DE19850986A1 - The genetic engineering of cells and their use for the prophylaxis and therapy of diseases - Google Patents

Abstract

The invention relates to a method for producing cells which are suitable for treating the human body. According to said method, (a) cells are taken from the human body; (b) the cells obtained in step (a) are transfected in vitro with at least one gene coding for a growth and/or differentiation factor, which gene is under the control of a promoter; so that the cells from step (b) are capable of differentiation in a desired manner once they are re-introduced into the human body. The invention also relates to cells which are obtained by said method and to their use for producing a medicament for treating diseases.

Description

The genetic engineering of cells and their use for prophylaxis and Therapy of diseases 1) Basics

Therapy with the help of cell transplantation is becoming increasingly popular Importance. For example, the transplantation of bone marrow cells is one established procedure for the treatment of leukemia or tumor diseases after high-dose chemotherapy. The transfer of cells into one In principle, the organism is problem-free if the cells to be transferred are without Impairment of their function and / or survivability in one Cell suspension can be transferred. Such cell suspensions are relative simply into the circulatory system, into a body cavity, into an organ or locally administer.

In cases where the cells to be transferred are in a differentiation stage are needed in which the production of a cell suspension to a significant damage to their function and survivability is one It is very difficult to administer these differentiated cells. This is for example, especially when cells are in the desired Differentiation stage grow strongly adhesive and their detachment from the cell culture vessel for the production of a cell suspension only with mechanical methods (e.g. Scraping) is possible with the addition of proteolytic enzymes.

Aware of these difficulties, a new process was developed to to be able to transplant such cells without any problems.

2) Summary of the invention

The invention relates to a method in which cells have at least one gene for a growth and / or differentiation factor and / or at least one gene for a receptor of a growth and / or differentiation factor get inserted and these cells are forced by the introduced gene differentiate in vitro or in vivo to a stage in which they have the desired function. In a special embodiment of the Invention, this function is associated with an integration of the transfected cell in a tissue association.

The invention particularly relates to cells in which at least one gene for a growth and / or differentiation factor and / or at least one gene for the associated receptor of the growth and / or differentiation factor has been inserted and the use of these cells for the purpose of prophylaxis or therapy of a disease.

The object of the invention are the mononuclear cells obtained from the bone marrow, lymphoid organs, body cavities, body exudates, blood, Blood vessels and connective tissue, in which at least one gene for a growth and differentiation factor and / or its receptor has been introduced for the Prophylaxis or therapy for a disease.

Such cells express and experience the gene or genes introduced into them by the expressed growth factor and / or receptor in the cell culture, especially after administration in an organism, a Further development towards a desired stage of differentiation.

According to the invention, the expression of the or in the cell introduced genes (s) placed under the control of promoters. This Promoters can be non-specific, cell type-specific, metabolic and / or be pharmacologically activatable and / or self-reinforcing.

Through the choice of the respective promoters, the growth and the Differentiation of the transduced cell can be directed as necessary.  

The cells according to the invention can already be used without further treatment Therapeutic or prophylactic can be used.

However, they can also be used as cellular vectors for gene therapy.

For this purpose, they are in vitro d. H. in cell culture, additional nucleotide sequences inserted, which are under the control of different promoters (e.g. cell type specific, cell cycle specific, metabolic, pharmacologically activatable and / or self-reinforcing) for prophylactically or therapeutically active proteins code or for enzymes for the activation of the precursor of a drug in a medicine. Examples of such nucleotide sequences are for prophylaxis and therapy of vascular diseases (EP A 0 777 739), of diseases of the central nervous system (EP A 0 777 740), of tumors (EP-A 0 804 601) and of Diseases caused by the immune system (EP-A 0 807 183) in the cited patent applications as well as in patent applications EP-A 0 859 058, EP-A 0 864 651 and DE 197 52 299.8 (not published) have already been described been.

The method claimed in this invention and by this method manufactured cells are new. State of the art is to in vitro in cell culture Differentiation by adding growth factors and the differentiated cells after mechanical or proteolytic detachment from the To use cell culture vessel.

For example, Asahara et al., Science 275, 964 (1997) human blood peripheral mononuclear cells, especially CD34 positive Cells, by adding serum and brain extract from bovine in the cell culture in Endothelial-like cells differentiated and for the study of their Properties used. These cells were used for injection into the organism however not used because of its differentiation stage as Endothelial cells and the associated strong adhesion to the Cell culture vessel a damage-free detachment and transfer into a Single suspension would have been difficult.  

Instead, mononuclear cells freshly isolated from the blood were injected by which, however, are known to be local in vivo Conditions in the respective organ in which they are located differentiate different cells and only partially (Asahara et al., 1997, p. o.) in endothelial cells.

The method of embossing cells towards differentiation claimed in this invention is to be distinguished from the method of dedifferentiating cells known from the literature. This dedifferentiation takes place by inserting a gene into a cell, which leads to an immortalization of this cell. Examples of such genes are:

• - IE 84 from CMV
  (Speir et al., Science 265, 391 (1994))
• - E1A from AV
  (Nevins, Science 258, 424 (1992))

3) Detailed description of the invention

The selection of the starting cells, the promoters and the nucleotide sequences coding for receptors and / or growth factors and Differentiation factors occur in accordance with the target for these cells Intended purpose, d. H. the state of differentiation sought in the organism of the cells and the therapeutic goal aimed at with these cells.

