DE10337770A1 - Endothelial-cell specific gene-transfer system, useful for treating tumors, comprises virus that includes vascular endothelial growth factor in its capsid - Google Patents

Abstract

Endothelial-cell specific gene-transfer system (A) for introduction of at least one gene (I) into an endothelial cell (EC) comprises a recombinant, replication-deficient virus that includes vascular endothelial growth factor (VEGF) in its capsid. An independent claim is also included for a method for preparing (A). ACTIVITY : Cytostatic. No details of tests for cytostatic activity are given. MECHANISM OF ACTION : Gene therapy.

Description

The Development of new concepts in tumor therapy has been available for many Years of medical research. Despite enormous Successes in certain areas of oncology can be found in others Areas just beginning. A major problem with older concepts, like chemo and radiotherapy, is the nonspecific application and thus the high load of the whole body or at least larger regions of the body. The operative removal is for many solid tumors the primary one Therapy, but usually needs to be coupled with other procedures to achieve long-term success. Furthermore, for some Tumors show that surgical removal is not just positive Has effects that could be shown to remove certain primary tumors promotes metastasis (Clark et al., 1989, Sugarbaker et al., 1977, Warren et al., 1977). The use of so-called anti-tumor factors is another concept, wherein Here, however, especially the problem of resistance development occurs thus limiting its use (Harris et al., 1996, Kerbel 1997).

tumorigenesis and metastasis are significantly dependent on angiogenesis (Folkman 1989, Kim et al. 1993, Millauer et al. 1994). Angiogenesis in Tumor tissue is stimulated among other things by tumor-specific Factors. Which includes Growth factors such as fibroblast growth Factor '(FGF) (Kandel et al. 1991) and the Vascular Endothelial Growth Factor (VEGF) (Cornali et al. 1996). In addition to the increased expression angiogenesefördernder Substances also became an increased expression of corresponding receptors in the Tumor endothelium observed. Here are among others, the 'Epidermal Growth Factor Receptor '(EGF-R),' Fibroblast Growth Factor-Receptor '(FGF-R) (Kandel et al., 1991), 'Plateled Derived Growth Factor Receptor (PDGF-R) 'fms-like Domain Receptor' (FLT1-R), 'Kinase Domaine Receptor' (KDR) and the 'Tyrosine Kinase Receptor' Tief1 and Tek / Tie2 (Strawn et al., 1996).

since For some time we are working on therapy concepts, which are specific on a ligation of the vascular Target tumor care. Approaches with angiogenesis inhibitors have already been done and seem promising (Böhm et al 1997, Kerbel 1997). When applying antibodies For example, a tumor regression could be detected in mice against VEGF- (Chesney et al 1998, Kim et al., 1993). A newer strategy is the specific intervention in the structure and function of the Tumor endothelium, to ultimately prevent the supply of the tumor. Here you can Gene transfer systems play an important role. Targeted expression certain proteins in the tumor endothelium are targeted via specific targeting or but specifically regulated expression done. When the latter does It is useful to note that tumor endothelium, unlike normal endothelium, does not represents dormant tissue and thus the use of certain cell cycle-dependent promoters can be used to express corresponding foreign proteins. The Implementation of this promising concept is currently hampered by the absence of a system that targets a specific endothelial cell taught.

The Human gene therapy is one of the youngest technologies in the world Oncology, the most promising therapeutic options offers. However, development is still in its infancy and the low Efficiency of somatic gene transfer and expression The main problems at the moment are the reasons for this Transfer systems, the specific targeting, the necessary regulatory Sequences for the expression in the individual cell types, as well as the switching off the host immune response.

Gene Therapy

• (1) ex vivo: Hierbei werden dem Körper Zellen (z.B. Blutzellen) ent nommen, welche dann nach Gentransfer wieder in den Körper eingebracht werden (retrovirale Vektoren).
• (2) in situ: In diesem Fall werden Gentransfersysteme lokal appliziert (z.B. adenovirale Vektoren in der Trachea und den Bronchien bei zystischer Fibrose).
• (3) in vivo: Bei dieser Art von Gentherapie werden Vektoren ins Blut injiziert und somit eine systemische Anwendung ermöglicht. Die letztere Kategorie ist wohl für die meisten klinischen Anwendungen die Technik der Wahl.

There are several categories of somatic gene therapy:

• (1) ex vivo: Here, the body cells (eg blood cells) ent taken, which are then introduced after gene transfer back into the body (retroviral vectors).
• (2) in situ: In this case, gene transfer systems are administered locally (eg adenoviral vectors in the trachea and the bronchi in cystic fibrosis).
• (3) in vivo: In this type of gene therapy, vectors are injected into the blood, allowing for systemic application. The latter category is probably the technology of choice for most clinical applications.

For gene transfer, either viral or non-viral systems (plasmid-liposome particles) have heretofore been used. Above all safety speaks for the non-viral transfer systems. Since the systems are either protein-free or contain only very limited proteins, in most cases the immune reactions are also considerably weaker (Farhood et al., 1994). Another advantage is the relatively high capacity for the incorporation of foreign DNA. The major disadvantage compared to the viral transfer systems lies in the targeting and inefficiency of the transfer (Crystal 1995). This inefficiency can only be compensated by the application of high numbers of particles, which may result in technical problems and toxic effects on the target cells (Buschle et al., 1995). Therefore, the viral transfer systems are more specific to the following is an important alternative in gene therapy.

