EP1144667A2 - Retroviral expression vectors on the basis of herv- long terminal repeat sequences - Google Patents

Abstract

Die vorliegende Erfindung betrifft retrovirale Expressionsvektoren mit zellspezifisch regulierbaren Promotoren. Die Vektoren sind beispielsweise zur zellspezifischen Expression therapeutisch wertvoller Gene im Rahmen einer Gentherapie einsetzbar. Die Erfindung beschreibt retrovirale Expressionsvektoren, enthaltend zumindest die nachfolgenden Elemente in funktioneller Anordnung: a) DNA-Sequenzen zur Verpackung der Vektor-RNA und zur zellspezifischen Expression von Proteinen oder Peptiden, die von heterologen DNA-Nukleotidsequenzen kodiert werden; b) eine oder mehrere für ein Protein oder Peptid kodierende DNA-Nukleotidsequenzen, dadurch gekennzeichnet, dass die DNA-Sequenzen zur zellspezifischen Expression eine zellspezifisch regulierbare Promotorregion aus einer humanen endogenen retroviralen DNA-Nukleotidsequenz (HERV) enthalten.

Description

 Retroviral expression vectors based on HERV-LTR sequences

The present invention relates to retroviral expression vectors with cell-specifically regulable promoters. The vectors can be used, for example, for cell-specific expression of therapeutically valuable genes in the context of gene therapy.

Retroviruses are RNA viruses in which the viral genes are encoded by a single-stranded RNA molecule. After the viruses have entered the cell, the viral RNA is converted into a double-stranded DNA molecule by reverse transcription. The DNA penetrates into the nucleus and integrates into the cellular chromosome. The integrated viral DNA form, the so-called provirus, forms the template for the expression of the viral genes.

The integration of the viral genome into the cellular chromosome is a mandatory part of viral replication and is mediated by virally encoded enzymes. It appears that, with a few exceptions, the presence of the retroviral genome in the cell, the expression of its genes, and the formation of virus particles do not or hardly affect the viability of the infected cell.

Retroviraier gene transfer is used to introduce functional genes, especially therapeutically valuable genes, into the cells without affecting the proliferation ability of the host cell. Because of their replication mode, retroviruses are suitable for such a gene transfer. In the simplest embodiment, at least some of the viral genes are replaced by a gene of interest and, with the aid of the efficient viral infection process, this gene of interest is transferred to the target cell.

Retroviral vectors are suitable for gene therapy because the infection by retroviruses is highly efficient and the retroviral vectors can be modified in such a way that they take up heterologous DNA and can integrate it stably into the host cell genome. A large number of retroviral vectors have been developed in recent years, and only for example is the review article by Günzburg et al. (1996) and Robbins et al. (1998). A possible preferred embodiment for retroviral vectors are so-called ProCon vectors, which were first described in WO 96/07748. For the purposes of disclosure, reference is made in full to this publication.

ProCon vectors carry heterologous promoter and optionally other regulatory elements in the 3'LTR, which, after infection, are duplicated and translocated to the 5'LTR in the target cell and which are able to regulate the expression of marker genes or therapeutic genes. These heterologous genes are not directly linked to the promoter, but are inserted into the interior of the vector.

ProCon vectors comprise a 5'LTR section of the structure U3, R, U5 and at least one coding and / or non-coding sequence and a 3'LTR area which comprises a completely or partially deleted U3 section, the deleted one U3 section was replaced by a polylinker sequence, followed by the R and U5 section.

These vectors are propagated with the help of a helper cell line, which produces large amounts of the viral proteins which are no longer synthesized by the expression vector itself. However, the helper cell line is no longer able to produce a replication-competent virus. This cell line is also referred to as a packaging cell line and comprises a cell line transfected with at least one second plasmid which carries those genes which enable packaging of the modified retroviral vector. We refer here e.g. to W092 / 10564, to which reference is made here in full.

The DNA coding for the modified retrovirus (expression vector) is transfected into the packaging cell line. Under these conditions, the modified retroviral genome including the inserted therapeutic genes or marker genes is transcribed and packaged into the retroviral particles (recombinant viral particles). This recombinant virus is then used to infect the target cells; the genome of the modified retrovirus, ie the expression vector, is integrated into the genome of the target cells, together with the marker genes or the therapeutic genes. A cell infected with the recombinant viral particles generated in this way can no longer produce a new vector virus since there are no further viral proteins in these cells. The in the hosts « cell-integrated DNA of the expression vector with the therapeutically valuable genes or the marker genes is integrated in the cellular DNA and can now be expressed in the cell.