3.1) Desired state of differentiation: endothelial cells a) Intended use

• - Substitution of endothelial cells in endothelial cell defects (e.g. after a Vascular dilation) or for the promotion of angiogenesis
• - Use as a cellular vector for gene therapy as detailed shown in patent applications DE 197 31 154.7 (not published) and DE 197 56 975.7 (not published)

b) Selection of the output cells

• - won mononuclear lines
• - from the blood z. B. from veins, capillaries, arteries, the umbilical cord or the placenta
• - from bone marrow cell suspensions
• - from spleen cell suspensions
• - from lymph node cell suspensions
• - from peritoneal cell suspensions
• - from pleural cell suspensions
• - from lymph
• - from connective tissue fluid (exiting e.g. on the surface of a superficially z. B. mechanically damaged epidermis)

Erythrocytes, granulocytes and other cell components are separated from these body fluids by density gradient centrifugation and thrombocytes by differential centrifugation according to the methods known to the person skilled in the art

• - mononuclear cells with surface markers CD34, CD11, CD13, CD14, CD64 and / or CD68 obtained from organs, body cavities or the blood

These cells are isolated using the methods known to those skilled in the art for example by immunoadsorption on supports which are combined with monoclonal Antibodies are coated specifically for the respective surface antigen.

• - endothelial cells

Endothelial cells can be, for example, using the methods known to the person skilled in the art from adipose tissue, by scraping veins or by detachment from Umbilical cord endothelium.  

c) Selection of the genes for growth and differentiation factors and for their Receptors

All growth factors and their receptors which are suitable for Proliferation and differentiation of endothelial cells.

These include, for example:

• - growth factors
• Vascular endothelial growth factor (VEGF) and other KDR or Flt ligands such as VEGF-B, VEGF-C, VEGF-D, neuropilin
• - fibroblast growth factor (FGFα, FGFβ)
• - epidermal growth factor (EGF)
• - Insulin-like growth factor (IGF-1, IGF-2)
• - β-endothelial cell growth factor (ECGF)
• - endothelial cell attachment factor (ECAF)
• - interleukin-3 (IL-3)
• - colony stimulating factor-1 (CSF-1)
• - GM-CSF
• - G-CSF, M-CSF
• - interleukin-4 (IL-4)
• - interleukin-1 (IL-1)
• - interleukin-8 (IL-8)
• - platelet derived growth factor (PDGF-AA, -AB, -BB)
• - interferon γ (IFNγ)
• - oncostatin M
• - B61
• - platelet derived endothelial cell growth factor (PDEGF)
• - stem cell factor (SCF)
• - transforming growth factor β (TGF-β)
• - angiogenin
• - pleiotrophin  
• - Flt-3 ligand (FL)
• - Tie-2 ligands such as angiopoietin-1
• - stromal derived factor-1 (SDF-1) and
• - TNFα
• - receptors
• - the VEGF receptor I (Fft)
• - the VEGF Receptor II (KDR)
• - the VEGF receptor III
• - the FGF receptors (-1, -2, -3, -4, -5)
• - the IGF receptor
• - the ECGF receptor
• - the ECAF receptor
• - the IL-3 receptor
• - the Oncostatin M receptor
• - the LIF receptor
• - the B61 receptor
• - the PDEGF receptor
• - the SCF receptor
• - the TGFβ receptor
• - the Tie-2 receptor
• - the SDF-1 receptor
• - the pleiotrophin receptor
• - the EGF receptor
• - the TNFα receptor and / or
• - the SDF-1 receptor
• - the PDGF receptor (α and β)

d) Selection of promoters

• - can be activated without restriction, specific for endothelial cells, can be activated metabolically, self-reinforcing and / or pharmacologically controllable and Combinations of these (see section 4)

3.2) Desired state of differentiation: osteoblasts a) Intended use

• - Promoting bone healing (e.g. with local or systemic Administration after broken bones)

b) Selection of the cells

• - mononuclear cells obtained as in 3.1. described
• - mononuclear cells with the surface markers CD34, CD11, CD13, CD14 and / or CD68 obtained as in 3.1. described
• - Fibroblasts obtained e.g. B. from the subcutis with the expert known methods

c) Selection of the genes for receptors of growth and differentiation factors and for growth factors and differentiation factors

All growth and differentiation factors which are suitable for Proliferation and differentiation of osteoblasts.

• - growth and differentiation factors
  These include in particular the "bone morphogenic proteins" (BPM), for example BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8 (Takahara et al. , Genomics 29, 9 (1995), Celeste et al., PNAS USA 87, 9843 (1990), Özkaynak et al., EMBO J. 9, 2085 (1990), Özkaynak et al., J. Biol. Chem. 267 , 25220 81992), Hino et al., Bichem. Biophys. Res. Commun. 223, 304 (1996), Ruppert et al., Eur. J. Biochem. 237, 295 (1996)), pleiotrophin (PTN) and Midkine (Kurtz et al., Crit. Rev. Oncogenesis 6, 151 (1995), Int. Dev. Bio. 37, 183 (1993)).

