WO2004057005A1

WO2004057005A1 - Vectors lacking an origin of replication functioning in mammalian cells and expressing growth factors

Info

Publication number: WO2004057005A1
Application number: PCT/FI2003/000980
Authority: WO
Inventors: Andres Männik; Urve Toots; Rein Sikut; Mart Ustav
Original assignee: Fit Biotech Oyj Plc
Priority date: 2002-12-20
Filing date: 2003-12-22
Publication date: 2004-07-08
Also published as: AU2003292280A1

Abstract

The invention relates to novel vectors, to DNA vaccines and gene therapeutics containing said vectors, to methods for the preparation of the vectors and DNA vaccines and gene therapeutics containing the vectors, and to therapeutic uses of said vectors. Specifically, the present invention relates to novel vectors comprising (a) an expression cassette of a gene of a nuclear-anchoring protein, which contains (i) a DNA binding domain capable of binding to a specific DNA sequence and (ii) a functional domain capable of binding to a nuclear component, (b) a multimerized DNA forming a binding site for the anchoring protein of a nuclear-anchoring protein, and (c) one or more expression cassettes of a DNA sequence encoding a growth factor of the vascular endothelial growth factor (VEGF) family, a receptor of the vascular endothelial growth factor receptor (VEGFR) family, a growth factor of the angiopoetin family, a member of an ephrin family or a member of an Eph-receptor family. In particular the invention relates to vectors that lack a papilloma virus origin of replication and an origin of replication functional in a mammalian cell.

Description

Vectors lacking an origin of replication functioning in mammalian cells and expressing growth factors

Field of the invention

[0001] The present invention relates to novel vectors, to DNA vaccines and gene therapeutics containing said vectors, to methods for the preparation of the vectors and DNA vaccines and gene therapeutics containing the vectors, and to therapeutic uses of said vectors. More specifically, the present invention relates to novel vectors comprising (a) an expression cassette of a gene of a nuclear-anchoring protein, which contains (i) a DNA binding domain capable of binding to a specific DNA sequence and (ii) a functional domain capable of binding to a nuclear component, (b) a multimerized DNA forming a binding site for the anchoring protein of a nuclear-anchoring protein, and (c) one or more expression cassettes of a DNA sequence encoding a growth factor of the vascular endothelial growth factor (VEGF) family, a receptor of the vascular endothelial growth factor receptor (VEGFR) family, a growth factor of the angiopoetin family, a member of an ephrin family or a member of an Eph- receptor family. In particular the invention relates to vectors that lack a papil- loma virus origin of replication. The invention also relates to vectors that lack an origin of replication functional in a mammalian cell. The invention further relates to methods for expressing a DNA sequence of interest in a subject.

Background of the invention

[0002] The formation of new blood vessels comprises two processes. In vasculogenesis primarily at the embryonic level, the differentation of mesemchymal cells to endothelial cells results in the formation of blood vessels. In angiogenesis both in the embryonic development and in, for instance, wound healing and neovascularization of tumors and metastases, new blood vessels are formed from pre-existing vessels. Both processes are regulated by a family of vascular endothelial growth factors (VEGFs), which act through specific vascular endothelial growth factor receptors (VEGFRs).

[0003] Growth factors belonging to a family of angiopoetins are also involved in maturation and regulation of vascular endothelial cells. Angiopoetins play an important role especially in the embryonic development of heart. Recently, some evidence suggests a role in development of arteries and veins for ephrins and Eph-receptors. [0004] In medicine, the growth factors and receptors can be used by taking advantage of their neovasculation inhibiting or inducing properties, as appropriate. So-called non-sprouting angiogenesis takes place in the heart and during wound healing, and for instance VEGF-A and its receptors and VEGFR- 3 participate in its regulation and control. Thus examples of the medical use of agonists of angiogenesis, such as VEGF, include for instance angiogenesis stimulation in cardiovascular disorders, such as myocardial infarct, in wound healing, and diabetic retinopathy. On the other hand, in tumor angiogenesis, antagonists of angiogenesis may be of advantage in efforts to inhibit formation of blood vessels nourishing the tumor and metastases.

[0005] Transfer of autologous or heterologous genes into animal or human organisms with the help of suitable vectors is emerging as a technique with immense potential to cure diseases with a genetic background or to prevent or cure infectious diseases. Several types of viral and non-viral vectors have been developed and tested in animals and in human subjects to deliver a gene/genes that are defective by mutations and therefore non-functional. Examples of such vectors include Adenovirus vectors, Herpes virus vectors, Ret- rovirus vectors, Lentivirus vectors and Adeno-associated vectors.

