JP2002537846A - Retroviral expression vector based on HERVLTR sequence - Google Patents

Abstract

  (57) [Summary] The present invention relates to a retrovirus expression vector having a cell-specific regulatable promoter. For example, the vectors may be useful for cell-specific expression of genes of therapeutic value in the context of gene therapy. The present invention provides a functional assembly comprising at least the following elements: a) a DNA sequence for the packaging of vector RNA and for cell-specific expression of a protein or peptide encoded by a heterologous DNA nucleotide sequence; b) a protein Or one or more DNA nucleotide sequences encoding a peptide, wherein said DNA sequence for cell-specific expression is a cell-specific regulatable promoter derived from a human endogenous retroviral DNA nucleotide sequence (HERV) A retroviral expression vector is described.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a retrovirus expression vector having a promoter that can be regulated in a cell-specific manner. The vectors are useful for cell-specific expression of genes of therapeutic value, for example, in connection with gene therapy.

[0002] Retroviruses are RNA viruses, whose viral genes are single-stranded RNA
Encoded by the molecule. After entry of the virus into the cells, the viral RNA is converted into double-stranded DNA molecules by reverse transcription. The DNA enters the nucleus and integrates into cellular chromosomes. The integrated form of the viral DNA, the so-called provirus, corresponds to the template for viral gene expression.

[0003] Integration of the viral genome into cellular chromosomes is an essential step in viral replication and is mediated by virally encoded enzymes. With few exceptions, the viability of the infected cells appears to have little or no effect on the presence of the retroviral genome, expression of that gene and virus particle formation in the cells.

[0004] Retroviral gene transfer is used to introduce a functional gene, particularly a therapeutically valuable gene, into a cell without affecting the ability of the host cell to grow. Due to their mode of replication, retroviruses are suitable for such gene transfer. In the simplest embodiment, at least a portion of the viral gene is replaced by the gene of interest and the gene of interest is introduced into the target cell by using an efficient viral infection method.

[0005] Retroviral infection occurs with high efficiency and is capable of modifying retroviral vectors, incorporating heterologous DNA, and allowing for stable integration into the host cell genome, resulting in retroviral infection. Viral vectors are suitable for gene therapy. In recent years, multiple retroviral vectors have been developed,
And by way of example, Gunzburg et al. (1996) and Robbins et al.
998).

A possible preferred embodiment for a retroviral vector is the so-called ProCo
n vector, which was first described in WO 96/07748. For disclosure, reference is made to this document fully.

[0007] The ProCon vector has a heterologous promoter element and, optionally, other regulatory elements in its 3 ′ LTR, which are replicated after infection and translocated to the 5 ′ LTR in target cells, and It is possible to regulate the expression of a marker or therapeutic gene. These heterologous genes are not directly linked to the promoter but are inserted inside the vector.

[0008] The ProCon vector comprises a 5 'LTR portion having the structure U3, R, U5, and at least one coding and / or non-coding sequence, and a completely or partially deleted U3 portion; The deleted U3 portion has been replaced with a polylinker sequence and includes the 3 'LTR region followed by the R-U5 portion.

[0009] The propagation of these vectors is performed by helper cell lines that produce large amounts of viral proteins that are no longer synthesized from the expression vector itself. However, helper cell lines are no longer able to produce replication competent viruses. The cell line is also referred to as a packaging cell line and includes a cell line transfected with at least a second plasmid having a gene capable of packaging the modified retroviral vector. In this regard, reference is made to WO 92/10564, which is fully incorporated herein.

[0010] The DNA (expression vector) encoding the modified retrovirus is transfected into a packaging cell line. Under these conditions, the modified retroviral genome, including the insertion therapy gene or marker gene, is transcribed and packaged into retroviral particles (recombinant viral particles), respectively. The recombinant virus is then used to infect target cells; the genome of the modified retrovirus,
That is, the expression vector is integrated into the target cell genome, and the integration occurs with the marker gene or therapeutic gene, respectively. Cells infected with the recombinant virus particles produced in this manner cannot produce new vector viruses because these cells have no other viral proteins. The DNA of the expression vector, which contains the therapeutically valuable gene or marker gene, respectively, integrated into the host cell, is present in the cellular DNA in an integrated form and can be subsequently expressed intracellularly. .

[0011] Preferably, the therapeutically valuable genes whose expression is achieved by such retroviral expression vectors are expressed in a cell and tissue specific manner. For this purpose, a cell-specific control sequence is introduced into the LTR sequence of the expression vector. For example, these cell-specific control sequences include a cell-specific regulatable promoter region, a cell-specific enhancer sequence, as well as a transcription factor binding site. The promoter is LT
Located at the U3 site of R.