Receptors

• - Receptors for BMP, PTN and Midkine.

d) Selection of promoters

• 1. can be activated without restriction, promoters of the genes for the BMP proteins, metabolically activatable, self-reinforcing and / or pharmacological controllable and combinations thereof (see section 4)

3.3) Desired state of differentiation: glial cells a) Intended use

• - Promote healing of CNS damage
• - cellular vector for gene therapy of diseases and / or damage of the CNS system, as shown in detail in the Patent application EP A 0 777 740

b) Selection of the cells

• - Mononuclear cells obtained as described in 3.1)
• - mononuclear cells with the surface markers CD34, CD11, CD13, CD14 and / or CD68, obtained as described in 3.1)

c) Selection of the genes for growth and differentiation factors and for the associated receptors Growth factors and differentiation factors

Suitable for the purposes of the invention are all growth factors which lead to Growth and differentiation of glial cells.

These include, for example:

• - the glia growth factor (GGF) (Trachtenberg et al., Nature 379, 174 (1996))
• - Neurotrophin-4 (NT-4; trk-C) (Funakoshi et al., Science 267, 1495 (1995))
• - the brain derived neurotrophic factor (BdNF; trk-B)
• - the ciliary neurotrophic factor (CNF)
• - the glia cell line derived neurotrophic factor (GDNF)  
• - the nerve growth factor (NGF; trk-A)

Receptors

Suitable for the purposes of the invention are all receptors for growth factors, which contribute to the proliferation and differentiation of glial cells.

For example:

• - the GGF receptor
• - the NGF receptor
• - the CNF receptor
• - the BdNF receptor
• - the NT-4 receptor
• - the GDNF receptor

d) Selection of promoters

• - Can be activated without restriction, can be activated specifically for glial cells, metabolically activatable, self-reinforcing and / or pharmacologically controllable and Combinations of these (see section 4).

3.4) Desired state of differentiation: synovial cells a) Intended use

• - Prevention and therapy of joint damage

b) cells

• - mononuclear cells isolated according to section 3.1)
• - blood cells with surface marks according to section 3.1)
• Fibroblasts obtained according to a method known to the person skilled in the art method

c) genes for growth factors and differentiation factors and their Receptors

• - growth factors and differentiation factors

These growth factors include all those proteins such as B. cytokines, Cytokine inhibitors, enzyme inhibitors, anti-adhesion molecules, antagonists of Oxygen radicals and growth factors for cartilage cells, which lead to a lead to anti-inflammatory response and differentiation of synovial cells.

These include, for example:

• - Transforming growth factor (TGF) β-1 and -2
• - Interleukin 10 (IL-10)
• - Insulin-like growth factor-1 (IGF-1)
• - Interleukin-4 (IL-4)
• - Interleukin Receptor Antagonist Protein (IRAP)
• - Inhibitors of metalloproteinases such as TIMP-1, -2, -3
• - Fibroblast Growth Factor (FGF)
• - interleukin-6 (IL-6)
• - Plasminogen Activator Inhibitor (PAI-1, -2)
• - Platelet derived growth factor (PDGF)
• - superoxide dismutase
• - Soluble (extracellular parts of) adhesion molecules such as CD18, ICAM-1, CD44
• - receptors

This includes all those receptors whose activation to one lead to anti-inflammatory response and differentiation of synovial cells.

These include, for example:

• - TGFβ receptors
• - IL-10 receptors
• - IGF-1 receptors
• - IL-4 receptors
• - bFGF receptors

d) promoters

• - not specific, lymphocyte and / or macrophages specific and / or Synovial cell specific (see section 4)

3.5) Desired state of differentiation: anti-inflammatory cells a) Intended use

• - Prophylaxis and therapy of inflammation, autoimmune diseases and Organ rejection

b) cells

• - mononuclear blood cells isolated according to section 3.1)
• - cells with surface markers isolated according to section 3.1)
• - fibroblasts

c) genes for growth factors and differentiation factors and / or their Receptors

Suitable for the purposes of the invention are all antiallergic or the antibody reaction or the cellular immune response inhibiting cytokines and their receptors.

These include, for example:

• - cytokines
• - interferons (IFNα, IFNβ, IFNγ)
• - Interleukin-4 (IL-4)
• - interleukin-6 (IL-6)
• - Interleukin-9 (IL-9)
• - Interleukin-13 (IL-13)
• - LIF
• - Oncostatin
• - Interleukin-10 (IL-10)
• - Interleukin-12 (IL-12)
• - TGFβ  
• - tumor necrosis factor α (TNFα)
• - TNFβ
• - Interleukin-1 receptor antagonist (IL-1RA)
• - receptors
• - Receptors for IFNα, -β, -γ
• - soluble IL-4 receptor
• - IL-4 receptor
• - IL-6 receptor
• - soluble IL-6 receptor
• - soluble IL-2 receptor
• - IL-10 receptor
• - IL-12 receptor
• - IL-13 receptor
• - TNFα receptor
• - TNFβ receptor
• - TGFβ receptor

d) Selection of promoters

• - Can be activated without restriction, specific for lymphocytes and / or macrophages activatable, metabolically activatable, self-reinforcing and / or pharmacologically controllable and combinations thereof (see section 4).

3.6) Desired state of differentiation: cells involved in inflammation a) Intended use

• - Supporting inflammatory and rejection reactions, for example in the context of infections or tumor diseases.

b) cells

• - mononuclear blood cells isolated according to section 3.1)
• - cells with surface markers isolated according to section 3.1)
• - fibroblasts

c) genes for growth and differentiation factors and / or their receptors

Suitable for the purposes of the invention are all cytokines and / or their receptors which promote an antibody-mediated or cellular immune response.