[0006] Known DNA vectors used for the expression of genes and DNA-sequences associated in vasculogenesis and angiogenesis usually suffer from a major disadvantage that the expression obtained is short lived: The vectors tend to disappear from the transfected cells little by little and are not transferred to daughter cells in a dividing cell population, resulting in only temporary immune responses in subjects immunized with such DNA vectors. Another major drawback is that in addition to the expression of the desired gene, the DNA vectors may express other foreign proteins from DNA-sequences necessary for the production stage, some of which may contain sequences, which are homologous with those of the recipient and the host's genome may effect the integration of the vector into the host's genome, resulting in adverse reactions in the recipient.

[0007] US patent application Serial Number 10/138,098 discloses improved DNA vectors capable for a long-term expression of a gene of interest. These vectors carry (i) an expression cassette of a DNA sequence encoding a nuclear-anchoring protein, and (ii) multiple copies of high affinity binding sites for said nuclear-anchoring protein, and spread in proliferating cells. As a result, the number of vector-carrying cells increases even without the replica- tion of the vector. When the vector additionally carries a gene or genes of interest, the number of such cells that express a gene or genes of interest similarly increases without the replication of the vector. Also, the known vectors lack viral origin of replication. Specifically, they lack an origin of replication functional in a mammalian cell.

[0008] Genes and DNA sequences of interest mentioned as useful in said US patent application include a large number of possible genes and DNA sequences. However, genes and DNA sequences associated in vasculogenesis and angiogenesis are not specifically mentioned.

Short description of the invention

[0009] Surprisingly it was discovered that DNA-vectors disclosed in US patent application Serial Number 10/138,098 containing as a gene of interest a DNA sequence encoding a growth factor of the vascular endothelial growth factor (VEGF) family expressed higher amounts of desired proteins as compared to the expression levels of the proteins exemplified in said US patent application, and significantly higher amounts that reference vectors, a CMV vector and a NGVL vector.

[0010] The present invention relates to expression vectors comprising:

(a) a DNA sequence encoding a nuclear-anchoring protein opera- tively linked to a heterologous promoter, said nuclear-anchoring protein comprising (i) a DNA binding domain which binds to a specific DNA sequence, and (ii) a functional domain that binds to a nuclear component, or a functional equivalent thereof;

(b) a multimerized DNA sequence forming a binding site for the nuclear anchoring protein, and

(c) one or more expression cassettes of a DNA sequence encoding a growth factor of the vascular endothelial growth factor (VEGF) family, a receptor of the vascular endothelial growth factor receptor (VEGFR) family, a growth factor of the angiopoetin family, a member of an ephrin family or a member of an Eph-receptor family, and/or a functional fragment or a mutant thereof, wherein said vector lacks an origin of replication functional in a mammalian cell.

[0011] The vectors of the invention may also contain one or more expression cassettes of a DNA sequence encoding a member of hypoxia indu- cible factors (HIFs), a member of fibroblast growth factors (FGFs), a member of insulin-like growth factors (IGFs), a hepatocyte growth factor (HGF), a member of platelet-derived growth factors (PDGFs), and/or functional fragments or mutants thereof,

[0012] The present invention also relates to a method for providing a protein to a subject, said method comprising administering to the subject a vector as described above, wherein said vector does not encode Bovine Papil- loma Virus protein E1 , and wherein said subject does not express Bovine Papilloma Virus protein E1.

[0013] The present invention further relates to a method for the preparation of a vector as described above comprising: (a) cultivating a host cell containing said vector; and (b) recovering the vector, and optionally (c) comprising before step (a) a step of transforming said host cell with said vector.

Drawings

[0014] Figure 1 shows the schematic map of plasmid s6wtVEGF165.

[0015] Figure 2 shows the schematic map of plasmid p3VEGF165.

[0016] Figure 3 shows the schematic map of plasmid pCMV- VEGF165.

[0017] Figure 4 shows the schematic map of plasmid sδwtEGFP- luc.

[0018] Figure 5 shows the schematic map of plasmid p3EGFP-luc. [0019] Figure 6 shows the schematic map of plasmid pCMVEGFP- luc.

[0020] Figure 7 shows the expression of the VEGF protein from the vectors of the invention and from reference vectors at two concentration levels in Jurkat cells.