When viral gene transfer systems are suitable:

(A) Retroviral vectors

Retroviral Vectors have been the preferred gene transfer vehicles to date (Anderson 1984), and currently about 60% of all clinical trials are based these vectors. The vectors are traditionally derived from the murine leukemia viruses (MuLV) and contain all for the integration of necessary sequences of the LTR regions and that for the packaging responsible element. The regions of the genes for viral proteins are through replaced the foreign genes and their control sequences. The vectors become expressed in helper cell lines containing a copy of the complete retrovirus genome contained, but with a defect in the sequence. Become from the cells thus provided all the necessary proteins, whereas only The transgenic RNA can be packed as it has its own region has. The recombinant replication-deficient virions can be expressed via the envelope proteins Adsorb to cells and bring the genome into the cytoplasm (fusion). The transgenic RNA is transcribed intracellularly into DNA and integrated into the host cell genome, this leads to permanent modification of the target cell genome (Mulligan 1993). This can be beneficial when eliminating hereditary or chronic defects in the genome but also negative effects on the cells as well as the Have an organism by overexpressing the inserted genome and thus toxic to the cell. It could also be the proviral DNA destroy a tumor suppressor gene or an oncogene through the Long Terminal Repeats (LTRs) promoter sequence to be activated. The absorption capacity of the vectors is approximately 8 kilobase pairs (Kb) (Verma and Somia 1997). Clinical studies with retroviral vector systems are based primarily on the ex vivo approach. It predominantly cells are transduced which have a high expression level of the natural Have receptors of MuLV and are capable of dividing. The advantage of retroviral Vectors is the stable integration of foreign genes, which, the passing on to ensure the daughter cells in cell division. The vectors must also be can not be applied multiple times, eliminating the elimination the immune system is limited. The main problems of the retroviral Vectors are nonspecific targeting and therefore conditional inefficient transfer into the specific target cells. by virtue of The relatively nonspecific targeting requires extremely high particle numbers be applied. We are currently trying to do the specific targeting to improve by keeping the natural ones Receptor binding domain through foreign domains replaced. However, there are problems in the folding of the surface protein ultimately, the ability to merge and thus the transfer impair can. Maintaining long-lasting expression is another Problem which, however, applies to all previously used systems.

(B) Adenoviral vectors

Adenovian (Serotype 2 and 5) have been the most prevalent among all DNA viruses to date used for gene transfer. Adenoviral vectors have many positive ones Properties, how big insertion capacity (<35 kilobases), efficient gene transfer for a variety of human cells, gene transfer into dividable and non-dividable cells and raising to high titers in culture. They have been especially so far for in situ applications used. Adenovirus vectors usually do not integrate into the Host cell genome. It could be shown that the vectors in non-dividable cells for some Time remaining. The first vectors were based on a deletion in the E1 and E3 regions, making the vectors replication-deficient (E1) and allowed the insertion of foreign sequences (E3). The great Disadvantage of these vectors lies in the mediation of a very strong Immune response, which among other things adenoviraler to a residual expression Proteins is recycled, as well as the absence of the E3 region, which immunosuppressive functions has (Ali et al., 1994). Following were vectors with additional Deletions to the so-called 'gutless vectors' (all viral genes deleted) developed. Animal experiments, however, give different results in relation on immunogenicity, stability foreign gene expression and persistence (Haecker et al., 1996, Chen et al. 1997, Kaplan et al. 1997). As things stand today the specific design of the vectors to gain a lot of importance, being before all immune stimulating genes switched off and possibly immunosuppressive Genes in addition be incorporated. The appropriate combination could be vectors in the future which have low toxicity and no or only a minor one Induce in the immune response. Disadvantageous for the use of adenoviral Vectors is also the strong prevalence of antibodies in the population. For this reason, was considered pulled, simultaneously with the vector application an immunosuppressive therapy initiate.

(C) adeno-associated viral vectors

The ability of adeno-associated viruses (AAV) (Parvoviridae) to integrate relatively site-specific (chromosome 19) and stably into the host cell genome in vitro, as well as the fact that AAV are apathogenic to humans, and that reactivation from latency to the presence dependent on helper viruses (adenovirus or herpesvirus) make these viruses suitable vectors for gene therapy approaches. In AAV vectors, the Genes for the structural proteins or all coding sequences replaced by corresponding foreign sequences. The chimeric genome is packaged in capsids in helper cells that constitutively synthesize the deleted viral functions and are infected with helper viruses. After infection of the cells, the chimeric genome can then be integrated into the host cell genome by means of the LTR elements and the desired products can be expressed under suitable conditions. Unfortunately, the AAV vectors available today do not mediate site-specific integration, but efficient gene transfers have been demonstrated for some cells (Fisher et al 1997, Miller 1997, Xiao and Samulski 1996). The advantages of AAV vectors are in the safety of the system and gene transfer into non-dividable cells. The drawbacks of the system are the low capacity for uptake of foreign DNA (4.8 kilobase pairs) and the high doses of infection needed to accomplish efficient gene transfer. Another disadvantage is that the preparation of the vehicle results in problems with helper virus contamination. Like the adenoviral vectors, AAV vectors also elicit strong immune responses, and about 80% of patients already have specific antibodies to AAV due to previous infection (Verma and Somia 1997).