These include, for example:

• - growth factors
• - Interleukin-1
• - Interleukin-2
• - Interleukin-4
• - Interleukin-5
• - Interleukin-6
• - LIF
• - Interleukin-7
• - Interleukin-8
• - Interleukin-11
• - GM-CSF
• - M-CSF
• - G-CSF
• - IFNα, -β, -γ
• - receptors
• - IL-1 receptor
• - IL-2 receptor
• - IFNα, -β, -γ receptor
• - IL-3 receptor
• - IL-5 receptor
• - IL-6 receptor
• - GM-CSF receptor
• - M-CSF receptor
• - Integrin beta 2 proteins

d) promoters

• - Can be activated without restriction, specific for lymphocytes and / or macrophages activatable, metabolically activatable, self-reinforcing and / or pharmacologically controllable and combinations thereof (see section 4).

4) Choice of promoters

For the purposes of the invention, nucleotide sequences are considered as promoter sequences use which, after binding transcription factors, the transcription of a at the 3 'end adjacent transgenes, such as a gene for a receptor of a growth factor or differentiation factor or one Activate genes for a growth factor or differentiation factor. For the purpose of the invention is in the cell of the invention at least one promoter sequence inserted. This promoter sequence can be combined with at least one other Promoter sequence can be combined. The choice of which to combine with The promoter sequence depends on the disease to be treated. So it can Promoter sequence unrestricted, endothelial cell specific, under certain metabolic conditions, such as inducible by hypoxia or inducible by a drug, virus-specific and / or cell cycle-specific can be activated. Such promoters were already in the patent applications EP-A 0 804 601; EP A 0 777 739; EP-A 0 807 183; EP A 0 777 740; EP A 0 753 580; EP A 0 857 781, EP-A 0 790 313; EP-A 0 860 445; EP-A 0 864 651 and EP-A 0 805 209. Reference is made to these patent applications taken. The promoter sequences to be selected include, for example:

4.1) Promoters and activator sequences that can be activated without restriction

like for example

• - the promoter of RNA polymerase III
• - the promoter of RNA polymerase II
• - the CMV promoter and enhancer
• - the SV40 promoter

4.2) Metabolically activatable promoter and enhancer sequences

such as the hypoxia-inducible enhancer (Semenza et al., PNAS 88: 5680 (1991), McBurney et al., Nucl. Acids Res. 19: 5755 (1991)).

4.3) Promoters that can be activated specifically for the cell cycle

Such are, for example, the promoter of the cdc25B gene, the cdc25C gene, the Cyclin A gene, the cdc2 gene, the B-myb gene, the DHFR gene, the E2F-1 gene or else binding sequences for or occurring during cell proliferation activated transcription factors. These binding sequences include, for example Binding sequences for c-myc proteins. To these binding sequences are monomers or multimers of the nucleotide sequence called Myc E-Box (5'- GGAAGCAGACCACGTGGTCTGCTTCC-3 '(SEQ ID NO .: 1); Blackwood and Eisenmann, Science 251, 1211 (1991)).

4.4) Self-reinforcing and / or pharmacologically controllable Promoters

In the simplest case, the combination of the same or different Promoters can be inducible, for example in the form of a promoter Promoter that can be activated or deactivated in the form of the tetracycline Operator in combination with a corresponding repressor.

According to the invention, however, the promoter can also be self-reinforcing with or without a pharmacologically controllable promoter unit.

Such self-reinforcing and / or pharmacologically controllable promoters have already been described in patent application EP A 0 848 061, to which explicit reference is made.

4.5) Promoters that can be activated specifically for endothelial cells

These include promoters or activator sequences from promoters or Enhancers of such genes, which are preferred for proteins formed in endothelial cells encode.  

For the purposes of the invention, promoters of the genes for the following proteins are to be used, for example:

• - Brain-specific, endothelial glucose-1 transporter
• - Endoglin
• - VEGF receptor-1 (flt-1)
• - VEGF receptor-2 (flk-1, KDR)
• - VEGF receptor-3
• - tie-1 or tie-2
• - B61 receptor (corner receptor)
• - B61
• - Endothelin, in particular Endothelin B or Endothelin-1
• - Endothelin receptors, especially the endothelin B receptor
• - Mannose-6-phosphate receptors
• - von Willebrand factor
• - IL-1α, IL-1β
• - IL-1 receptor
• - Vascular Cell Adhesion Molecule (VCAM-1)
• - Interstitial Cell Adhesion Molecule (LCAM-3)
• - synthetic activator sequences
• - platelet endothelial cell adhesion molecule (PECAM)

As an alternative to natural endothelial cell-specific promoters use synthetic activator sequences from oligomerized binding sites for transcription factors that are preferentially or selectively active in endothelial cells, consist. An example of this is the transcription factor GATA-2, whose Binding site in the endothelin-1 gene is 5'-TTATCT-3 '(Lee et al., Biol. Chem. 16188 (1991)), Dormann et al., J. Biol. Chem. 1279 (1992) and Wilson et al., Mol. Cell Biol. 4854 (1990).

4.6) Promoters that can be activated specifically for glial cells

Promoters and activator sequences that are activated in glial cells are e.g. B. gene regulatory sequences from genes, for example for the following proteins encode: the Schwann cell-specific protein periaxin, glutamine synthetase, the Glial cell-specific protein (GFAP), the glial cell protein S100, IL-6, CNTF, 5-HAT receptors, TNFα, IL-10, insulin-like growth factor Receptor I and II or VEGF.