[0021] Figure 8 shows the expression of the luciferase from the vectors of the invention and from reference vectors at three concentration levels in Jurkat cells.

Detailed description of the invention

[0022] The present invention is based using a DNA sequence encoding a vascular endothelial growth factor (VEGF), a vascular endothelial growth factor receptor (VEGFR), an angiopoetin, an ephrin or an Eph-receptor or functional fragments thereof as the gene of interest in vectors a DNA containing a DNA sequence encoding a nuclear-anchoring protein operatively linked to a heterologous promoter, said nuclear-anchoring protein comprising (i) a DNA binding domain which binds to a specific DNA sequence, and (ii) a functional domain that binds to a nuclear component, or a functional equivalent thereof, a multimerized DNA sequence forming a binding site for the nuclear anchoring protein. These vectors are described in detail in US patent application Serial Number 10/138,098, which is incorporated herein by reference.

[0023] Surprisingly it was observed that additional advantages in terms of expression yields over those specifically exemplified for HIV protein in said application could be achieved. Additionally, significantly better expression yields could be obtained as compared to reference vectors specifically disclosed for expressing vascular endothelial growth factors in international patent application WO01/4141674.

[0024] The term "nuclear-anchoring protein" as used in the present invention refers to a protein, which binds to a specific DNA sequence and is capable of providing a nuclear compartmentalization function to the vector, i.e., to a protein, which is capable of anchoring or attaching the vector to a specific nuclear compartment. In certain embodiments of the invention, the nuclear- anchoring protein is a natural protein. Examples of such nuclear compartments are the mitotic chromatin or mitotic chromosomes, the nuclear matrix, nuclear domains like ND10 and POD etc. Examples of nuclear-anchoring proteins are the Bovine Papilloma Virus type 1 (BPV1 ) E2 protein, EBNA1 (Epstein-Barr Virus Nuclear Antigen 1 ), and High Mobility Group (HMG) proteins etc. The term "functional equivalent of a nuclear-anchoring protein" as used in the present invention refers to a protein or a polypeptide of natural or non-natural origin having the properties of the nuclear-anchoring protein.

[0025] In certain other embodiments of the invention, the nuclear- anchoring protein of the invention is a recombinant protein. In certain specific embodiments of the invention, the nuclear-anchoring protein is a fusion protein, a chimeric protein, or a protein obtained by molecular modeling. A fusion protein, or a protein obtained by molecular modeling in connection with the present invention is characterized by its ability to bind to a nuclear component and by its ability to bind sequence-specifically to DNA. In a preferred embodiment of the invention such a fusion protein is encoded by a vector of the invention, which also contains the specific DNA sequence, to which the fu- sion/chimeric protein binds. Nuclear components include, but are not limited to chromatin, the nuclear matrix, the ND10 domain and POD. In order to reduce the risk of interference with the expression of genes endogenous to the host cell, the DNA binding domain and the corresponding DNA sequence is preferably non-endogenous to the host cell/host organism. Such domains include, but are not limited to, the DNA binding domain of the Bovine Papilloma Virus type 1 (BPV1 ) E2 protein, Epstein-Barr Virus Nuclear Antigen 1 (EBNA1 ), and High Mobility Group (HMG) proteins (HMG box).

[0026] The vectors of the invention contain one or more expression cassettes of a DNA sequence encoding a growth factor of the vascular endothelial growth factor (VEGF) family, a receptor of the vascular endothelial growth factor receptor (VEGFR) family, a growth factor of the angiopoetin family, a member of an ephrin family, a member of an Eph-receptor family. Examples of encoded proteins include vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), angiopoetin, ephrin or Eph-receptor and/or functional fragments thereof. Specific examples of DNA sequences encoding VEGFs include, but are not limited to those encoding VEGF or VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PIGF). Examples of DNA sequences encoding VEGFRs include, but are not limited to those encoding VEGFR-1 , VEGFR-2, and VEGF-3. Examples of DNA sequences encoding angiopoietins include, but are not limited to, Ang-1 and Ang-2. The vectors of the invention may also contain one or more expression cassettes of a DNA sequence encoding a member of hypoxia indu- cible factors (HIFs), a member of fibroblast growth factors (FGFs), a member of insulin-like growth factors (IGFs), a hepatocyte growth factor (HGF), a member of platelet-derived growth factors (PDGFs),

[0027] According to the present invention, the expression cassette of a gene of a nuclear-anchoring protein, which contains a DNA binding domain capable of binding to a specific DNA sequence and a functional domain capable of binding to a nuclear component, such as an expression cassette of a gene of a chromatin-anchoring protein, like BPV1 E2, comprises a heterologous eukaryotic promoter, the nuclear-anchoring protein coding sequence, such as a chromatin-anchoring protein coding sequence, for instance the BPV1 E2 protein coding sequence, and a poly A site. Different heterologous, eukaryotic promoters, which control the expression of the nuclear-anchoring protein, can be used. Nucleotide sequences of such heterologous, eukaryotic promoters are well known in the art and are readily available. Such heterologous eukaryotic promoters are of different strength and tissue-specificity. In a preferred embodiment, the nuclear anchoring protein is expressed at low levels.