Therapeutically valuable genes that are expressed using such retroviral expression vectors should preferably be expressed in cell- and tissue-specific form. For this purpose, cell-specific regulatory sequences are used in the LTR sequence of the expression vector. These cell-specific regulatory sequences include, for example, cell-specifically regulatable promoter sections, cell-specific enhancer sequences and transcription factor binding sites. The promoters are in the U3 section of the LTR.

Cellular promoter sequences or promoter sequences of exogenous retroviruses are currently used in retroviral vectors.

Cellular promoters are often dependent on additional signal structures that can be located at a great distance upstream or downstream of the promoter. It has therefore repeatedly proven difficult to isolate strong, tissue-specific cellular promoter sequences and to clone them in retroviral vectors. Promoters of exogenous retroviruses have the advantage that they contain all the necessary regulatory elements in a small space in the retroviral LTR and are therefore largely transcribed independently of the neighboring DNA sequences of the integration site. A serious disadvantage, however, is that, although they are very strong, they are not tissue-specific and are usually expressed equally strongly in all cell types.

It is therefore an object of the present invention to provide new retroviral expression vectors which, although taking advantage of the retroviral promoters, concentrate all the signal structures required for transcription in a narrow space within the U3 and R region, but avoid the associated disadvantages.

This object is achieved according to the invention in that a cell-specifically regulatable promoter section from a human endogenous retroviral DNA nucleotide sequence (HERV) is used in retroviral expression vectors. These promoter sequences of human endogenous retroviral viruses are already present in the host cell, and according to found that they are outstandingly suitable for regulating the cell-specific expression of marker genes and therapeutically valuable genes.

Endogenous retroviruses (ERV) can be found in the genome of all cells in an organism. They are transmitted vertically via germline cells and can be reactivated by environmental influences. The human genome consists of approximately 2% endogenous retroviruses and retroviral sequences, with solitary HERV-LTRs with 20,000-40,000 copies per genome being represented (Tab. 1) (Leib-Mösch et al., 1993; Wilkinson et al., 1994 , Patience et al., 1997).

Since HERV sequences were integrated into the genome of primates 30 - 40 million years ago, it can be assumed that in the course of evolution most pathogenic sequences have been eliminated from the provirus by mutations and rearrangements or changed in such a way that they are no longer a disadvantage for the organism. Retroviral vectors that are constructed from such sequences have the advantage over vectors from animal viruses that no new viral sequences have to be introduced into the genome. Even by recombination with HERV sequences already present in the genome, no new types of retroviruses can arise, e.g. the case would be if retroviruses of other species are used as vectors. For this reason, the use of these sequences to construct retroviral vectors can minimize the security risk. Furthermore, homologous regions contained in the genome could be used for tissue-specific integration of the retroviral vectors into specific locations of a chromosome.

HERV elements have taken on a number of cellular functions during evolution. For example, promoter and enhancer elements of HERV LTRs are used to control the transcription of cellular genes (Kato et al., Feuchter-Murthy et al., 1993; Di Christofano et al., 1995). An example of the use of LTR regulatory elements for tissue-specific expression of a cellular gene is the human amylase gene. This gene is controlled by the LTR of a HERV-E element and is therefore only specifically expressed in the salivary glands (Ting et al., 1992). In addition, Schulte and co-workers (1996) have shown that the insertion of an endogenous retrovirus into the 5 'untranslated region of the pleiotrophin gene is responsible for its trophoblast-specific activity - is literal (Schulte et al., 1996). In other cases, polyA signals from HERV-LTRs can also serve to polyadenize cellular transcripts (Mager, 1989; Goodchild et al., 1992).

The great advantage of using retroviral promoters is that, as already stated above, all signal structures required for transcription are localized in a narrow space within the U3 and R areas of the LTR, since retroviruses are as independent as possible of the environment in which they are integrated must remain transcriptional. Since these HERV promoters have persisted in the primate genome for millions of years, they have adapted in the course of evolution so that, like cellular promoters, they are cell type-specific, thus combining the advantages of cellular and retroviral promoters.