4.7) Promoters that can be activated specifically for synovial cells

Promoter and activator sequences that are activated in synovial cells are for example the promoter sequences of genes coding for matrix Metalloproteinases (MMP) such as e.g. B. MMP-1 (interstitial collagenase), MMP-3 (Stromelysin / transin) or tissue inhibitors of metal proteinases (TIMP), such as TIMP- 1, -2, -3.

4.8 Lymphocyte and / or macrophage-specific activators that can be activated

Promoters and activator sequences found in lymphocytes and / or macrophages activated are, for example, the promoter and actuator sequences of the genes coding for cytokines, cytokine receptors and adhesion molecules and receptors for the Fc fragment of antibodies such. B. IL-1 receptor, IL-1α, IL-1β, Il-2 IL-2 Receptor, IL-3, IL-3 receptor (α-subunit), IL-3 receptor (β-subunit), Il-4, IL-4 Receptor, IL-5, IL-6, IL-6 receptor, interferon regulatory factor 1 (IRF-1), the Promoter of IRF-1 is activated by IL-6 as well as by IFNγ or IFNβ, IFNγ Responsive Promoter, IL-7, IL-8, IL-10, IL-11, IFNγ, GM-CSF, GM-CSF- Receptor (α-chain), IL-13, LIF, macrophage-colony stimulating factor (M-CSF) - Receptor, Type I and II Macrophage Scavenger Receptors, MAC-1 (Leukocyte functional antigen) LFA-1α (leukocyte functional antigen) or p150.95 (Leukocyte function antigen).

4.9) Combination of the same or different promoters

The combination of the same promoters is carried out, for example, by successive linkage of several promoters in the reading direction of 5 ' after 3 'of the nucleotide sequence.

For combining the same or different promoters however, technologies are preferably used which are already used in the Patent applications EP-A 0 802 985; EP-A 0 790 313; EP-A 0 860 445; EP-A 0 864 651; EP-A 0 805 209; EP-A 0 848 063 and EP-A 0 848 061 are described in detail were. These patent applications are within the scope of this invention expressly referred. Examples of such technologies are:

4.10) Chimeric promoters

A chimeric promoter is the combination of an upstream activator sequence that can be activated in a cell-specific, metabolic or virus-specific manner with a downstream promoter module which contains the nucleotide sequence CDE-CHR or E2FBS-CHR, to which suppressive proteins bind, which hereby activates the upstream activator sequence in G Can inhibit ₀ and G ₁ phase of the cell cycle (EP-A 0 802 895; Lucibello et al., EMBO J., 12 (1994)).

Further studies on how it works, especially the Promoter element CDE-CHR, showed that the by the CDE-CHR element cell cycle dependent regulation of an upstream activator sequence is largely dependent on the activation sequence of Transcription factors with glutamine-rich activation domains is activated (Zwicker et al., Nucl. Acids Res., 3822 (1995)).

Such transcription factors include, for example, Sp1 and NF-Y.

This consequently limits the use of the promoter element CDE CHR for chimeric promoters. The same is true for the promoter element E2F-BS Assume CHB of the B-myb gene (Zwicker et al., Nucl. Acids Res. 23, 3822 (1995)).  

4.11) Hybrid promoters

The hybrid promoters have already been described in patent application EP-A 0 848 063. For the combination of the endothelial cell-specific promoter with at least one further promoter, for example, a gene construct is selected which contains the following components in total:
The nucleotide sequence of the endothelial cell-specific promoter in a form in which at least one binding site is mutated for a transcription factor. This mutation blocks the initiation of the transcription of the effector gene.

A transgene that codes as an effector gene for an active ingredient.

At least one more non-specific, cell-specific, virus-specific, by Tetracycline and / or cell cycle-specific activatable promoter or Enhancer sequence, which is the transcription of at least one gene for at least activated a transcription factor which is mutated in such a way that it binds to the bind mutated binding site (s) in the endothelial cell-specific promoter and can activate it.

In an exemplary embodiment of this invention, the mutation in the Promoter sequence e.g. B. a mutation of the TATA box of the cdc25B promoter represent.

The mutation of the TATA can be, for example, TGTATAA. Through this mutation the DNA binding site of the normal TATA box binding protein (TBP) are no longer recognized and the effector gene can no longer be efficiently transcribed. Accordingly, the nucleic acid sequence which codes for the TBP must have a Have commutation. This commutation binds the TBP to the mutated one TATA box (e.g. on TGTATAA) and thus leads to efficient transcription of the Effector gene. Such commutations of the TBP gene are for example from Strubin and Struhl (Cell, 721 (1992)) and by Heard et al. (EMBO J., 3519 (1993)) have been described.  

4.12) Multiple promoters in combination with a nuclear retention Signal and a nuclear export factor

This technology is already described in detail in patent application EP-A 0 805 209 have been described. Reference is made to this patent application.

According to the invention, such a promoter contains the following components:

• - A first endothelial cell-specific, activatable promoter or enhancer sequence that activates the basal transcription of a transgene.
• - A transgene that codes as an effector gene for an active ingredient.
• - A nuclear retention signal (NRS) whose cDNA at the 5 'end matches the 3'- End of the structural gene (b) is linked indirectly or directly.
• - Preferably the transcription product of the nuclear retention Signals a binding structure for a nuclear export factor.
• - Another non-specific, cell-specific, virus-specific, metabolic and / or promoter or enhancer sequence that can be activated specifically for the cell cycle, which activates the basal transcription of a nuclear export factor.
• - A nucleic acid encoding a nuclear export factor (NEF) that is the transcription product of the nuclear retention signal binds and thereby the transport of the transcription product of the transgene from the cell nucleus mediated.