[0028] The multimerized DNA binding sequences, i.e., DNA sequences containing multimeric binding sites, as defined in the context of the present invention, are the region, to which the DNA binding dimerization domain binds. The multimerized DNA binding sequences of the vectors of the present invention can contain any suitable DNA binding site, provided that it fulfills the above requirements.

[0029] In a preferred embodiment, the multimerized DNA binding sequence of a vector of the present invention can contain any one of known 17 different affinity E2 binding sites as a hexamer or a higher oligomer, as a oc- tamer or a higher oligomer, as a decamer or higher oligomer. Oligomers containing different E2 binding sites are also applicable. Specifically preferred E2 binding sites useful in the vectors of the present invention are the BPV1 high affinity sites 9 and 10, affinity site 9 being most preferred. When a higher oligomer is concerned, its size is limited only by the construction circumstances and it may contain from 6 to 30 identical binding sites. Preferred vectors of the invention contain 10 BPV-1 E2 binding sites 9 in tandem. When the multimerized DNA binding sequences are comprised of different E2 binding sites, their size and composition is limited only by the method of construction practice. Thus they may contain two or more different E2 binding sites attached to a series of 6 to 30, most preferably 10, E2 binding sites. The Bovine Papilloma Virus type 1 genome contains 17 E2 protein- binding sites, which differ in their affinity to E2. The E2 binding sites are described in Li et al. [Genes Dev 3(4) (1989) 510-526], which is incorporated by reference in its entirety herein

[0030] Alternatively, the multimerized DNA binding sequences may be composed of any suitable multimeric specific sequences capable of inducing the cooperative binding of the protein to the plasmid, such as those of the EBNA1 or a suitable HMG protein. 21x30bp repeats of binding sites for EBNA- 1 are localized in the region spanning from nucleotide position 7421 to nucleo- tide position 8042 of the Epstein-Barr virus genome. These EBNA-1 binding sites are described in the following references: Rawlins et al., Cell 42(3) (1985) 859-868; Reisman et al., Mol Cell Biol 5(8) (1985) 1822-1832; and Lupton and Levine, Mol Cell Biol 5(10) (1985) 2533-2542, all three of which are incorporated by reference in their entireties herein.

[0031] The position of the multimerized DNA binding sequences relative to the expression cassette for the DNA binding dimerization domain is not critical and can be any position in the plasmid. Thus the multimerized DNA binding sequences can be positioned either downstream or upstream relative to the expression cassette for the gene of interest, a position close to the promoter of the gene of interest being preferred.

[0032] The vectors of the invention also contain, where appropriate, a suitable promoter for the transcription, of the gene or genes or the DNA sequences of interest, additional regulatory sequences, polyadenylation sequences and introns. Preferably the vectors may also include a bacterial plasmid origin of replication and one or more genes for selectable markers to facilitate the preparation of the vector in a bacterial host and a suitable promoter for the expression the gene for antibiotic selection.

[0033] The selectable marker can be any suitable marker allowable in DNA vaccines, such a kanamycin or neomycin, and others. In addition, other positive and negative selection markers can be included in the vectors of the invention, where applicable.

[0034] The vectors of the invention are preferably amplified in a suitable bacterial host cell, such as Escherichia coli. The vectors of the invention are stable and replicate at high copy numbers in bacterial cells. If a vector of the invention is to be amplified in a bacterial host cell, the vector of the invention contains a bacterial origin of replication. Nucleotide sequences of bacterial origins of replication are well known to the skilled artisan and can readily be obtained.

[0035] Upon transfection into a mammalian host in high copy number, the vector of the invention spreads along with cell divisions and the number of cells carrying the vector increases without the replication of the vector, each cell being capable of expressing the factor of interest. The vectors of the invention result in high expression of the desired factor. For instance, as demonstrated in Example 1 , significantly improved expression yields of VEGF and a long-lasting expression pattern were observed with the vectors of the invention as compared to reference vectors.