Here, a cellular promoter sequence or an exogenous retroviral promoter sequence is inserted into a retroviral vector. Cellular promoters often require additional signal structure, which may be located far upstream or downstream from the promoter. Therefore, it has always been found to be difficult to isolate strong, tissue-specific cellular promoter sequences and clone them into retroviral vectors. The exogenous retroviral promoter contains all necessary regulatory regions in a limited region within the retroviral LTR, and thus can be transcribed essentially independently of the DNA sequence flanking the integration site. It has the advantage of being possible. However, a severe disadvantage is that they are strong, but not tissue-specific, and are generally expressed with equal strength in all cell types.

[0013] It is therefore an object of the present invention to take advantage of the retroviral promoter, which concentrates all the signal structures required for transcription in a limited region within the U3 and R regions, but at the same time relates to it. It is to provide a novel retroviral expression vector that avoids disadvantages.

[0014] In accordance with the present invention, this object has been achieved by inserting a cell-specific regulatable promoter portion derived from a human endogenous retroviral DNA nucleotide sequence (HERV) into a retroviral expression vector. These promoter sequences of the human endogenous retrovirus are already present in the host cell and, according to the invention, they are very suitable for controlling cell-specific expression of marker genes and genes of therapeutic value. Has been found.

[0015] Endogenous retroviruses (ERVs) can be found in the genome of all cells of an organism. They move vertically through the germ line and can be reactivated by environmentally induced conditions. About 2% of the human genome consists of endogenous retroviruses and retroviral sequences,
The LTR is present in an amount of 20,000-40,000 copies per genome (Table 1) (Leib-Mosch et al., 1993; Wilkinson et al., 1994).
Patience et al., 1997).

[0016]

[Table 1]

Since the HERV sequence was already integrated into the primate genome 30,000-40 million years ago, during evolution, most of the pathogenic sequences were mutated and rearranged, respectively. Can be assumed to have been removed or modified from the provirus and no longer disadvantageous to the organism. Compared to vectors derived from animal viruses, retroviral vectors constructed from such sequences have the advantage that no new viral sequences need to be introduced into the genome. In addition,
Recombination with HERV sequences already present in the genome does not give rise to new retroviruses, as might be the case when using other species of retroviruses as vectors. For this reason, the use of these sequences in the construction of a retroviral vector can minimize safety risks. Further, the homologous region contained in the genome can be used for tissue-specific integration of the retroviral vector into a specific site of the chromosome.

[0018] During the course of evolution, the HERV element has accepted several cellular functions. For example, the promoter and enhancer elements of the HERV LTR are used to regulate the transcription of cellular genes (Kato et al., Feuchter-Murthy et al.
993; Di Christofano et al., 1995). One example of the use of LTR regulatory elements for tissue-specific expression of cellular genes is the human amylase gene. This gene is regulated by the LTR of the HERV-E element and is specifically restricted in this manner to be expressed only in salivary glands (Ting et al., 199).
2). In addition, Schulte and co-workers (1996) have shown that insertion of an endogenous retrovirus into the 5 'untranslated region of the pleiotrophin gene is responsible for its trophoblast-specific activity (Schulte). Et al., 1996).
In another example, the polyA signal of the HERV LTR may also be used to polyadenylate cellular transcripts (Mager, 1989; Gooddc).
hill et al., 1992).

The main advantage of using a retroviral promoter is that, as mentioned above, the retrovirus must maintain its transcriptional activity very independent of the region surrounding the integration site, as described above. This is due to the fact that all signal structures are located in restricted regions within the U3 and R regions of the LTR. Because these HERV promoters have persisted in the primate genome for millions of years, they are active during evolution in a cell type-specific manner similar to cellular promoters, and thus cellular and retroviral promoters It has been adapted to combine the advantages of

HERV is transcribed starting from the classical RNA polymerase II promoter (Wilkinson et al., 1994). This promoter is located within the LTR region. Thus, the HERV transcript does not contain any complete copy of the provirus. To offset the loss of transcriptional regulatory elements, these elements have evolved the mechanism of reverse transcription. This will play back the missing arrays on both sides of the element,
Thereby, the LTR is reproduced next. In addition to the promoter sequence, HERV LT
R is also responsible for multiple distinct binding sites of transcription factors (
Seifart et al., 1998).