(D) Rhabdoviral vectors:

Vesicular Stomatitis virus (VSV) is a rhabdovirus, it causes extreme cytopathic infections in most animal cells as well human T cells in culture, but is usually non-pathogenic to humans (Wagner and Rose 1996). VSV has a non-segmented negative RNA genome that replicates in the cytoplasm of the infected cell. By the viral RNA polymerase transcribes five different mRNAs, which in turn have five structural proteins (N, P, M, G, L) encode. Only one of these proteins, the glycoprotein (G) is present in the viral membrane and thus responsible for the catalyzing Fusion of the viral membrane with the cellular membrane (Florkiewicz and Rose 1984, Riedel et al. 1984).

(E) Other viral vectors

to Time are also vectors based on the herpes simplex virus and some smallpox viruses in the research that are currently published However, data are still under-founded to make statements about To make use of somatic gene transfer (Breakefield and De Luca 1991, Fink and Gloriose 1997, Jacoby et al. 1997).

For the efficient and careful application to the patient will be mainly viral vector systems which allow for specific target cell targeting. For the Use in tumor therapy will be mainly a gene transfer system With endothelial cell targeting sought to increase the vascular supply of the tumors too prevention.

One naturally Ligand that binds highly specifically to endothelium is the vascular Endothelial Growth Factor (VEGF). VEGF is made from a variety of cell types expressed. About 'splicing' four different monomeric forms of the VEGF, the two smaller ones (VEGF121, VEGF165) secretory and the two major forms (VEGF189, VEGF206) cell-associated proteins (Cornali et al., 1996). The two secretory isoforms differ in their biological Activity. VEGF165 has much stronger mitogenic effect on endothelial cells and gets better from the endothelium bound as VEGF121 (Keyt et al., 1996, Soker et al., 1997). The The biologically active form of VEGF is a disulfide-linked compound Homodimer, which binds to specific receptors on endothelial cells binds. As receptors, the 180KDa FLT receptor (Shibuya et al. 1990, de Vries et al. 1992) and the 200KDa KDR receptor (Terman et al., 1992) which primarily expresses on endothelial cells in their expression are limited. VEGF leads to an increase the vascular permeability and the Endothelial cell proliferation, which among other things also angiogenesis promotes (Cornali et al., 1996). It could be shown that many tumor cells increasingly produce VEGF and thus its own neovascularization promote (Ferrara 1995, Plate et al., 1992, 1993). VEGF plays a big role in that in angiogenesis as well as in the growth of various tumors (Strawn et al., 1996). However, there is currently no viral gene transfer system known to mediate a specific endothelial cell targeting.

task The invention therefore provides a viral gene transfer system for endothelial cell-specific targeting to provide, which has low toxicity and for introduction at least one gene in endothelial cells of an organism suitable is.

The The object is achieved by providing replication-deficient pseudotype viruses, which instead of a virus protein at least one specific protein for binding to receptors of the host organism, preferably the Vascular Endothelial Growth Factor (VEGF) as receptor binding site carry containing transmembrane protein dissolved.

• a) Bereitstellen eines rekombinanten replikationsdefizienten Virus
• b) Bereitstellen eines eukariotischen Expressionsvektors, der den VEGF-ORF exprimiert
• c) Konstruktion des rekombinanten replikationsdefizienten Virus (a) in einer Zellinie bei gleichzeitiger Transfektion mit dem eukariotischen Expressionsvektors (b)
• e) Infektion mit einem Virus, das das in die Endothelzelle einzuschleusende Gen trägt.

The process for producing an endothelial cell-specific gene transfer system comprises the steps:

• a) providing a recombinant replication-deficient virus
• b) providing a eukaryotic expression vector expressing the VEGF ORF
• c) Construction of the recombinant replication-deficient virus (a) in a cell line with simultaneous transfection with the eukaryotic expression vector (b)
• e) infection with a virus that carries the gene to be introduced into the endothelial cell.

The Advantages of the viral gene transfer system according to the invention lie in the fact that the replication-deficient pseudotype viruses are unstable or also stable - as a specific protein for binding to receptors of the host organism a chimeric one Transmembrane protein consisting of VEGF and CD8 carry with them bind highly specific to endothelial cells in the host organism and thus as a non-toxic gene transfer system for introduction at least of a gene in endothelial cells of an organism in gene and tumor therapy are suitable.

Surprisingly It was found that replication - deficient pseudotype viruses, the to no binding and fusion of the virus to the host cell membrane more are capable However, the infected cell can still rely on "budding," especially for manufacturing of the gene transfer system according to the invention are suitable.