[0036] As mentioned above, the vectors of the present invention carrying the mechanism of spreading in the host cell find numerous applica- tions as vaccines, in gene therapy, in gene transfer and as therapeutic immu- nogens. The vectors of the invention can be used to deliver a normal gene to a host having a gene defect, thus leading to a cure or therapy of a genetic disease.

[0037] The vaccines of the invention can be used as therapeutical vaccines in the treatment of cardiovascular disorders, such as myocardial in- farct, in wound healing, and diabetic retinopathy, to stimulate angiogenesis. Additionally, they can be used in tumor angiogenesis to inhibit formation of blood vessels nourishing the tumor and metastases. Other uses of the present vaccines are easily recognized by those skilled in the art.

[0038] The vaccines of the present invention contain a vector of the present invention or a mixture of said vectors in a suitable pharmaceutical carrier. The vaccine may for instance contain a mixture of vectors containing genes for different members of the VEGF family and/or the VEGFR, where applicable or any desired combination of genes.

[0039] The vaccines of the invention are formulated using standard methods of vaccine formulation to produce vaccines to be administered by any conventional route of administration, i.e. intramuscularily, intradermally and like. In a specifically preferred embodiment of the present invention the vectors of the invention are used as nucleic acids in medical devices, as disclosed in US patent application nr. 2003059463, the contents of which are incorporated herein by reference.

[0040] The invention is further illustrated by the following non- limiting examples.

Example 1

Comparison of the expression of VEGF from the vectors of the invention and reference vectors

[0041] The vector plasmids s6wtVEGF165 (Figure 1 ), p3VEGF165 (Figure 2), and plasmid pCMV-VEGF165 (Figure 3) were prepared by replacing a DNA-sequence encoding the NEV-protein by a cDNA encoding human VEGF₁₆₅ (Medline accession number M32977; SEQ ID. NO. 1 and 2) in a vector identified in US patent application Serial Number 10/138,098 as NNV-2wt.

[0042] To analyze the expression properties of the vectors Jurkat cells (human T-cell lymphoblasts; about 2 millions per sample) were trans- fected by electroporation with the equimolar amounts of GTU vectors p3VEGF165 and s6wtVEGF165 or with reference vectors pNGVLhuVEGF165 and pCMVVEGF165 disclosed in WO01/41674. In all transfections 50 micro- grams of the salmon sperm carrier DNA was added. For negative control the cells were transfected with carrier DNA only. Two concentration series were performed:

[0043] Low:

100ng p3VEGF165

96ng s6wtVEGF165

70ng pNGVLhuVEGF165

63ng pCMVVEGF165

[0044] High:

1000ng p3VEGF165

960ng s6wtVEGF165

700ng pNGVLhuVEGF165

630ng pCMVVEGF165

[0045] The samples were plated in 4ml of IMDM medium containing 10% of fetal calf serum (FCS).

[0046] Two days post-transfection, 1 ml of cell suspension was collected from each sample, the cells were pelletted, washed twice with phosphate buffered saline (PBS) and stored as dry pellet at -20°C for analysis of the amount of the total protein. The total protein analysis was used as a value, which correlates with the cell number. The medium of the remaining culture was collected and stored at -20°C for analysis of the VEGF concentration. The cells were resuspended and plated in 11 ml of the fresh medium.

[0047] Five days post-transfection, 1 ml of cell suspension was collected from each sample, the cells were pelleted, washed twice with PBS and stored as dry pellets at -20°C for analysis of the amount of the total protein. The medium of the remaining culture was collected and stored at -20°C for analysis of the VEGF concentration.

[0048] In the analysis, samples of the dry cell pellets were lysed in 100 microliters of the 1% sodium dodecyl sulphate (SDS), clarified by centrifu- gation and the total protein concentration in lysates was measured by Pierce BCA assay kit.

[0049] VEGF concentrations in 1 :80 dilutions of the medium samples were measured using human VEGF immunoassay kit (R&D Systems, Cat no. DVE00). The results are shown in Figure 1. In the calculation of the VEGF charts set forth in Figure 1 the total protein concentrations were not taken into concentration. However, less then two times differences between the samples were obtained in both time points.

[0050] Significantly improved expression yields of VEGF and a long- lasting expression pattern were observed with the vectors of the invention as compared to reference vectors.

Example 2.