The transcription of the HERVs is started from a classic RNA polymerasell promoter (Wilkinson et al., 1994). This promoter is located within the LTR region. The HERV transcript therefore does not consist of the full copy of the provirus. In order to compensate for the loss of the transcription control elements, these elements have developed the mechanism of reverse transcription, with which the lost sequences are regenerated on both sides of the elements, which in turn results in the LTRs. In addition to promoter sequences, HERV-LTRs also contain a large number of different binding sites for transcription factors (Seifarth et al., 1998), which are responsible for the tissue specificity of the expression.

Although there is some structural similarity between HERVs and exogenous animal retroviruses such as MLV or MMTV, HERV sequences and especially promoters of HERVs have never been considered as possible candidates for the development of retroviral expression vectors. On the contrary, they have so far only been regarded as disruptive factors in connection with gene therapy (Patience et al., 1997). It was feared that they could interfere with the therapeutic vector in the target cell by recombinations due to sequence homologies. So far, these fears have not been confirmed experimentally, but with the development of very efficient human packaging cell lines, the problem of co-packaging and the unwanted transmission of potentially infectious HERV sequences has arisen. Therefore, the possible packaging of expressed HERV sequences in virions based on MLV was examined in detail. Patience et al. (1998) identified mRNA transcripts from several different HERV families such as HERV-K and HERV-H in human packaging cell lines. However, even with a highly sensitive RT-PCR test, none of these sequences could be detected in the MLV vector particles released by the cells.

According to these findings, packaging and transmission of HERV sequences and ultimately HERV-based vectors in MLV packaging systems seemed to be excluded. Although HERV genes have a sequence homology to MLV genes of 50-65%, the areas essential for packaging and infection, in particular the packaging signal located between the 5 'LTR and the gag region and the LTR itself, do not have any recognizable sequence homology to the corresponding MLV sequences. Efficient HERV packaging systems, on the other hand, are also currently unimaginable, since no cell lines are known to date that produce HERV particles in sufficient quantities.

The present invention thus solves the problem of retroviral expression vectors which control cell and tissue-specific expression of foreign genes (gene of interest) by providing expression vectors which, in a functional arrangement, contain at least the following elements:

a) DNA sequences for packaging the vector RNA and for cell-specific expression of proteins or peptides which are encoded by heterologous DNA nucleotide sequences,

b) one or more heterologous DNA nucleotide sequences coding for a protein or peptide (transcription unit); the DNA sequences for cell-specific expression are characterized in that they comprise a cell-specifically regulatable promoter region which originate from a human endogenous retroviral virus, in particular the LTR sequence of this virus;

The promoter region from a HERV sequence can encompass the entire LTR region of the HERV. In further embodiments of the invention, however, the promoter region only comprises the U3 region or the R-U3 region of the HERV-LTR. In a further preferred embodiment of the invention, in addition to these regions, the untranslated region between the 5 'LTR and the gag genes is also included in the promoter section. According to the invention it was found that sequences are also located in this area which control cell-specific expression of proteins or peptides, ie are at least partly responsible for this cell-specific expression.

The promoters are sections of the DNA which are necessary for the start of the transcription of the assigned structural genes. The promoter contains the starting point of the transcription, the recognition and binding site for the RNA polymerase. The promoter can also comprise further sequences to which regulatory proteins can bind and thereby specifically regulate the initiation of transcription. Examples of such proteins are transcription factors and repressors. Examples of these regulatory elements of transcription activity are the CAAT box, the GC box and the TATA box. The promoters are recognized by a type II polymerase.

The promoter sections for cell-specific expression of foreign proteins from HERVs can optionally be combined with further sequences which originate from exogenous retroviruses and which promote cell-specific expression. A combination with regulatory sequences from cellular genes is also conceivable to support cell-specific expression.

The retroviral expression vector according to the invention further contains at least DNA sequences for packaging the vector by a packaging helper cell line. The packaging DNA sequences are located between the 5 'LTR and the gag gene. Such packaging signals are present in all retroviral vectors and are therefore known to the person skilled in the art. Examples of packaging sequences are listed in Mann et al., 1985, and Rein, 1994, and the literature cited therein. Full reference is made to this literature.