One example for Replication-deficient pseudotype viruses is a recombinant rhabdoviral Vesicular stomatitis virus (VSV), which is the complete G protein gene is missing (Schnell et al., 1997) and thus no binding and fusion the virus at the host cell membrane is more possible. In contrast to the Retroviruses can damage this replication-deficient VSV virus despite the Absence of the G protein by budding the infected cell (Mebatsion and Conzelmann 1996).

According to the invention is in replication-deficient pseudotype viruses at least one specific protein for binding to a receptor of the host organism, in particular the chimera Transmembrane protein VEGF / CD8 stably or unstably inserted, so that the pseudotype viruses after budding by VEGF a specific Target cell targeting to the endothelial cell receptor of endothelial cells enable.

The successful expression of VEGF on the plasma membrane of the cell is inventively a chimera Transmembrane protein, hereafter called the VEGF / CD8 chimera, realized.

The the following drawing explains the steps for preparing the VEGF / CD8 chimera:

• A: 1040 Basenpaar-Abschnitt des offenen Leserahmens von CD8 (aus der Sequenz Littman et al. 1985). F1 bezeichnet eine 66 bp upstream-Region, das ATG-Startcodon liegt an Position 66, E bezeichnet eine 543 bp externe Domäne, M einen 80 bp Membrananker, C eine 84 bp cytoplasmatische Domäne, das Stopcodon befindet sich an Position 773, und F2 bezeichnet eine 267 bp flankierende downstream-Region. Mit Pfeilen sind die Positionen für Schnittstellen durch Restriktionsenzyme (HindIII, EcoRV und ApaI) eingezeichnet.
• B: Abschnitt des offenen Leserahmens von CD8 (aus der Sequenz Littman et al. 1985) nach Restriktionsspaltung mit den Restriktionsenzymen HindIII und ApaI. Es sind von der ursprünglichen Position 66 bis 773 noch die Domänen E, M und C erhalten, F1 und F2 fehlen.
• C: Austausch der externen CD8 Domäne E gegen den ORF von VEGF nach Restriktionsspaltung mit den Restriktionsenzymen HindIII und EcoRV. Die cytoplasmatische Domäne C und der Membrananker M des CD8-ORF bleiben erhalten.
• D: zeigt schematisch eine Kontrollsequenz, die nur den VEGF-ORF trägt, ohne die cytoplasmatische Domäne C und den Membrananker M des CD8-ORF.

1 schematically shows the cloning steps for the preparation of the VEGF / CD8 chimera;

• A: 1040 base pair section of the open reading frame of CD8 (from the sequence Littman et al., 1985). F1 denotes a 66 bp upstream region, the ATG start codon is at position 66, E denotes a 543 bp external domain, M an 80 bp membrane anchor, C an 84 bp cytoplasmic domain, the stop codon is located at position 773, and F2 denotes a 267 bp flanking downstream region. Arrows indicate the positions for restriction enzyme sites (HindIII, EcoRV and ApaI).
• B: Section of the open reading frame of CD8 (from the sequence Littman et al., 1985) after restriction cleavage with the restriction enzymes HindIII and ApaI. They are from the original position 66 bis 773 still the domains E, M and C obtained, F1 and F2 missing.
• C: Replacement of the external CD8 domain E against the ORF of VEGF after restriction cleavage with the restriction enzymes HindIII and EcoRV. The cytoplasmic domain C and the membrane anchor M of the CD8 ORF are retained.
• D: shows schematically a control sequence which carries only the VEGF ORF, without the cytoplasmic domain C and the membrane anchor M of the CD8 ORF.

1 shows schematically the production of nucleotide sequences which code for a chimeric transmembrane protein (VEGF / CD8) and are cloned into a eukaryotic expression vector, eg pcDNA3.

Since CD8, like VEGF, forms dimers (Gao et al 1997, Muller et al 1997), the donor of a hydrophobic domain is chosen according to the invention as the carboxy-terminal membrane anchor of a type I transmembrane protein, preferably CD8. In 1A schematically shows the open reading frame (ORF) of CD8. The starting source used for the PCR amplification of the CD8 ORF is the sequence of Littman et al. Used in 1985. The CD8 ORF consists of a 66 bp flanking upstream region (F1), the ATG start codon at position 66, a 543 bp external domain (E), an 80 bp membrane anchor (M), an 84 bp cytoplasmic domain (C). , a stop codon at position 773 and a 267 bp flanking downstream region (F2). With arrows are in 1 the positions for restriction enzyme sites (HindIII, EcoRV and ApaI) are plotted.

The external domain (E), the membrane anchor (M) and the cytoplasmic domain (C) of the CD8 ORF are excised with the restriction enzymes HindIII and ApaI in the standard method from the CD8 ORF ( 1B ) and into a eukaryotic expression vector, for example, pcDNA 3.0 (Invitrogen), using conventional standard laboratory methods and materials, such that the expression is under the control of a suitable promoter, for example the CMV and T7 promoter (the vector is named pcDNA-CD8 below). For cloning with VEGF, the secretory VEGF165 is preferably used because it has a high binding affinity to the endothelial receptors FLT and KDR.