Comparison of the expression of luciferase from the vectors of the invention and reference vectors

[0051] The vector plasmids s6wtEGFP-luc (Figure 4), 3EGFP-luc (Figure 5), and pCMVEGFP-luc (Figure 6) were prepared by replacing a DNA- sequence encoding the NEV-protein by a DNA encoding luciferase in a vector identifided in US patent application Serial Number 10/138,098 as NNV-2wt. NNV-2wt.

[0052] To analyze the expression properties of the vectors Jurkat cells (about 2 millions per sample) were transfected by electroporation with equimolar amounts of GTU vectors p3EGFP-luc and s6wtEGFP-luc or with reference vectors pNGVLEGFP-luc and pCMVEGFP-luc (). In all transfections 50 micrograms of the salmon sperm carrier DNA was added. For negative control the cells were transfected with carrier DNA only. Three concentration series were performed:

[0053] Low:

50ng p3EGFP-luc

49ng s6wtEGFP-luc

39ng pNGVLEGFP-luc

36ng pCMVEGFP-luc

[0054] Medium:

100ng p3EGFP-luc

97ng s6wtEGFP-luc

77ng pNGVLEGFP-luc

71 ng pCMVEGFP-luc

[0055] High:

1000ng p3EGFP-luc

770ng pNGVLEGFP-luc

712ng pCMVEGFP-luc [0056] Samples were plated in 5 ml of IMDM medium containing 10% of FCS.

[0057] Two days post-transfection, 2 ml of cell suspension were collected from each sample and replaced with the 2.5 ml of fresh medium. The cells were pelleted, washed twice with PBS and lysed in 100 microliters 1^*CCLR buffer (included in the Luciferase Assay System, Promega).

[0058] Four days post-transfection fresh medium was added to the cultures (up to 12ml). Five days post-transfection, 8 ml of cell suspension were collected from each sample and replaced with 8 ml of fresh medium. The cells were pelleted, washed twice with PBS and lysed in 400 microliters 1*CCLR buffer. Seven days post-transfection, 10 ml of cell suspension were collected from each sample and replaced with the 8 ml of fresh medium. The cells were pelleted, washed twice with PBS and lysed in 400 microliters 1*CCLR buffer.

[0059] In the analysis, the total protein concentrations of the samples were analysed first. The samples were diluted 4 times with water and Pierce BCA assay kit was used for determination.

[0060] The luciferase activity was measured using the Luciferase Assay System kit (Promega) and plate reading luminometer. Different dilutions of the samples in 1^*CCLR buffer were used to verify that all measurements are done at a linear range. The results are shown in Figure 2. The values were corrected in view of the total protein concentrations.

[0061] Significantly improved expression yields of luciferase and a long-lasting expression pattern were observed with the vectors of the invention as compared to reference vectors.

Claims

Claims:

1. An expression vector comprising:

(c) one or more expression cassettes of a DNA sequence encoding a protein selected from a group containing the vascular endothelial growth factor (VEGF) family, a receptor of the vascular endothelial growth factor receptor (VEGFR) family, a growth factor of the angiopoetin family, a member of an ephrin family or a member of an Eph-receptor family, a member of hypoxia inducible factors (HIFs), a member of fibroblast growth factors (FGFs), a member of insulin-like growth factors (IGFs), a hepatocyte growth factor (HGF), and a member of platelet-derived growth factors (PDGFs), wherein said vector lacks an origin of replication that can function in a mammalian cell.

2. An expression vector according to claim 1 wherein the protein is selected from a group containing VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PIGF).

3. An expression vector according to claim 1 wherein the protein is selected from a group containing VEGFR-1 , VEGFR-2, and VEGF-3.

4. An expression vector according to claim 1 wherein the protein is selected from a group containing Ang-1 and Ang-2.

5. An expression vector according to claim 1 wherein the protein is selected from a group containing hypoxia inducible factors (HIFs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), a hepatocyte growth factor (HGF), and platelet-derived growth factors (PDGFs),

6. A vaccine comprising at least one vector according to any of claims 1 to 5.

7. A method for providing a protein to a subject, said method comprising administering to the subject the vector of any one of claims 1 to 5, wherein said vector does not encode Bovine Papilloma Virus protein E1 , and wherein said subject does not express Bovine Papilloma Virus protein E1.

8. A method for the preparation of a vector of any one of claims 1 to comprising:

(a) cultivating a host cell containing said vector; and

(b) recovering the vector, and optionally

(c) comprising before step (a) a step of transforming said host cell with said vector.