The gene coding for the nuclear retention signal is preferably selected from the group comprising the Rev-Responsive Element (RRE) of HIV-1 or HIV-2, the RRE-equivalent retention signal from retroviruses, or the RRE- equivalent retention signal of HBV.

The nuclear export factor is preferably a gene selected from the Group comprising the Rev gene of the viruses HIV-1, HIV-2, Visna-Maidi virus, Caprine arthritis encephalitis virus, the virus of the infectious anemia of the horse, the Immunodeficiency virus of the cat, of retroviruses, of HTLV or the gene of hnRNP-A1 protein or the gene of the transcription factor TFIII-A.  

4.13) Activator-responsive promoter unit

Activator-responsive promoter units are in patent application EP-A 0 805 209 have already been described in detail. Reference is made to this patent application taken.

An activator-responsive promoter unit consists of the following components:

• - One or more identical or different promoter or enhancers sequence (s), for example, cell cycle-specific, cell proliferation-dependent, metabolic, endothelial cell specific or virus specific or both cell cycle specific as well as metabolic, endothelial cell specific or virus can be activated specifically (so-called chimeric promoters)
• - One or more identical or different activator subunit (s), which each located downstream of the promoter or enhancer sequences and is activated by these in their basal transcription
• - An activator-responsive promoter, which by the expression products is activated by one or more activator subunit (s).

In a preferred embodiment, activators according to the invention responsive promoter units binding sequences for chimeric transcription factors from DNA binding domains, protein-protein interaction domains and Represent transactivation domains. All mentioned in the registration Transcription factor binding sites can be single (monomers) or in multiple Copies (multimers, for example up to 10 copies) are available.

An example of an activator-responsive promoter activated by two assets The LexA operator represents gate subunits in conjunction with the SV40 promoter. The first activator subunit comprises the cDNA for the LexA DNA binding protein coding for amino acids 1-81 or 1-202, the 3 'end of which is linked to the 5 'end of the cDNA for the Gal80 protein (amino acids 1-435).

The second activator subunit comprises the cDNA of the Gal80 binding domain of Gal4 protein coding for amino acids 851-881, the 3 'end of which is linked  with the 5 'end of the cDNA of the SV40 large T antigen coding for the Amino acids 126-132, the 3 'end of which is linked to the 5' end of the cDNA for the Transactivation domain of the VP16 of HSV-1 coding for amino acids 406-488.

Another example of an activator-responsive promoter activated by two Activator subunits relate the binding sequence for the Gal4 protein to the SV40 promoter.

The first activation unit comprises the cDNA for the DNA binding domain of the Gal4- Proteins (amino acids 1-147), the 3 'end of which is linked to the 5' end of the cDNA for the Gal80 protein (amino acids 1-435).

The second activation subunit comprises the cDNA for the Gal80 binding domain of Gal4 (amino acids 851-881), the 3 'end of which is linked to the 5' end of the Nuclear localization signal of SV40 (SV40 large T; amino acids 126-132), the 3 'end of which is linked to the 5' end of the cDNA for the Transactivation domain of the VP16 of HSV-1 coding for amino acids 406-488.

Another example of two activator subunits that activate the activator-responsive promoter, consisting of the binding sequence for the Gal4 protein and the SV40 promoter, is shown

• - A first activation unit that the cDNA for the cytoplasmic domain of CD4 T cell antigen (amino acids 397-435), the 5 'end of which is linked with the 3 'end of the cDNA for the transactivation domain of the VP16 of HSV-1 (Amino acids 406-488), the 5 'end of which is in turn linked to the 3' end the cDNA of the nuclear localization signal of SV40 (SV40 large T; Amino acids 16-132) and
• - The second activation unit comprising the nuclear cDNA Localization signal from SV40 (SV40 large T; amino acids 126-132), the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), the 3'-  End is linked to the 5 'end of the cDNA for the CD4 binding sequence of p56 Ick protein (amino acids 1-71).

5) Examples to illustrate the subject of the invention

The following examples describe what the genetic engineering of CD14 ⁺ cells to endothelial cells could look like.

a) Isolation of CD14 ⁺ cells

Peripheral blood mononuclear cells (PBMC) are made from healthy Donors using Ficoll density gradient centrifugation according to the According to the manufacturer (Ficoll-Paque, Pharmacia) isolated.

The PBMCs obtained are washed twice in cold PBS and washed with anti CD14 antibody-coupled magnetic bead (CD14 Micro Beads, Miltenyi Biotec) for 15 min. Incubated at 4 ° C, then washed with cold PBS and in PBS suspended containing 0.002% EDTA and 1% human serum albumin.

The CD14 ⁺ cells are detached from the column using the Vario MACS Separation kit (Mitenyi Biotec) in accordance with the manufacturer's instructions. The purity of the CD14 ⁺ cells thus produced is in the range between 70% and 95%.

b) Construction of the plasmid

In the reading direction from 5 'to 3', the following DNA sequence with the expert known method.