Although there are certain structural similarities between HERV and exogenous animal retroviruses, such as MLV or MMTV, HERV sequences and especially the HERV promoter are potential candidates for the development of retroviral expression vectors. Has never been regarded. In contrast, to date, they have only been regarded as disruptive in terms of gene therapy (Patence et al., 1997). Due to sequence homology, there has been concern that they may interfere with therapeutic vectors due to recombination in target cells. To date, it has not been possible to confirm these concerns by experimentation, but in developing highly efficient human packaging cell lines, co-packaging and accidental packaging of potentially infectious HERV sequences There was a problem with the introduction of. Therefore, a detailed study of the possibility of packaging the expressed HERV sequence into MLV-based virions has been performed. P
(1998) describe the mRs of several different HERV families, such as HERV-K and HERV-H, in human packaging cell lines.
The NA transcript was identified. However, even with the very sensitive RT-PCR test, none of these sequences could be detected in MLV vector particles released into cells.

According to these findings, the HERV sequence in the MLV packaging system
And, thus, also ultimately, the packaging and introduction of HERV-based vectors seemed out of the question. The HERV gene has 50-65% sequence homology with the MLV gene, but the LTR itself, especially with the regions essential for packaging and infection, and especially with the packaging signal located between the 5 'LTR and the gag region. Has no detectable sequence homology with the corresponding MLV sequence. However, since no cell line producing HERV particles in sufficient quantities is known to date, an efficient HERV packaging system is also not currently considered.

Thus, the present invention provides a functional assembly comprising at least the following elements: a) DNA for the packaging of vector RNA and for the cell-specific expression of proteins or peptides encoded by heterologous DNA nucleotide sequences Sequence; b) one or more heterologous DNAs encoding the protein or peptide
An expression vector comprising a nucleotide sequence (transcription unit), wherein the DNA sequence for cell-specific expression comprises a human endogenous retrovirus, and in particular a cell-specific regulatable promoter region from the LTR sequence of said virus. Provides a solution to the problem of retroviral expression vectors that regulate cell and tissue-specific expression of foreign genes (genes of interest).

[0024] The promoter region of the HERV sequence may include the entire LTR region of HERV. However, in another aspect of the invention, the promoter region comprises a HERV LTR
Only the U3 region or the RU3 region. In another preferred embodiment of the invention, in addition to these regions, the promoter region also contains the untranslated region between the 5 'LTR and the gag gene. In accordance with the present invention, it has also been found that this region also includes sequences that modulate, respectively, the cell-specific expression of the protein or peptide, ie, at least contribute to said cell-specific expression.

A promoter is a partial region of DNA required to initiate transcription of a corresponding structural gene. A promoter contains a transcription initiation site, a recognition and binding site for RNA polymerase. The promoter may also bind regulatory proteins,
And, thereby, may also include other sequences that specifically regulate transcription initiation. Examples of such proteins are transcription factors and repressors. Examples of said regulatory elements of transcription activity are the CAAT box, GC box and TATA box. The promoter is recognized by type II polymerase.

The promoter region for cell-specific expression of a foreign protein from HERV may be combined, if desired, with other sequences from an exogenous retrovirus that promote cell-specific expression. In addition, combinations with regulatory sequences from cellular genes that support cell-specific expression are contemplated.

Further, the retroviral expression vector of the present invention contains at least a DNA sequence for packaging the vector with a packaging helper cell line. The DNA sequence for packaging is located between the 5 'LTR and the gag gene. Such packaging signals are present on any retroviral vector and are therefore known to those skilled in the art. Examples of packaging signals are listed in the references cited therein, along with Mann et al., 1985, and Rein, 1994. This document is fully incorporated herein by reference.

[0028] The retroviral expression vector of the present invention comprises one or more transcription units encoding an amino acid sequence. Amino acid sequence refers to protein or peptide. Any sequence encoding the protein or peptide of interest may be inserted into the expression vector. For example, such a protein or peptide may be encoded by a marker gene, a gene of therapeutic value, a gene with antiviral function, an anti-tumor gene and / or a cytokine gene. This list may continue to any number. Genes that may be introduced into the retroviral expression vector are known to those skilled in the art. The type of gene to be inserted depends on the intended use of the vector of the invention.

For example, the vectors of the present invention may be used in gene therapy to introduce heterologous DNA into target cells, so that the disease is affected by specific therapy. Vector DN
A is introduced into the selected target cells, the heterologous DNA is expressed in the target cells, and D
Allow the product encoded by NA to be produced. This includes, in particular, genes for the expression of proteins that are not or no longer produced by the target cells or are not produced in sufficient quantity, so that a disease state develops. The present invention is modified not only in each of these naturally occurring proteins or peptides, but also in a manner that achieves the desired effects, such as higher enzymatic activity, blocking of viral binding sites, destruction of tumor cells by suicide genes, etc. Including those that are.