Around To obtain the VEGF165 ORF, RNA is firstly made from human fibroblasts (HFF) with the Trizol Reagent (GibcoBRL, Karlsruhe, Germany) Manufacturer's instructions extracted and reverse transcribed with Superscript II according to the manufacturer's instructions (Gibco BRL, Karlsruhe, Germany). Subsequently the entire VEGF ORF is made from the material of reverse transcription amplified by PCR, the specific primers being selected that at the 5'-end one HindIII and at the 3'-end an EcoRV recognition sequence wear. The DNA material of the PCR reaction is analyzed by sequence analysis, a routine method known to those skilled in the art, to the VEGF165 sequence by comparison with a known VEGF sequence from the Gen Bank (Leung et al. (1989) Science 246, 1306-1309).

In a further step, the external domain (E) of the CD8 ORF in the pcDNA-CD8 vector is replaced by the VEGF165 ORF (amino acids 1 to 215). The deletion of the external domain (E) of CD8-ORF from pcDNA-CD8 takes place by restriction cleavage of the plasmid pcDNA-CD8 with the restriction enzymes HindIII and EcoRV and subsequent purification. In the thus prepared plasmid, the amplification product, the VEGF ORF via the restriction sites HindIII and EcoRV in the plasmid pcDNA-CD8 cloned ( 1C ) and this results in the plasmid pcDNA-VEGF / CD8. As a control for further investigations, the VEGF ORF ( 1D ) is separately cloned into the eukaryotic expression vector, for example pcDNA3.0, to give the plasmid pcDNA-VEGF.

outgoing from the ones chosen here Restriction interfaces, however, it is also easy for the skilled person possible, select other combinations of restriction enzyme recognition sequences, and for each to attach suitable recognition sequences to the primers such that the wished Plasmid with the nucleic acid sequence for the chimera Transmembrane protein VEGF / CD8 is formed.

Of the Expression detection of pcDNA-VEGF / CD8 and pcDNA-VEGF becomes transient performed in HeLa cells. Using lipofectin transfection, expression becomes of pcDNA-VEGF / CD8 and pcDNA-VEGF via the CMV promoter. Detection is carried out with specific commercial antibodies in the immunofluorescence and in the immunoblot after the expert Standard procedure. Expression of VEGF / CD8 via the plasmid pcDNA3.0-VEGF / CD8 shows the localization at the plasma membrane, i. the VEGF / CD8 chimera is in anchored to the plasma membrane.

For the endothelial cell specific Targeting and introducing at least one gene or genetic Information in endothelial cells of an organism becomes recombinant noninfectious Virus particles are produced that are different from replication Viruses, for example the recombinant VSV pseudotype viruses (Schnell et al. 1997), which according to the invention instead of the VSV-specific Glycoproteins (G) on the surface stable or unstable VEGF or the chimeric transmembrane protein VEGF / CD8 express. Thus, the viruses bind specifically to VEGF receptors and allow an endothelium-specific targeting.

The recombinant VSV system of Schnell et al. (1997) is based on the Infection with a recombinant vaccinia virus encoding T7 polymerase (vTF-3) and subsequent Transfection with plasmids coding for the proteins of the VSV polymerase complex [pBS-N (nucleoprotein), pBS-P (phosphoprotein), pBS-L (polymerase); all under T7 promoter control) and for the glycoprotein (T7 promoter control) as well as another plasmid (pVSVFL), via which, provided the VSV genome becomes. about transient expression of viral proteins in the cell arise recombinant viruses present in the supernatant be discharged.

This VSV system is for its use for endothelial-specific targeting improved so that a gene mutant is present in which the VSV glycoprotein gene (G) is deleted and / or replaced by a foreign gene or even an additional foreign gene is installed. As a foreign gene, the first example is the Expression of the, Green Fluorescence Protein '(GFP) tested by recombinant infectious VSV viruses. It several passages are carried out where the infection is easily over the GFP expression can be controlled.

• 1. VSV-Genommutante pVSV-GFP die das vollständige VSV-Genom mit Glykoprotein (G) und zusätzlich das ,Green Fluorescence Protein' (GFP) trägt,
• 2. VSV-Genommutante pVSV-_ΔG-GFP mit Deletion des Glykoprotein (G) und zusätzlich das ,Green Fluorescence Protein' (GFP)
• 3. VSV-Genommutante pVSV-_ΔG + GFP + VEGF/CD8 mit Deletion des Glykoprotein (G) und zusätzlich das ,Green Fluorescence Protein' (GFP) und der VEGF/CD8 Chimäre

Three VSV gene mutants are prepared:

• 1. VSV genome mutant pVSV-GFP bearing the complete VSV genome with glycoprotein (G) and additionally the 'Green Fluorescence Protein' (GFP),
• 2. VSV gene mutant pVSV- _Δ G-GFP with deletion of the glycoprotein (G) and additionally the, Green Fluorescence Protein '(GFP)
• 3. VSV genome mutant pVSV- _Δ G + GFP + VEGF / CD8 with deletion of the glycoprotein (G) and additionally the 'Green Fluorescence Protein' (GFP) and the VEGF / CD8 chimera

All VSV genome mutants are initially in recombinant system tested for infectiousness.