The retroviral expression vector of the present invention contains one or more transcription units that code for an amino acid sequence. The amino acid sequence stands for a protein or peptide. Any sequence that encodes any protein or peptide of interest can be inserted into the expression vector. Such proteins or peptides can be encoded, for example, by marker genes, therapeutically valuable genes, genes with an antiviral effect, antitumor genes and / or cytokine genes. This list could be supplemented as desired. The inserted into the retroviral expression vector The genes are known to the person skilled in the art. The type of genes used depends on the intended use of the vector according to the invention.

The vectors according to the invention can be used, for example, in gene therapy to transfer heterologous DNA into target cells in order to make diseases accessible to a specific therapy. The vector DNA is introduced into the selected target cells so that the heterologous DNA is expressed in the target cell and the product encoded by the DNA is produced. These include in particular those genes for the expression of proteins which are not produced in the target cell or are no longer produced or are produced in insufficient quantities, so that a disease state arises. The invention includes not only those proteins or peptides that occur naturally, but also those that have been modified in such a way that a desired effect is achieved, for example a higher activity of an enzyme, the blocking of the binding site of viruses, destruction of tumor cells through suicide genes etc.

The DNA nucleotide sequences coding for a protein or a peptide are generally heterologous DNA which codes for RNA and proteins which are normally not produced in vivo by the cell in which the proteins or peptides are expressed. It can also be called foreign DNA. Any proteins, for example enzymes, hormones and antibodies, are included. The retroviral expression vectors provided according to the invention are therefore designed such that they can express proteins of interest in human cells.

The promoter regions used according to the invention are selected from HERV sequences which come from the known HERV families. Examples include HERV-K, HERV-H, HERV-E, HERV-L, HERV-T, HERV-R, HERV-I, HERV-P, ERV9, HERV-W.

Of course, other HERV families which are not yet known can of course also be screened in order to find as yet unknown promoter sequences which control cell-specific expression.

LTR sequences from HERVs which are preferred according to the invention and which can be used for the tissue-specific expression of proteins and peptides of interest are set out in the appendix disclosed. They can be used in retroviral expression vectors in order to achieve the object of the invention. Of course, only parts of these LTRs can be selected by methods known per se in order to keep the sequences used in the vector as small as possible. The appropriate fragments can be selected using various deletion mutants. Further modifications of these LTR sequences are also possible, for example point mutations, insertions, additions, exchange of several nucleotides etc. in order to increase the efficiency of the tissue-specific expression and to adapt it to the desired function.

In a preferred embodiment of the invention, the ProCon vectors described at the outset are used. Such ProCon vectors include a 5 'LTR portion of structure U3-R-U5, one or more sequences encoding a protein or peptide, and optionally non-coding sequences, and a 3' LTR portion comprising a partial or Completely deleted U3 section, the deleted U3 section comprising at least the HERV-LTR sequences used according to the invention, followed by the R-U5 section. Further details are described, for example, in WO96 / 07748 and WO96 / 28564. Full reference is made here to these writings.

According to the invention, a strategy was developed in order to detect cell-specific promoter sequences. This strategy is explained in more detail in the description below. Of course, other methods for finding cell-specific HERV-LTR sequences are of course also conceivable and applicable. The invention is therefore not restricted to the following exemplary embodiments.

The retroviral expression vectors according to the invention are packaging-deficient, ie they are unable to produce virus particles without the aid of a packaging helper cell line. The invention therefore also comprises a retroviral vector system which contains a retroviral expression vector as described in the present invention and a packaging cell line with at least one retroviral or recombinant retroviral construct which codes for the packaging proteins of the retroviral expression vector. Such packaging cell lines are known and described per se. For example, reference is made here to the murine packaging cell line PA317 (Salier et al., 1998). In the following, the invention is first described in general and then using exemplary embodiments.