In general, the DNA nucleotide sequence encoding a protein or peptide, respectively, is typically expressed in vivo by the cell in which the protein or peptide is expressed.
Heterologous DNA encoding RNA and proteins not produced in vo. It may also be referred to as foreign DNA. This includes any proteins, such as enzymes, hormones, and antibodies. Therefore, the retrovirus expression vector provided in the present invention is designed to express a target protein in human cells.

The promoter region used according to the present invention is selected from HERV sequences from known HERV families. Examples of these are HERV-K, HERV
-H, HERV-E, HERV-L, HERV-T, HERV-R, HERV-
I, HERV-P, ERV9, and HERV-W.

It should be understood that other, currently unknown, HERV families can be screened to find promoter sequences that are currently unknown and control cell-specific sequences.

[0033] Preferred HERV-derived LTR sequences according to the invention that may be used for tissue-specific expression of proteins and peptides are disclosed in the annex. These may be introduced into a retroviral expression vector to achieve the purpose according to the present invention. However, it should be understood that it is also possible, by methods known per se, to select only those parts of the LTR which keep the sequence inserted in the vector as small as possible. Useful fragments may be selected using a variety of deletion mutants. In addition, other mutations of these LTR sequences, such as point mutations, insertions, additions, substitutions, etc. of some nucleotides, are also possible to increase the efficiency of tissue-specific expression and adapt to the desired function. .

In a preferred embodiment according to the present invention, the ProCon as described initially
Vectors are used. Such a ProCon vector has the structure U3-RU-U5
Comprising one or more sequences encoding a protein or peptide and optionally non-coding sequences, and a partially or completely deleted U3 region, wherein the deleted U3 portion comprises , Comprising at least the HERV-LTR sequence used according to the invention, followed by the RU5 region, 3 '.
Includes LTR region. Further details are described, for example, in WO 96/07748 and WO
96/28564. These documents are fully incorporated herein by reference.

In accordance with the present invention, strategies have been developed to locate promoter sequences with cell-specific functions. This strategy is described in more detail in the description below. In principle, it must be understood that other methods of discovering HERV LTRs that act in a cell-specific manner may also be considered and used. Therefore, the present invention is not limited to the following examples.

The retroviral expression vector according to the invention is defective in packaging, ie it is not possible to produce virus particles without the assistance of a packaging helper cell line. Accordingly, the present invention also includes a retroviral expression vector as described in the present invention, and a packaging cell line comprising at least a retrovirus or a recombinant retroviral construct encoding a packaging protein of the retroviral expression vector. Also includes retroviral vector systems. Such packaging cell lines are known per se and have been described. See, for example, the murine packaging cell line PA317 (Saller et al., 1998).

In the following, the invention will be described in general terms and thereafter with reference to examples. In accordance with the present invention, the applicability of human endogenous retroviruses to develop tissue-specific vectors for gene therapy was investigated. For this purpose, first, HERV
The tissue specificity of the pol transcript was examined in different cell lines, such as T cells, keratinocytes and breast cancer cells, using a "reverse dot blot" method. In this test, various HER
It has been found that the expression pattern of the V family generally depends on the cell type.
To isolate HERV LTRs with transcriptional activity from different cell lines and tissues,
A primer has been developed that can be used to specifically amplify the U3 / R region from mRNA preparation. With the isolated LTR sequence, the individual members of the already known LTR were inserted into the expression vector. Following transient transfection of the reporter plasmid, LTR promoter activity was tested in different cell lines via luciferase activity or eGFP fluorescence, respectively. The promoter activity of individual HERV LTRs was found to differ distinctly depending on the cell line tested. In some tests, HERV isolated from astrocytes and hepatocytes was found to be particularly active in lung fibroblasts (LCS).
-H LTR promoter region was inserted into two retroviral promoter conversion vectors (pLESN and pLX) and tested in packaging cell lines,
Packaging efficiency was evaluated and tested for the occurrence of promoter conversion after infection of target cells. FACS analysis was performed to detect transcriptional activity in target cells.

Thus, the HERV promoter sequence (U3 / R) that mediates tissue-specific expression
Regions) have been described that can be identified and isolated. Subsequently, these sequences were tested for tissue specificity and promoter activity in a variety of human cell lines in a transient transfection assay. Finally, the appropriate sequence was selected and cloned into a promoter conversion vector (ProCon vector), thereby examining its utility for constructing a tissue-specific vector for gene therapy.
The preparation of the retroviral expression vector according to the invention is carried out using recombinant techniques known per se. Such techniques are described, for example, in Sambrook et al., 198
9, and Perbal, 1984. For the construction of the ProCon vector, see WO 96/07748, already mentioned at the beginning of the relevant literature.