• - the DNA binding sequence from LexA
  (Nucleotide sequence 5'-TACTGTATGTACATACAGTA-3 '(SEQ ID NO .: 2); Brent et al., Nature 612, 312 (1984))
• - the von Willebrand factor (vWF) promoter
  (Nucleotide sequence -487 to +247; Jahroudi and Lynck, Mol. Cell. Biol. 14, 999 (1994))
• - DNA coding for VEGF receptor II (KDR)
  (Yin et al., Mammalian Genome 9, 408 (1998))
• - the vWF promoter
• - the binding domain of LexA
  (Amino acid 1-81; Kim et al., Science 255, 203 (1992))
• - the transactivation domain of HSV-1 VP16
  (Amino acids 406 to 488; Triezenberg et al., Genes Developm. 2, 718 (1988), Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995))
• - the polyadenylation signal of SV40
  (Elder et al., Annu. Rev. Genet. 15, 295 (1981))

This nucleotide sequence is shown schematically in FIG. 1:
This nucleic acid sequence ( FIG. 1) is cloned into a pxP2 plasmid vector (Norden, BioTechniques 6, 454 (1988)).

The respective components of the nucleic acid construct are linked together suitable restriction sites connected to the ends of the different Components are introduced using PCR amplification. The connection of the components takes place with the help of restriction-specific enzymes and with the Help from DNA ligases.

c) Transfection of CD14 ⁺ cells

The CD14 ⁺ cells are adjusted to a concentration of 1 × 10 ⁷ / ml culture medium [medium 199 with 20% fetal calf serum and penicillin / streptomycin / amphothericin (Gibco-BRL)], sown in 60 mm culture dishes and with a complex from the plasmid shown in b) (coding for VEGF receptor) and Superfect (from Quiagen, Düsseldorf) for 10 min. incubated at 37 ° C.

The complex is manufactured according to the manufacturer of Superfect (Quiagen).  

The success of the transaction is determined by proof of VEGF Receptor II (DR) m-RNA using RT-PCR. The RT-PCR is carried out as described by Sewing et al., J. Cell Sci. 104, 545 (1993).

d) Cultivation of VEGF Receptor II (KDR) expressing CD14 cells in vitro

VEGF Receptor II (KDR) expressing CD14 cells are adjusted to 1-2 × 10 ⁶ / ml and in the culture medium "Medium 199" (Gibco), which 10 ng / ml VEGF (Pepro Tech, London, England) and 20% fetal calf serum had been added, incubated for 7 days.

After 7 days, the cells are immunocytochemically compared to the expert known methodology of alkaline-phosphatase-anti-alkaline phosphatase (APAAP) - Method using specific antibodies (see Table 1) examined. In addition, m-RNAs specific for CD14, CD31, CD36 and vWF are generated using the RT-PCR (Sewing et al., J. Cell Sci. 104, 545 (1993)) was detected.

The following results were achieved:
The majority of the cells in the cell culture already differentiated into cells after 7 days which have the characteristic surface markers of endothelial cells (see Table 2).

Table 1

Table 2

Claims (16)

1. Process for the production of cells which are suitable for the treatment of the human body in which

• a) cells are removed from the human body;
• b) the cells from step (a) are transfected in vitro with at least one gene for a growth and / or differentiation factor which is under the control of a promoter;