According to the invention, the suitability of human endogenous retroviruses for the development of tissue-specific vectors for gene therapy was investigated. For this purpose, the tissue specificity of the HERV pol transcription in various cell lines such as T cells, keratinocytes and breast cancer cells was first checked in a "reverse dot blof" procedure. The expression patterns of the various HERV families were found to be entirely dependent on the cell type To isolate the transcriptionally active HERV LTRs from different cell lines and tissues, primers were developed with which the U3 / R regions from mRNA preparations can be specifically amplified.The isolated LTR sequences and individual representatives of already known LTRs were incorporated into expression vectors. The activity of the LTR promoters was tested after transient transfection of the reporter plasmids via luciferase activity or via eGFP fluorescence in different cell lines, and it was found that the promoter activities of the individual HERV LTRs vary significantly depending on the cell line tested -H LTR, which from A Strocytes and liver cells had been isolated and had proven to be particularly active in lung fibroblast cells (LC5) in several experiments, were inserted into two retroviral promoter conversion vectors (pLESN and PLX), tested in packaging cell lines, the packaging efficiency examined and checked to see if Infection of the target cells a promoter conversion has taken place. FACS analyzes were carried out to demonstrate the transcription activity in the target cells.

A method has thus been described with which HERV promoter sequences (U3 / R region), which mediate tissue-specific expression, can be identified and isolated. The tissue specificity and promoter activity of these sequences was then tested in a transient transfection assay in various human cell lines. Finally, suitable sequences were selected, cloned into a promoter conversion vector (ProCon vector) and their suitability for the construction of tissue-specific vectors for gene therapy was checked. The retroviral expression vectors according to the invention are produced by recombinant techniques known per se. Such techniques are described, for example, in Sambrook et al., 1989, and Perbai, 1984. tion of the ProCon vectors, reference is made to WO 96/07748 already mentioned at the outset and the literature associated therewith.

3. Results

3.1 Analysis of HERV transcription in different cell types

To examine HERV transcription in different cells, a method (reverse dot blot hybridization) was used in the first step, which was originally developed for the detection of HERV expression in mononuclear cells of peripheral blood (Herrmann and Kalden, 1994). Hereby, cloned and characterized HERV po. Gene fragments from human genomic DNA were fixed on a membrane and hybridized with radioactive HERV-po / gene probes. The probes were amplified by RT-PCR from mRNA from different lines using degenerate oligonucleotides that are homologous to a highly conserved region of retroviral po. Genes (Shih et al., 1989; Donehower et al., 1990). With this method, we obtained a characteristic hybridization pattern with each cell line examined so far, which was a first indication of a tissue-specific expression of the HERV elements.

3.2 Isolation of LTR U3 regions of expressed HERVs

The tissue-specific expression of a retrovirus is primarily determined by its U3 region. All regulatory sequences such as promoter, enhancer and the binding sites for various cellular transcription factors are located in this area. For this reason, primers were developed with which these HERV sequences could be isolated from mRNA of different cell lines by RT-PCR (Tab. 2; Fig.1). About 30 different HERV-LTRs were cloned in this way. Part of these sequences were tested in a reporter plasmid for promoter activity and tissue specificity.

In the first approach, a polydT primer was combined for the PCR with a primer that is complementary to the polypurine tract (PPT) of the retroviral RNA (Fig. 1). The PPT tract is a conserved region in the untranslated area between the env gene and the U3 Region of the 3'-LTR. The PPT region is used as the primer binding site for the synthesis of the plus strand during the reverse transcription of the retrovirus (Sorge and Hughes, 1982).

Database analyzes identified PPT sequences from different HERV families and divided them into different groups by comparing their homologies. From the consensus sequences of the individual groups, oligonucleotides were synthesized as primers for the RT-PCR. The mRNA was prepared from various cell lines: epithelial cells (HeLa, HaCaT), fibroblast cells (LC5), T cells (H9, HUT78), lymphoblasts (CML), gnome cells (85HG66, U373), pancreatic cells (MiaPaCa2, Pand), liver cells ( Chang Liver) and breast cancer cell lines (T47-D, MCF7). In addition, cDNA gene banks (Clontech) from various human tissues (brain, heart, liver, kidney, lung, pancreas, placenta, Skeiett muscle) were used for the RT-PCR.