[0039] 3. Results 3.1 Analysis of HERV transcripts in different cell types To examine HERV transcripts in different cells, in a first step a method originally developed to detect HERV expression in peripheral blood mononuclear cells (reverse dot) Blot hybridization) (Herrmann and Kalden, 1994). In this method, a HERV pol gene fragment cloned and characterized from human genomic DNA is immobilized on a membrane and radiolabeled HERV
Hybridized with the pol gene probe. Probes were amplified from mRNAs of different cells using RT PCR and degenerate oligonucleotides homologous to highly conserved regions of the retroviral pol gene (Shih et al., 1989;
Donehower et al., 1990). Using this method, we obtained a characteristic hybridization pattern in all cell lines examined so far,
This was the first result showing tissue-specific expression of the ERV element.

3.2 Expression Isolation of the LTR U3 Region of HERV The tissue-specific expression of a retrovirus is mainly determined by its U3 region.
This region localizes all regulatory sequences, such as promoters, enhancers, and binding sites for various cellular transcription factors. For this reason, primers have been developed that can be used to specifically isolate these HERV sequences from mRNAs of different cell lines by RT PCR (Table 2; FIG. 1). In this manner, about 30 different HERV LTRs were cloned. In part, these sequences were tested in reporter plasmids for promoter activity and tissue specificity.

[0041]

[Table 2]

In a first approach, for PCR, a poly dT primer was combined with a primer complementary to the polypurine extension (PPT) of retroviral RNA (FIG. 1).
). PPT extension is a conserved part of the untranslated region between the env gene and the U3 region of the 3 'LTR. During retroviral reverse transcription, the PPT region is used as a primer binding site for positive strand synthesis (Sorge and Hughes,
1982).

Database analysis identified PPT sequences of different HERV families,
By comparing their homology, they were classified into different groups. Oligonucleotides were synthesized from individual groups of consensus sequences as primers for RT PCR. MRNA was prepared from different cell lines: epithelial cells (HeLa, HaCa
T), fibroblasts (LC5), T cells (H9, HUT78), lymphoblasts (CM
L), glioma cells (85HG66, U373), pancreatic cells (MiaPaCa2
, Panc1), hepatocytes (Chang liver), and breast cancer cell lines (T47-D,
MCF7). In addition, cDNA libraries (Clontech) of various human tissues (brain, heart, liver, kidney, lung, pancreas, placenta, skeletal muscle) are also available from RT PCR.
Used in.

Subsequently, the obtained fragments were cloned, sequenced and analyzed by database comparison. Among the PCR fragments obtained using the PPT and poly dT primers,
By homology comparison, two LTRs were assigned to the HERV-H and HERV-K families. By using poly dT primers in these PCR samples, a number of sequences that did not show any homology to the known retroviral LTR and further did not contain any promoter structural elements were amplified. For this reason, from the conserved region of the U3 region of the HERV-K and HERV-H families,
From the R region, another sequence was selected for primer synthesis (Mold et al.,
1997) (FIG. 1, Table 1). After separating the resulting PCR product on an agarose gel, it was transferred to a nitrocellulose filter and treated with a different HERV L
TR (HERV-K-p1167, HERV-H-H6, HERV-E, HER
VL) and hybridized with the probe prepared from the LTR region. Thereafter, the hybridizing fragment was cloned into a vector (pZERO, Invitrogen) and sequenced. Using this method, several HERs listed in Table 3 were used.
It was possible to isolate the VLTR.

[0045]

[Table 3]

HERV-K isolated from human brain and heart tissue and from T47-D cells
The LTR shows very strong sequence homology to the 3 ′ LTR of HERV-K10. In contrast, the HERV-H LTR showed much higher sequence variation. HERV-
H31, HERV-H3, HERV-HCM1, HERV-HCM4, HERV
-HMP23 is homologous to the HERV-H6 LTR isolated from Mager et al., And the other HERV-H sequences are the velvet monkey, marmoset (marmoset) isolated from Anderssen et al. (1997).
set) and HERV-H LTRs of human origin. The HERV-W LTR isolated from T47-D cells was clone CL6 (Komurian).
-Pradel, 1998).

