isolated host cell comprising an expression cassette of any one of claims 1 to 30 or an expression vector of any one ofclaims 31 to 35. 15 37. The expression cassette of any one ofclaims 1 to 30 or the expression vector of any one of claims 31 to 35 for use as a medicament for the treatment ofa disorder. The expression cassette of any one of claims 1 to 30 or the expression vector of any one of claims 31 to 35 for use in gene therapy. 39. An in vitro method for the expression ofa polypeptide, comprising transfecting a host cell with the expression cassette of any one of claims 1 to 30 or the expression vector of any one ofclaims 31 to 35 and recovering the polypeptide. 25 40. The method of claim 39, wherein the expression cassette or the expression vector is stably transfected. 41. The method of claim 39, wherein the expression te or the expression vector is transiently transfected. 42. Use of an sion te of any one of claims 1 to 30 or an expression vector of any one of claims 31 to 35 for the expression of a heterologous polypeptide in an isolated mammalian host cell. 1001476446 43. The expression vector of any one of claims 1, 3, 32 or 33, substantially as before described.