The fragments obtained were then cloned, sequenced and analyzed using database comparisons. From the PCR fragments obtained with the PPT and polydT primers, two LTRs could be assigned to the HERV-H and HERV-K families via homology comparisons. By using polydT primers in these PCR approaches, numerous sequences were amplified that did not contain any homologies to known retroviral LTRs and, furthermore, did not contain any promoter structural elements. For this reason, further sequences from conserved areas of the U3 region and from the R region of HERV-K and HERV-H families were selected for primer synthesis (Mold et al., 1997), (Fig. 1, Tab. 1). The resulting PCR products were transferred to nitrocellulose filters after separation in an agarose gel and hybridized with probes prepared from the LTR regions of various HERV-LTRs (HERV-K-pl167, HERV-H-H6, HERV-E, HERV-L) were. The hybridizing fragments were then cloned into a vector (pZE-RO, Invitrogen) and sequenced. Various HERV-LTRs, which are listed in Table 3, were isolated using this method.

The HERV-K-LTRs, which were isolated from human brain and heart tissue as well as from T47-D cells, show very high sequence homologies to the 3 ^' LTR of HERV-K10. The HERV-H LTRs, on the other hand, show much higher sequence variabilities. HERV-H31, HERVHS, HERV-HCM1, HERV-HCM4, HERV-HMP23 are homologous to that of Mager et al. isolated HERV-H-H6 LTR, the other HERV-H sequences show homologies to that of Anderssen et al. (1997) isolated HERV-H LTRs from monkeys, marmosets and humans. The HERV-W LTRs isolated from T47-D cells are related to the LTR of clone CL6 (Komurian-Pradel, 1999).

3.3 Analysis of the expression of HERV promoters in a transient luciferase assay

To analyze the promoter activity and the tissue specificity of the isolated HERV-LTRs, these were first cloned into a luciferase reporter plasmid (pBL, Butz, K., DKFZ, Heidelberg). This vector contains the Photinus pyralis luciferase gene fused to the SV40 polyA signal from pBLCAT2 (Hoppe-Seyler et al., 1991).

The individual vector constructs were transiently transfected into different cell lines. After 48 hours, the luciferase activity from the cell lysate was measured with the luciferase assay kit from Promega and, after comparing the β-galactosidase activity or the Renilla luciferase activity, determined as relative luciferase activity. The promoter activities of the LTRs were in epithelial cells (HeLa, HaCaT), fibroblast cells (LC5), T cells (H9, HUT78), glioma cells (85HG66, U373), liver cells (Chang Liver), pancreatic cells (MiaPaCa2, Pand) and breast cancer cell lines (T47 -D, MCF7).

The results are shown in Figures 2a - 2f. The HERV-H-H6 LTR therefore has the strongest promoter of all endogenous LTRs examined. The HERV-K LTR from placenta is particularly active in HeLa cells. This LTR is only very weakly active in all other cell lines. HERV-K-T47-D also showed a strong activity in HeLa cells, this LTR was also active in HaCat cells and pancreatic cells. The HERV-L LTR has strong promoter activity in liver cells and is weakly active in T cells and pancreatic cells. The HERV-T-S71A and HERV-E LTRs were not active in any celline tested. So far, no activity of a HERV-LTR has been observed in CML cells.

The cloned HERV-H LTRs (HERV-H1, HERV-H8, HERV-H13, HERV-H19, HERV-H-H6, Table 3) were almost all active in 85HG66 lines, with HERV-H 1 and HERV-H8 showed the highest activity in this cell line (without figure). HERV-H19 was very active in HeLa cells. The HERV-HCM1 LTR showed the highest promoter activity in all cell lines and was particularly active in lung fibroblasts (LC5) (Fig. 3).

3.4 Construction of HERV hybrid vectors and verification of the activities of HERV promoters in these vectors

The functionality of human endogenous retroviral LTR sequences in retroviral vectors was tested in two different promoter conversion vectors (ProCon). Hybrid HERV / MLV vectors were constructed using two MLV-based vectors pLESN-MMTV (Fig. 7) and pLX-MMTV (Fig. 8). These vectors contain the EGFP gene as reporter gene, which is expressed from the 5 'LTR (different, depending on whether before or after the promoter conversion), and a neomycin gene, which is expressed from an SV40 promoter. In addition, the vector pLX-MMTV contains a prokaryotic replication original, which allows the provirus to be recloned for further molecular characterization.