3.3 Analysis of HERV Promoter Expression in Transient Luciferase Assays For analysis of the promoter activity and tissue specificity of the isolated HERV LTR, these were first luciferase reporter plasmids (pBL, Butz, K., D
KFZ, Heidelberg). This vector is pBLCAT2
Ptilinus fused to the SV40 polyA signal of Photinu
s.pyralis) (Hoppe-Seyle)
r et al., 1991).

The individual vector constructs were transiently transfected into different cell lines. Forty-eight hours later, luciferase activity from cell lysates was measured using a Promega luciferase assay kit and, after normalization for β-galactosidase activity or Renilla luciferase activity, respectively, determined as relative luciferase activity. . LTR promoter activity was determined by epithelial cells (HeLa, HaCaT), fibroblasts (LC5), T cells (H9,
HUT78), glioma cells (85HG66, U373), hepatocytes (Chang
Liver), pancreatic cells (MiaPaCa2, Panc1), and breast cancer cell line (T4
7-D, MCF7).

The results are shown in FIGS. 2a-2f. According to these results, of all the endogenous LTRs tested, the HERV-H-H6 LTR has the strongest promoter.
HERV-KLTR from placenta is particularly active in HeLa cells. In all other cell lines, the present LTR shows very weak activity. Also, HeL
In a cells, HERV-K-T47-D showed strong activity, and the LTR was also active in HaCaT cells and pancreatic cells. The HERV-L LTR has strong promoter activity in hepatocytes and weak activity in T cells and pancreatic cells. HERV-T-S71A and HERV-ELTR were not active in any of the cell lines tested. To date, in CML cells, H
No activity was observed for any of the ERV LTRs.

Almost all of the cloned HERV-H LTR (HERV-H1
HERV-H8, HERV-H13, HERV-H19, HERV-H H6
, Table 3) was active in 85HG66 cells, but HERV-H1 and HERV
-H8 showed the highest activity in this cell line (data not shown). 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).

[0051] 3.4 The functionality of the HERV hybrid vector construct and a human endogenous retrovirus LTR sequences in monitoring retroviral vectors in our Keru HERV promoter activity in these vectors in two different promoters conversion vectors (ProCon), Tested. For this purpose, two vectors based on MLV, pLESN-MMTV (
7) and pLX-MMTV (FIG. 8) to obtain a hybrid HERV / MLV
The vector was constructed. These vectors contain the neomycin gene expressed from the SV40 promoter with the EGFP gene as a reporter gene, expressed from the 5 'LTR (different amounts depending on whether measured before or after promoter conversion). In addition, the vector pLX-MMTV contains a prokaryotic origin of replication, which allows the provirus to be recloned for further molecular characterization.

To construct a HERV hybrid vector, in each case the MMTV LTR
Was replaced by the HERV-HCM1 LTR (FIG. 7). For this purpose, the LTR was first amplified by PCR from the vector pBL-HERV-H using specific primers containing additional sequences for the restriction enzymes MluI and SacII. These fragments were then inserted into a vector whose 3'U3 region had been deleted. After transfection into the packaging cell line, the EGFP reporter gene is first expressed from the MLV promoter (FIG. 9a). After successful infection of the target cell and successful promoter conversion by the reverse transcriptase in the target cell, the reporter gene is under the transcriptional 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 parent vectors pLESN-MMTV and pLX-MMTV, were added to the amphotropic packaging cell line PA317. Transfected. The resulting retroviral vector particles were then used for infection of cell lines CrfK and LC5.

The infected cell line was cloned and selected cellular clones were examined for the presence of the vector construct and for the occurrence of promoter conversion. For this purpose, chromosomal DNA was prepared from infected and uninfected cells and analyzed by PCR. MLV
Primers were selected from the HERV-H region (P1), together with the U3 (P5) and R (P2) regions, and combined with primers from the EGFP region and used for PCR (FIG. 9a). The PCR product was hybridized with a HERV-H specific probe (FIG. 9b). After amplification using primers P1 and P3, pLX HERV
DNA infected with -H particles hybridizes to the HERV-H probe
A 1 kb PCR product was generated. When the DNA of cells infected with pLX and pLX HERV-H was amplified using the MLV U3-specific primer (P2 / P3), a PCR product having a size of about 900 bp that did not show hybridization to the HERV-H probe was obtained. occured. Amplification using the MLV R primer (P5 / P3) did not yield a PCR product that hybridized to the HERV-H probe. These results indicate that promoter conversion has occurred and that the MLV promoter in the 5 'LTR has been replaced by the HERV promoter.