To do this, a BHK-21 or endothelial cell line is infected with Modified Vaccinia Virus Ankara (MVA), a recombinant vaccinia virus expressing T7 RNA polymerase (Carroll and Moss 1997). The transfection is then carried out according to the method described by Schnell et al. Method 1997 described with the plasmids (pBS-N, pBS-P, pBS-L) and the plasmid pVSV _Δ G + GFP wherein viruses produced which are 'pseudotype' for the wild type VSV-G. Because the viruses carry no glycoprotein on the surface, they are not infectious. In the experiment, this is made clear by the fact that these cells do not shine, even though the viruses have the GFP protein.

To produce recombinant replication-deficient VSV viruses carrying VEGF, in particular the VEGF / CD8 chimera, BHK21 or HeLa cells are infected with MVA and transfected as described. In addition to the previously described plasmids (pBS-N, pBS-P, pBS-L, pVSV- _Δ G + GFP) plasmid pcDNA-VEGF / CD8 is cotransfected. This results in recombinant viruses that carry the VEGF / CD8 molecule on the surface, but no VSV-G. These viruses preferentially bind to endothelial cells because they carry the two VEGF receptors (FLT-1 and KDR) on the cell surface. In order to increase transient expression of the VEGF / CD8 chimera for the production of recombinant replication-deficient VSV viruses, a cell line stably expressing the VEGF / CD8 chimera is preferred. Particularly preferred is a Hela cell line is used, since this normally has no VEGF receptors.

In an embodiment The invention consists of the endothelial cell-specific gene transfer system Recombinant replication - deficient pseudotypic viruses found on the surface Wear VEGF that previously was not stable (transient) in the membrane of the Cell is inserted. When the viruses leave the cell through budding, wear VEGF in the virus envelope.

In an alternative embodiment The invention consists of the endothelial cell-specific gene transfer system from recombinant replication - deficient pseudotype viruses, which the surface VEGF previously stably inserted into the genome of the virus and subsequently in the virus envelope is installed.

In a form of use according to the invention It is proposed that this method of endothelial-specific targeting especially in gene and tumor therapy. With the help of endothelium cell-specific Pseudotypic viruses will be at least one foreign gene in endothelial cells a patient infiltrated there to defective genes in this patient turn off to exchange defective genes for intact genes or to the cells to form a therapeutically or prophylactically to induce active protein.

The introduced Gene can be in the form of nucleic acid coding for the appropriate gene, optionally with the appropriate Regulatory elements, such as promoters provided. In preferred embodiment will be introduced Gene inserted into the virus genome.

Of the The term patient refers equally to humans and vertebrates. With that you can the endothelial cell-specific pseudotype viruses in human and veterinary medicine be used. The endothelial cell-specific pseudotype viruses and the therapeutically effective gene or genes contained therein the patient, as part of a pharmaceutically acceptable composition either orally, rectally, parenterally intravenously, intramuscularly or subcutaneously, intracisternal, intravaginal, intraperitoneal, intravascular, local (Powder, ointment or drops) or administered in spray form.

dosage forms for the local Administration of the vaccine of this invention include ointments, Powders, sprays or inhalants.

embodiments

1. Expression of the VEGF / CD8 chimera

1A schematically shows the 1040 bp open reading frame (ORF) of human CD8 (according to the sequence of Littman et al., 1985). It consists of a 66 bp upstream region (F1), the ATG start codon is located at position 66, a 543 bp external domain (E), an 80 bp membrane anchor (M), an 84 bp cytoplasmic domain (C ), the stop codon is in position 773 and a 267 bp flanking downstream region (F2). The positions for restriction enzyme sites (HindIII, EcoRV and ApaI) are indicated by arrows.

The 543 bp external domain (E), the 80 bp membrane anchor (M) and the 84 bp cytoplasmic domain (C) of the CD8 gene are cloned with the restriction enzymes HindIII and ApaI from the plasmid described by Littman with the CD8 gene cut out ( 1B ) and into a eukaryotic expression vector, for example the pcDNA 3.0 (Invitrogen), so that the expression is under the control of the CMV and T7 promoter (pcDNA-CD8).

In a reaction mixture of 20 ul to 10 μl CD8 DNA starting clone (source see Littman et al., 1985) or to 10 μl PCR amplified CD8 DNA (RNA isolated from T cells) per 1 μl restriction enzymes HindIII and ApaI with 2 μl reaction buffer, 6 μl H ₂ O added and incubated for 2 h at 37 ° C.

The next step is to replace the external domain (E) of CD8 from pcDNA-CD8 with the VEGF165 ORF (amino acids 1 to 215) ( 1C ).

For this purpose, first the RNA of human VEGF165 from human fibroblasts (HFF) with Trizol Reagent according to the manufacturer (GibcoBRL, Karlsruhe, Germany) isolated and carried out a reverse transcription PCR (RT-PCR): In a volume of 20 ul 1 μg total RNA with 40 U of Superscript II and 1 μg of oligo- (dT) ₁₈ according to the manufacturer (Gibco BRL, Karlsruhe, Germany) reverse transcribed.