To construct the HERV hybrid vectors, the MMTV-LTR was replaced by the HERV-HCM1 LTR (Fig. 7). For this purpose, the LTR was first amplified by means of PCR from the vector pBL-HERV-H with specific primers which contained additional sequences for the restriction enzymes Mlul and Sacll. These fragments were then inserted into the 3 ^' U3 deleted vectors. The reporter gene EGFP is first expressed from the MLV promoter after transfection into the packaging cell line. (Fig. 9a) After infection of the target cells and successful promoter conversion by reverse transcription in the target lines, the reporter gene is under the transcription control of the HERV LTR.

The HERV-Hybrid vector constructs pLESN-HERV-H (Fig. 7) and pLX-HERV-H (Fig.8), as well as the origin vectors pLESN-MMTV and pLX-MMTV were transfected into the amphotrophic packaging cell line PA317. The resulting retroviral vector particles were then used to infect the CrfK and LC5 cell lines.

The infected cell lines were cloned and the selected cell clones were examined to determine whether they contained the vector constructs and whether the promoter conversion had taken place. To this end, chromosomal DNA was prepared from infected and uninfected cells and analyzed by PCR. The primers were selected from the MLV U3 (P5) and R (P2) region and from the HERV-H region (P1) and used in combination with a primer from the EGFP region for a PCR (Fig. 9a). The PCR products were hybridized with HERV-H specific probes (Fig. 9b). After amplification with the primers P1 and P3, the DNA which was infected with the pLX HERV-H particles resulted in a PCR product of 1.1 kb which hybridized with the HERV-H probe. Amplification with the MLV U3-specific primers (P2 / P3) with DNA from cells infected with pLX and with pLX HERV-H resulted in PCR products with a size of approximately 900 bp which did not hybridize with the HERV-H probe. No PCR product which hybridized with the HERV-H probe was obtained from the amplification with the MLV R primers (P5 / P3). These results show that promoter conversion has taken place and the MLV promoter of the 5 ^' LTR has been replaced by the HERV promoter.

The promoter activity of the HERV LTR in the retroviral vectors was determined after integration into the DNA of the target cells by means of FACS analyzes by measuring the EGFP fluorescence (FIG. 10). The activity of the starting vector pLX-MMTV was compared with the HERV vector pLX-HERV-H (H6) before and after induction with dexamethasone. The vector with the MMTV LTR can be activated by dexamethasone. The HERV LTR vector is not activated by dexamethasone, but it is approximately 10 times more active than the dexamethasone-stimulated MMTV hybrid vector.

3.5 Influence of regulatory elements in the R and U5 region on the promoter activity of HERV sequences.

In order to test which sequence region is required for a functional HERV promoter, the influence of additional LTR sequences which are located outside the U3 region in the LTR was investigated using some examples. For this purpose, the activity of the U3 region was compared with the activity of the corresponding U3-R fragments in the luciferase assay of 7 HERV-K LTRs (HERV-K-T47D, L5, L50, L8, L9, L48 and L20 / 49) . Surprisingly, it was found that the different R regions can influence the promoter in the U3 region in very different ways. For group 1 LTRs (L5, L50, L8, L9), the presence of R sequences leads to a significant increase in the pro- motor activity (Fig.4a). In contrast, in group 2 LTRs (L20 / L49), the activity of the HERV promoter is reduced by the R region (FIG. 4b). The HERV-K-T47D promoter (Fig. 5) and the L48 promoter (not shown) are not significantly influenced by the corresponding R sequences. Interestingly, however, in the case of the HERV-K-T47D-LTR, sequence regions that are located downstream of the U3-R region and include the U5 region, as well as the 3 ′ untranslated region and the beginning of the gag gene, have a clear activating effect (Fig. 5).

Sequence analysis of the various R regions tested showed that Group 1 LTRs in the R region have a binding site for the transcription factor SP1 that is missing in the Group 2 R region LTR (FIG. 6). In contrast, the group 2 R region contains a potential binding part for the factor TFS3, which acts as a transcription repressor. This shows that the activity of HERV promoters can be modified by incorporating additional regulatory elements such as transcription factor binding sites, enhancer sequences or negatively regulating elements.