After integration into target cell DNA, the HERV LTR in the retroviral vector
Promoter activity was determined by FACS analysis via measurement of EGFP fluorescence (FIG. 10). For this purpose, the activity of the starting vector pLX-MMTV was determined by comparing the HERV vector pLX-HERV-H (H6) before and after induction of dexamethasone.
Compared to Vectors containing the MMTV LTR can be activated by dexamethasone. Vectors containing the HERV LTR are not activated by dexamethasone, but their activity is higher by a factor of 10 compared to dexamethasone-stimulated MMTV hybrid vectors.

3.5 Effect of control regions in the R and U5 regions on HERV sequence promoter activity To determine which sequence regions are required for a functional HERV promoter,
The effect of additional LTR sequences located outside the U3 region in the LTR was examined in several cases. For this purpose, seven HERV-K LTRs (HERV-K-T47
D, L5, L50, L8, L9, L48, and L20 / 49) U3 regions were compared for activity in the luciferase assay to the activity of the corresponding U3-R fragment. Surprisingly, it has been found that different R regions are able to affect the promoter in the U3 region in a very different manner. Group 1 LTR (L5
, L50, L8, L9), the presence of the R sequence resulted in a significant increase in promoter activity in all cell lines tested (FIG. 4a). In contrast, LTR (L2
0 / L49), HERV promoter activity is reduced by the R region (FIG. 4).
b). The HERV-K-T47D promoter (FIG. 5) and the L48 promoter (not shown) are substantially unaffected by their respective R sequences. Interestingly,
In the case of the HERV-K-T47D LTR sequence, the region located downstream of the U3-R region and including the 3 'untranslated region and the start of the gag gene together with the U5 region has a clear activating effect (Fig. 5).

[0057] Sequence analysis of the different R regions tested revealed that the Group 1 LTR has a binding site for the transcription factor SP1 in the R region that is missing in the R region of the Group 2 LTR. (FIG. 6). In contrast, the R region of Group 2 contains a potential binding site for the factor TFS3, which acts as a transcription repressor. This indicates that HERV promoter activity may be modified by the insertion of additional regulatory regions, such as transcription factor binding sites, enhancer sequences, or negative regulatory regions. References

[0058]

[Table 4]

[0059]

[0060]

[0061]

[Procedure amendment]

[Submission date] October 29, 2001 (2001.10.20)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Added

[Contents of correction]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows an RT PCR strategy for U3 / R region isolation of transcribed HERV sequences.

FIG. 2g shows the relative promoter activity of different HERV LTRs in various cell lines.

FIG. 3a shows relative promoter activity plotted against cell lines. FIG. 3b
Indicates relative promoter activity plotted against cell lines.

FIG. 4 shows that the LTR-R region regulated HERV-K-T47D-related LTR promoter activity. FIG. 4A shows relative promoter activity plotted against activated, cell lines. FIG. 4B shows relative promoter activity plotted against inhibition, cell line.

FIG. 5 shows that sequences downstream of the LTR-R regulate promoter activity of the HERV-K-T47D-related LTR. Relative promoter activity plotted against cell lines.

FIG. 6 shows a control region in the R region of the HERV-K T47D LTR.

FIG. 7 shows the retroviral ProCon vector, pLESN-MMTV and pLESN-HERV-H.

FIG. 8 shows the retroviral ProCon vector, pLX-MMTV and pL.
X-HERV-H is shown.

FIG. 9 shows: a) Promoter conversion of ProCon hybrid vector b) Detection of correct promoter conversion by PCR and hybridization using HERV-H and psi probes.

FIG. 10. a) Two ProCon vectors, pLX-MMTV and pLX-HERV.
B) HERV-H LT compared to MMTV LTR after infection with CrfK cells
3 shows the promoter activity of R.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 105 A61P 43/00 111 111 C12N 7/00 C12N 5/10 C12R 1:93 7/00 C12N 15/00 ZNAA // (C12N 7/00 5/00 B C12R 1:93) (72) Inventor Leip-Mesh, Christine DE-80809 München, Nadistrasse 23 (72) Inventor Shane, Ulrike Federal Republic Day-80335 München, Dachau Strasse 73 (72) Inventor Baost, Corinna Federal Republic Day-69190 Wald Wolff, Schlossweg 30a (72) Inventor Salar, Robert Michael Federal Republic Day −80636 Mün Hen, Utästrasse 22 F term (reference) 4B024 AA01 BA32 CA04 DA02 EA02 FA02 FA06 GA11 HA12 HA17 4B065 AA90X AA97X AA97Y AB01 AC14 BA02 BA25 CA24 CA44 4C084 AA13 NA14 ZB011 ZB211 ZB261 ZB331 ZB31 ZB31 ZB31 ZB31 ZB33