so that the cells from step (b) have the ability to differentiate in a desired manner after return to the human body. 2. The method according to claim 1, characterized in that the cells Step (b) after returning to the human body Integrate tissue bandage. 3. The method according to any one of claims 1 or 2, characterized in that in step (b) the gene for a growth and / or differentiation factor and / or for a receptor of a growth and / or differentiation factor as Part of a nucleic acid construct that is under control is transfected a promoter both the gene for the growth and / or Differentiation factor and / or for said receptor, as well prophylactically or therapeutically effective proteins or enzymes for the Activation of drug precursors can express. 4. The method according to any one of claims 1 to 3, characterized in that the promoter is cell type-specific, cell cycle-specific, metabolic or is pharmacologically activated. 5. The method according to any one of claims 1 to 4, characterized in that the rows selected from step (a) are selected from the group containing mononuclear cells from blood, veins, capillaries, arteries, the umbilical cord, the Placenta, suspensions of cells from bones, spleen, lymph nodes, and lymph Connective tissue fluid, peritoneal cell suspensions, pleural cell suspensions, Endothelial cells and fibroblasts.   6. The method according to claim 5, characterized in that the mononuclear cells contain surface markers selected from the group CD34, CD11, CD13, CD14 and CD68. 7. The method according to claim 6, characterized in that the desired Differentiation corresponds to that of an endothelial cell and the gene for growth and / or differentiation factor is selected from the group comprising Vascular endothelial growth factor (VEGF) and other KDR or Flt ligands, (VEGF-B, VEGF-C, VEGF-D, Neuropilin), fibroblast growth factor (FGFα, FGFβ), Epidermal growth factor (EGF), insulin-like growth factor (IGF-1, IGF-2), β- endothelial cell growth factor (ECGF), endothelial cell attachment factor (ECAF), Interleukin-3 (IL-3), Colony stimulating factor-1 (CSF-1), GM-CSF, G-CSF, M-CSF, Interleukin-4 (IL-4), Interleukin-1 (IL-1), Interleukin-8 (IL-8), Platelet derived growth factor (PDGF-AA, -AB, -BB), interferon γ (IFNγ), oncostatin M, B61, platelet derived endothelial cell growth factor (PDEGF), star cell factor (SCF), transforming growth factor β (TGF-β), angiogenin, pleiotrophin, Flt-3 ligand (FL), Tie-2 ligands (Angiopoietin-1, Angiopoietin-2), Stromal derived factor-1 (SDF-1) and TNFα; and the gene for the receptor of the growth and / or differentiation factor is selected from the group containing VEGF receptor I (Flt), VEGF Receptor II (KDR), VEGF Receptor III, FGF Receptors (-1, -2, -3, -4, -5), IGF Receptor, ECGF receptor, ECAF receptor, IL-3 receptor, Oncostatin M receptor, LIF receptor, B61 receptor, PDEGF receptor, SCF receptor, TGFβ receptor, Tie- 2 receptor, SDF-1 receptor, pleiotrophin receptor, EGF receptor, TNFα receptor and / or SDF-1 receptor and the PDGF receptor (α and β). 8. The method according to claim 6, characterized in that the desired Differentiation corresponds to that of an osteoblast and the gene for one The growth and / or differentiation factor is selected from a group containing BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, Pleiotrophin and midkine; and the gene for the receptor of a growth and / or differentiation factor is selected from the group containing the Receptors for BMP 1-8, PTN and Midkine.   9. The method according to claim 6, characterized in that the desired Differentiation corresponds to that of a glial cell and the gene for a growth and / or differentiation factor is selected from a group containing glia growth factor (GGF), neurotrophin-4 (NT-4; trk-C), brain derived neurotrophic factor, Ciliary neurotrophic factor (CNF), glia cell line derived neurotrophic factor (GDNF) and nerve growth factor (NGF; trk-A) and the gene for the receptor of The growth and / or differentiation factor is selected from the group containing the GGF receptor, NGF receptor, CNF receptor, BdNF receptor, NT-4 Receptor and the GDNF receptor. 10. The method according to claim 6, characterized in that the desired Differentiation corresponds to that of a synovial cell and the gene for one The growth and / or differentiation factor is selected from a group containing transforming growth factor (TGF) β-1 and 2, interleukin 10 (IL-10), Insulin-like growth factor-1 (IGF-1), interleukin-4 (IL-4), interleukin receptor Antagonist protein (IRAP), inhibitors of metalloproteinases such as, for example TIMP-1, -2, -3, fibroblast growth factor (FGF), interleukin-6 (IL-6), plasminogen Activator inhibitor (PAI-1, -2), platelet derived growth factor (PDGF), Superoxide dismutase, soluble extracellular parts of CD18, ICAM-1, CD44 and that Gene selected for the growth and / or differentiation factor receptor is from the group containing TGF-β receptors, IL-10 receptors, IGF-1 Receptors, IL-4 receptors and bFGF receptors. 11. The method according to claim 6, characterized in that the desired Differentiation corresponds to that of an anti-inflammatory cell and the gene for a growth and / or differentiation factor is selected from the group containing cytokines, interferons (IFNα, IFNβ, IFNγ), interleukin-4 (IL-4), interleukin-6 (IL-6), Interleukin-9 (IL-9), Interleukin-13 (IL-13), LIF, Oncostatin, Interleukin-10 (IL-10), Interleukin-12 (IL-12), TGFβ, tumor necrosis factor α (TNFα), TNFβ and the Interleukin-1 receptor antagonists (IL-1 RA); and the gene for the receptor of Growth and / or differentiation factor is selected from the group containing the soluble IL-4 receptor, IL-4 receptor, IL-6 receptor, soluble IL-6 Receptor, soluble IL-2 receptor, IL-10 receptor, IL-12 receptor, IL-13  Receptor, TNFα receptor, TNFβ receptor, TGFβ receptor and receptors for IFNα, -β, -γ. 12. The method according to claim 6, characterized in that the desired Differentiation that corresponds to a cell involved in inflammation and that Gene for a growth and / or differentiation factor is selected from a group containing interleukin-1, interleukin-2, interleukin-4, interleukin-5, Interleukin-6, LIF, Interleukin-7, Interleukin-8, Interleukin-11, GM-CSF, M-CSF, G- CSF and IFNα, -β, -γ; and the gene for the receptor of growth and / or Differentiation factor is selected from the group containing the IL-1 Receptor, IL-2 receptor, IFNα, -β, γ receptor, IL-3 receptor, IL-5 receptor, IL-6 Receptor, GM-CSF receptor, M-CSF receptor, integrin beta 2 proteins. 13. The method according to claim 6, characterized in that the cells from step (a) are CD 14-positive, peripheral mononuclear cells of the blood (PBMC) which contain the following elements with a plasmid in the 5'-3 'direction:

• 1. the DNA binding sequence of LexA
  (Nucleotide sequence 5'TACTGTATGTACATACAGTA-3 '; 312 (1984))
• 2. the coding sequence for the von Willebrand factor (vWF) promoter (nucleotide sequence -487 to +247; (1994))
• 3. DNA coding for VEGF receptor II (ICDR)
• 4. the vWF promoter
• 5. the coding sequence for the binding domain of LexA (amino acid 1-81);
• 6. the coding sequence for the transactivation domain of HSV-1 VP16
  (Amino acids 406 to 488);
• 7. the polyadenylation signal of SV40

be transfected. 14. Cell obtainable by a method according to any one of claims 1 to 13. 15. The cell of claim 14 for use in gene therapy.   16. Use of a cell according to claim 14 for the manufacture of a medicinal product for the treatment of endothelial cell effects, to promote angiogenesis, for bone healing, to promote healing of damage to the CNS, for Prophylaxis and therapy of joint damage, inflammation, Autoimmune diseases, organ rejection and to support Inflammation and rejection reactions in infections or Tumor diseases.