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Claims

claims

1. Retroviral expression vector containing at least the following elements in a functional arrangement:

a) DNA sequences for packaging the vector RNA and for cell-specific expression of proteins or peptides which are encoded by heterologous DNA nucleotide sequences; b) one or more DNA nucleotide sequences coding for a protein or peptide,

characterized in that the DNA sequences for cell-specific expression contain a cell-specifically regulable promoter region from a human endogenous retroviral DNA nucleotide sequence (HERV).

2. Expression vector according to claim 1, characterized in that the DNA sequences for cell-specific expression originate from the LTR area and optionally the non-translated area between the 5 'LTR and the gag area from HERVs.

2. Vector according to claim 1 or claim 2, characterized in that the entire LTR region, the U3 region or the R and U3 region comes from a human endogenous retroviral nucleotide sequence.

3. Vector according to one or more of the preceding claims, characterized in that the nucleotide sequences coding for one or more proteins or peptides are selected from one or more elements of the group consisting of marker genes, therapeutic genes, antiviral genes, antitumor genes and cytokines .

. Vector according to one or more of the preceding claims, characterized in that the cell-specifically regulatable promoter region originates from the LTR region of a cell-specifically expressed endogenous human retroviral nucleotide sequence.

5. Vector according to one or more of the preceding claims, characterized in that the human endogenous retroviral line-specifically regulable promoter sequences are selected from one or more promoter sequences of HERV families of the group consisting of HERV-K, HERV-H, HERV-E, HERV-L, HERV-T, HERV-R, HERV-I, HERV-P, ERV9. HERV-W.

6. Vector according to one or more of the preceding claims, characterized in that the promoter region comprises, in addition to the TATA box, further recognition and binding sites for regulatory proteins.

7. Vector according to claim 7, characterized in that the recognition and binding sites for regulatory proteins include the GC box, the CAAT box, enhancer sequences and repressor sequences as well as hormone-sponsible sequence motifs and that optionally additional recognition and binding sites for regulatory proteins Proteins from the LTR region of exogenous retroviruses and / or from cellular genes are contained.

8. Vector according to one or more of the preceding claims, characterized in that the vector is a promoter conversion vector comprising a 5 'LTR section of the structure U3-R-U5, one or more sequences selected from coding and non-coding sequences , and a 3 'LTR section comprising a partially or completely deleted U3 section, the deleted US section being replaced by a cell-specifically regulatable promoter region from a HERV-LTR sequence, followed by the R-U5 section.

. mRNA or RNA of a retroviral expression vector according to one or more of the preceding claims.

10. prokaryotic line or eukaryotic cell containing a retroviral expression vector according to one or more of the preceding claims.

11. Eukaryotic cell containing a retroviral expression vector according to one or more of the preceding claims in an integrated form.

12. Use of a cell-specifically regulatable promoter section from a human endogenous retroviral DNA nucleotide sequence for controlling the expression of foreign genes in retroviral expression vectors, preferably ProCon vectors.

13. Use of an expression vector according to one or more of the preceding claims for the expression of foreign genes in gene therapy.

14. Virion containing a retroviral expression vector RNA derived from an expression vector DNA according to one or more of the preceding claims.

15. A method for producing a virion according to claim 15 for introducing one or more nucleotide sequences coding for a protein or peptide, characterized in that the retroviral expression vector according to one or more of the preceding claims is introduced into a suitable packaging cell line under conditions such that the virion is formed and is released from the packaging cell line.

16. A method for introducing nucleotide sequences coding for one or more proteins or peptides into a eukaryotic cell, characterized in that the cell is infected with a virion according to claim 15 under conditions such that the nucleotide sequences coding for the protein or peptide in the chromosomal DNA of the eukaryotic cell is inserted.

17. The method according to claim 17, characterized in that the eukaryotic cell is a mammalian cell.

18. The method according to claim 18, characterized in that the mammalian cell is a human cell.

19. Retroviral vector system, comprising a retroviral expression vector according to one or more of the preceding claims and a packaging line with at least one retroviral or recombinant retroviral construct which codes for the packaging proteins of the retroviral expression vector.

20. Retroviral vector system according to claim 20, characterized in that the packaging line contains retroviral or recombinant retroviral constructs which code for those retroviral proteins which are not encoded by the retroviral expression vector.