Claims (20)

[Claims] 1. A functional assembly comprising at least the following elements: a) a DNA sequence for the packaging of vector RNA and for the cell-specific expression of a protein or peptide encoded by a heterologous DNA nucleotide sequence; b) One or more DNA nucleotide sequences encoding a protein or peptide, wherein said DNA sequences for cell-specific expression are cell-specific regulatable from a human endogenous retroviral DNA nucleotide sequence (HERV) A retroviral expression vector, comprising: a promoter region. 2. The method according to claim 1, wherein said DNA sequence for cell-specific expression comprises L of HERV.
The expression vector according to claim 1, wherein the expression vector is derived from a TR region and, if desired, an untranslated region between the 5 'LTR and the gag region. 2. The vector according to claim 1, wherein the entire LTR region, U3 region, or R and U3 regions are derived from a human endogenous retroviral nucleotide sequence. 3. The method according to claim 1, wherein the nucleotide sequence encoding one or more proteins or peptides is one or more of the group consisting of a marker gene, a therapeutic gene, an antiviral gene, an antitumor gene, and a cytokine gene. A vector according to one or more of the preceding claims, selected from the elements. 4. The vector according to one or more of the preceding claims, wherein said cell-specific regulatable promoter region is derived from the LTR region of an endogenous human retrovirus nucleotide sequence that is expressed cell-specifically. . 5. The human endogenous retrovirus cell-specific regulatable promoter sequence is HERV-K, HERV-H, HERV-E, HERV-L, H
ERV-T, HERV-R, HERV-I, HERV-P, ERV9, HERV
The vector according to one or more of the preceding claims, wherein the vector is selected from one or more promoter sequences of the HERV family of the group consisting of -W. 6. The vector according to one or more of the preceding claims, wherein said promoter region further comprises a recognition and binding site for a regulatory protein in addition to a TATA box. 7. The recognition and binding site of a regulatory protein comprises a hormone response element motif together with a GC box, a CAAT box, an enhancer sequence and a repressor sequence, and optionally, an exogenous retroviral LTR.
8. The vector according to claim 7, which comprises a further recognition and binding site for a regulatory protein from a region and / or from a cellular gene. 8. The 5 ′ LTR according to claim 8, wherein the vector has the structure U3-RU-5.
One or more sequences selected from a portion, a coding and a non-coding sequence,
And a partially or completely deleted U3 region, wherein the deleted U3 region is HE
4. The promoter-converting vector according to one or more of the preceding claims, wherein the promoter-converting vector comprises a 3 'LTR portion replaced by a cell-specific regulatable promoter region from the RV LTR sequence, followed by the RU5 region. vector. 9. The mRNA or RNA of a retroviral expression vector according to one or more of the preceding claims. 10. A prokaryotic or eukaryotic cell comprising a retroviral expression vector according to one or more of the preceding claims. 11. A eukaryotic cell comprising, in an integrated form, a retroviral expression vector according to one or more of the preceding claims. 12. The use of a cell-specific regulatable promoter region derived from a human endogenous retroviral DNA nucleotide sequence to control foreign gene expression in a retroviral expression vector, preferably in a ProCon vector. 13. Use of an expression vector according to one or more of the preceding claims for expressing foreign genes in gene therapy. 14. A virion comprising a retroviral expression vector RNA derived from the expression vector DNA according to one or more of the preceding claims. 15. A method of preparing a virion according to claim 15, for introducing one or more nucleotide sequences encoding a protein or peptide, wherein the method comprises preparing one or more of the preceding claims. The method wherein the retroviral expression vector described is introduced into a suitable packaging cell line under conditions such that virions are formed and released from the packaging cell line. 16. A method for introducing a nucleotide sequence encoding one or more proteins or peptides into a eukaryotic cell, wherein the nucleotide sequence encoding the protein or peptide is located on the chromosomal DNA of the eukaryotic cell. 16. The method wherein the cells are infected by the virions of claim 15 under conditions such that they are inserted. 17. The method of claim 17, wherein the eukaryotic cell is a mammalian cell. 18. The method according to claim 18, wherein said mammalian cell is a human cell. 19. A packaging cell comprising a retroviral expression vector according to one or more of the preceding claims, and at least one retrovirus or recombinant retroviral construct encoding a packaging protein of the retroviral expression vector. Retroviral vector systems, including strains. 20. The retroviral vector system of claim 20, wherein the packaging cell line comprises a retroviral or recombinant retroviral expression vector that encodes a retroviral protein that is not encoded by the retroviral expression vector.