Subsequently, the amplification of VEGF-ORF with primers carried the HindIII or EcoRV interfaces: In a reaction mixture of 100 .mu.l are according to standard protocol: (Taq DNA, Boehringer Mannheim) 50 ul H ₂ O, 20 ul 5 × buffer, 8 μl of NTP, 8 μl of Mg, 5 μl of DTT, 1 μl of template (RNA), 1 μl of enzyme and 3.5 μl each of the two primers:
3.5 μl Primer 1: 5'-GAC AAG CTT ATG AAC TTT CTG TCT-3 '
3.5 μl Primer 2: 5'-GAC GAT ATC TCC CCG CCT CGG CTT GTC-3 '

1 μl of the transcribed cDNA is amplified in the PCR system 2400 thermocycler (Perkin Elmer, Weiterstadt, Germany) with the abovementioned, for VEGF165-specific primers, with the following parameters being set:
Denaturation for 45 sec at 95 ° C,
Annealing for 60 sec at 60 ° C,
Extension for 60 sec at 72 ° C.

The PCR product (the VEGF165 ORF) is heavily amplified after 35-40 cycles, gel chromatographically analyzed and sequenced.

For the preparation of a plasmid encoding a chimeric transmembrane protein from VEGF 165 and CD8, the external domain (E) of pcDNA-CD8 is cleaved with the restriction enzymes HindIII and EcoRV in the following 20 μl reaction mixture at 37 ° C. for 2 h:
10 μl pcDNA-CD8 output clone
1 μl HindIII, 1 μl EcoRV, 2 μl reaction buffer,
6 μl H ₂ O

PCR product and plasmid are according to standard protocol of QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) purified in a pre-preparation approach.

Subsequently, the cloning of VEGF165 takes place via the cloning sites HindIII and EcoRV in the prepared pcDNA-CD8 to pcDNA-VEGF / CD8). The ligation takes place at 37 ° C. for 8 h in the following reaction mixture:
6 μl H ₂ O
2 μl reaction buffer, 2 μl pcDNA-CD8 starting clone
10 μl VEGF / CD8 of the PCR mixture
0.5 μl ligase

As a control, VEGF ORF ( 1D ) is cloned into the eukaryotic expression vector pcDNA3.0 (pcDNA-VEGF).

2. Expression detection the VEGF / CD8 chimera in HeLa cells

Of the Expression detection is transient in HeLa cells. To do this the plasmid constructs are transformed into HeLa cells by lipofectin transfection and their expression is detected by standard immunofluorescence protocol.

For transfection HeLa cells are 250 μl FCS-free Dulbecco Medium with 2.5 μl Lipofectin and 250 μl FCS-free Dulbecco Medium with 1-2 μg DNA (pc3.0 DNA Vector with VEGF or VEGF / CD8 incubated for 1 h at room temperature. Then be both media mixed and for Incubated again at room temperature for 45 min. The media mix will then Carefully pipetted on the cells to be transfected. The transfection the cells are done for 4-6 h at 37 ° C. Then be for 8-12 h at 37 ° C 500 μl 10% FCS Dulbecco medium added.

Of the Detection is carried out with specific commercial antibodies in of immunofluorescence. The cells are permeated and treated with paraformaldehyde for 30 min at 4 ° C fixed. The visualization of the membrane insertion of VEGF / CD8 becomes with indirect immunofluorescence, performed with goat anti-human IgG (VEGF) and a second fluorescein isothiocyanate (FITC) labeled rabbit anti-goat IgG (Dako A / S, Denmark) for 30 min at 4 ° C. For the documentation becomes the photographic equipment of Zeiss Axiopot Microscope and Spot Camera (Diagnostic Instrument, Version 2.1.2 for Windows 95/98 / NT) and Spot 32 programs used.

When Result shows that in the control (plasmid pcDNA-VEGF) VEGF165 released from the cell and detected in HeLa cells can, the chimera Transmembrane protein VEGF / CD8, however, is in the plasma membrane inserted.

3. Construction of VEGF / CD8 pseudotype viruses

• 1. VSV-Genommutante pVSV-GFP, die das vollständige VSV-Genom mit Glykoprotein (G) und zusätzlich das ,Green Fluorescence Protein' (GFP) trägt.
• 2. VSV-Genommutante pVSV-_ΔG-GFP mit Deletion des Glykoprotein (G) und statt dessen das Green Fluorescence Protein (GFP).
• 3. VSV-Genommutante pVSV-_ΔG + GFP + VEGF/CD8 mit Deletion des Glykoprotein (G) und zusätzlich das Green Fluorescence Protein (GFP) und die VEGF/CD8 Chimäre.

First, the method described by Schnell et al. 1997 modified recombinant VSV genome and VSVΔG genome, so that in addition to the G protein, a foreign gene, for example, for the Green Fluorescence Pro tein (GFP), is incorporated (VSV-GFP) or instead of G protein the. Green Fluorescence Protein (GFP) is advertised (VSVΔG-GFP)

• 1. VSV genome mutant pVSV-GFP bearing the complete VSV genome with glycoprotein (G) and additionally the 'Green Fluorescence Protein' (GFP).
• 2. VSV gene mutant pVSV- _Δ G-GFP with deletion of the glycoprotein (G) and instead the green fluorescence protein (GFP).
• 3. VSV gene mutant pVSV- _Δ G + GFP + VEGF / CD8 with deletion of the glycoprotein (G) and additionally the Green Fluorescence Protein (GFP) and the VEGF / CD8 chimera.

The GFP gene is inserted into the genome of the VSV between the G gene and L gene with the cloning methods known to those skilled in the art, and in the case of the deleted VSV (VSV _Δ G) between the M gene and L gene via the Interfaces XhoI and NheI are inserted (Schnell et al., 1997).

The Infection of BHK-21 cell line is done with the recombinant expressing T7 RNA polymerase Vaccinia virus MVA.

VSV gene mutant pVSV-GFP

transfection according to the Schnell et al. 1997 given method with the plasmids (pBS-N, pBS-P, pBS-L) and pVSVG + GFP

VSV genome mutant pVSV- _Δ G-GFP

transfection according to the Schnell et al. 1997 given method with the plasmids (pBS-N, pBS-P, pBS-L) and pVSVΔG + GFP

VSV gene mutant pVSV- _Δ G + GFP + VEGF / CD8

transfection according to the Schnell et al. 1997 given method with the plasmids (pBS-N, pBS-P, pBS-L) and pVSVΔG + GFP and cotransfection with the plasmid pcDNA3.0-VEGF / CD8.

The detection of viruses for infectiousness and their localization is carried out by single step infection and measurement of the expression of GFP. For this purpose, 1-3 ml of virus supernatant is applied to endothelial cells and monitored whether the viruses penetrate into the cells and these fluoresce in the case of VSV genommutante pVSV-GFP and VSV genommutant pVSV- _Δ G + GFP + VEGF / CD8.

In an embodiment The invention consists of the endothelial cell-specific gene transfer system Recombinant replication - deficient pseudotypic viruses found on the surface Wear VEGF that previously was not stable (transient) in the membrane of the Cell is inserted. When the viruses leave the cell by budding wear VEGF in the virus envelope.

One important advantage in the corresponding application in a transient Integration is the ease of use as there is no encapsulation of the VEGF into the genome.

In an alternative embodiment The invention consists of the endothelial cell-specific gene transfer system from recombinant replication - deficient pseudotype viruses, which the surface Wear VEGF, which previously stably inserted into the membrane of the cell and when leaving the virus from the cell, the VEGF taken becomes.

advantage in the corresponding application with a stable integration: there are more new viruses that can reach a greater impact radius and specifically bind only endothelial cells.

In a further alternative embodiment The invention consists of the endothelial cell-specific gene transfer system from recombinant replication - deficient pseudotype viruses, which the surface Instead of a proprietary protein, carry VEGF, which was previously stable Viral genome is inserted.

Of the Specialist knows many analogies or can do this with the help of usual Experiments on the specific applications described here Determine invention. Such correspondences are more intentional Wise within the scope of the following claims.

Claims (13)

Endothelial cell specific gene transfer system for introduction at least one gene in endothelial cells, consisting of recombinant replication-deficient viruses present in the viral envelope of the vascular endothelial Growth Factor (VEGF). Endothelial cell-specific gene transfer system according to claim 1, characterized in that VEGF composed a chimeric protein from VEGF and part of the transmembrane protein CD8. Endothelial cell-specific gene transfer system according to claim 1 and 2, characterized in that, VEGF is the secretory VEGF165 is. Endothelial cell-specific gene transfer system according to one of the preceding claims, since characterized in that the portion of the CD8 protein comprises the nucleic acids of the membrane anchor and the cytoplasmic domain. Endothelial cell-specific gene transfer system according to a of the preceding claims, characterized in that in the recombinant replication deficient Viruses a Hüllproteingen is deleted. Endothelial cell-specific gene transfer system according to a of the preceding claims, characterized in that the recombinant replication deficient Viruses VEGF stable or unstable on the shell wear. Endothelial cell-specific gene transfer system according to a of the preceding claims, characterized in that the inserted gene inserted into the viral genome becomes. Process for producing an endothelial cell-specific Gene transfer system, characterized by the steps: a) Provide a recombinant replication-deficient virus b) Provide a eukaryotic expression vector expressing the VEGF ORF c) Construction of the Recombinant Replication-Deficient Virus (a) in a cell line with simultaneous transfection with the eukaryotic Expression vector (b) e) Infection with a virus that causes carries into the endothelial cell to be inserted gene. Method according to claim 8, characterized in that the eukaryotic expression vector, which expresses the VEGF ORF for a chimera Protein composed of VEGF and the transmembrane protein CD8 coded. Method according to claim 8, characterized in that the VEGF gene of the secretory ORF VEGF165 is. Method according to claim 8 and 9, characterized in that the CD8 gene is the membrane anchor and the cytoplasmic domain of the CD8 gene. Use of an endothelial-specific gene transfer system according to one of the preceding claims for the preparation of a medicament for the introduction at least of a gene in endothelial cells. Medicament, characterized in that it is a Endothelial-specific gene transfer system according to one of the preceding claims having.