CA2349497A1 - The genetic determination of genes and its use for the prophylaxis and therapy of diseases - Google Patents
The genetic determination of genes and its use for the prophylaxis and therapy of diseases Download PDFInfo
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- CA2349497A1 CA2349497A1 CA002349497A CA2349497A CA2349497A1 CA 2349497 A1 CA2349497 A1 CA 2349497A1 CA 002349497 A CA002349497 A CA 002349497A CA 2349497 A CA2349497 A CA 2349497A CA 2349497 A1 CA2349497 A1 CA 2349497A1
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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
WO 00128010 PCTlEP99107902 Description The genetic marking of cells and their use for the prophylaxis and therapy of disorders 1 ) Basis Therapy with the aid of transplantation of cells is increasingly gaining in importance. Thus the transplantation of, for example, bone marrow cells is an established process for the treatment of leukemias or oncoses after high-dose chemotherapy. In principle, the transfer of cells to a body is problem-free if the cells to be transferred can be converted into a cell suspension without impairment of their function andlor their survival ability.
Cell suspensions of this type are relatively simple to administer to the circulation, to a body cavity, to an organ or locally.
In cases in which the cells to be transferred, however, are needed in a stage of differentiation in which the production of a cell suspension leads to considerable damage to their function and survival ability, administration of these differentiated cells is possible only with very great difficulty. This is, for example, particularly the case when cells grow in strongly adhesive form in the desired stage of differentiation and their detachment from the cell culture vessel for the production of a cell suspension is only possible using mechanical methods (e.g. scraping off), if appropriate with addition of proteolytic enzymes.
With knowledge of these difficulties, a novel process has been developed in order to be able to transplant cells of this type without problems.
Cell suspensions of this type are relatively simple to administer to the circulation, to a body cavity, to an organ or locally.
In cases in which the cells to be transferred, however, are needed in a stage of differentiation in which the production of a cell suspension leads to considerable damage to their function and survival ability, administration of these differentiated cells is possible only with very great difficulty. This is, for example, particularly the case when cells grow in strongly adhesive form in the desired stage of differentiation and their detachment from the cell culture vessel for the production of a cell suspension is only possible using mechanical methods (e.g. scraping off), if appropriate with addition of proteolytic enzymes.
With knowledge of these difficulties, a novel process has been developed in order to be able to transplant cells of this type without problems.
2) Summary of the invention The invention relates to a process in which cells have inserted at least one gene for a growth and/or differentiation factor andlor at least one gene for the associated receptor of the growth andlor differentiation factor and these cells are forced by the inserted gene to differentiate in vitro or in vivo to a stage in which they have the desired function. In a particular embodiment of the invention, this function is associated with an integration of the transfected cell into a tissue group.
The invention relates in particular to cells in which at least one gene for a growth andlor differentiation factor and/or at least one gene for the associated receptor of the growth and/or differentiation factor has been inserted and to the use of these cells for the purposes of the prophylaxis or therapy of a disorder.
The invention additionally relates to mononuclear cells obtained from the bone marrow, lymphatic organs, body cavities, body exudates, blood, blood vessels and connective tissue, into which at least one gene for a growth and differentiation factor and/or its receptor has been introduced for the prophylaxis or therapy of a disorder.
Cells of this type express the genes) introduced into them and undergo, as a result of the expressed growth factor and/or receptor in the cell culture, but particularly also after administration to a body, further development up to a desired stage of differentiation.
According to the invention, the expression of the genes) introduced into the cell is placed under the control of promoters. These promoters can be nonspecifically, cell type-specifically, metabolically andlor pharmacologically activatable and/or self-potentiating.
As a result of the choice of the particular promoters, the growth and the differentiation of the transduced cells can be influenced as necessary. The cells according to the invention can even be used as a therapeutic or prophylactic without further treatment.
They can, however, also be used as cellular vectors for gene therapy.
For this, in vitro, i.e. in cell culture, additional nucleotide sequences are inserted into them which, under the control of different promoters (e.g, cell type-specifically, cell cycle-specifically, metabolically, pharmacologically activatable andlor self potentiating), code for prophylactically or therapeutically active proteins or else for enzymes for the activation of the precursors of a pharmaceutical into a phamnaceutical. Examples of nucleotide sequences of this type have already been described for the prophylaxis and therapy of vascular disorders (EP A 0 777 739), of disorders of the central nervous system (EP A 0 777 740), of tumors
The invention relates in particular to cells in which at least one gene for a growth andlor differentiation factor and/or at least one gene for the associated receptor of the growth and/or differentiation factor has been inserted and to the use of these cells for the purposes of the prophylaxis or therapy of a disorder.
The invention additionally relates to mononuclear cells obtained from the bone marrow, lymphatic organs, body cavities, body exudates, blood, blood vessels and connective tissue, into which at least one gene for a growth and differentiation factor and/or its receptor has been introduced for the prophylaxis or therapy of a disorder.
Cells of this type express the genes) introduced into them and undergo, as a result of the expressed growth factor and/or receptor in the cell culture, but particularly also after administration to a body, further development up to a desired stage of differentiation.
According to the invention, the expression of the genes) introduced into the cell is placed under the control of promoters. These promoters can be nonspecifically, cell type-specifically, metabolically andlor pharmacologically activatable and/or self-potentiating.
As a result of the choice of the particular promoters, the growth and the differentiation of the transduced cells can be influenced as necessary. The cells according to the invention can even be used as a therapeutic or prophylactic without further treatment.
They can, however, also be used as cellular vectors for gene therapy.
For this, in vitro, i.e. in cell culture, additional nucleotide sequences are inserted into them which, under the control of different promoters (e.g, cell type-specifically, cell cycle-specifically, metabolically, pharmacologically activatable andlor self potentiating), code for prophylactically or therapeutically active proteins or else for enzymes for the activation of the precursors of a pharmaceutical into a phamnaceutical. Examples of nucleotide sequences of this type have already been described for the prophylaxis and therapy of vascular disorders (EP A 0 777 739), of disorders of the central nervous system (EP A 0 777 740), of tumors
3 (EP A 0 804 601 ) and of disorders which are caused by the immune system (EP A 0 807 183) in the cited patent applications and also in the Patent Applications EP A 0 859 058, EP A 0 864 651 and DE19752299.8.
The process claimed in this invention and the cells prepared by this process are novel. The prior art is to carry out the differentiation in vitro by addition of growth factors in cell culture and to use the differentiated cells after mechanical or proteolytic detachment from the cell culture vessel.
For example, peripheral mononuclear cells, in particular CD34-positive cells, from human blood were differentiated into endothelial cell-like cells in cell culture by Asahara et al., Science 275, 964 (1997) by addition of serum and brain extract of cattle and used for the investigation of their properties.
These cells were not used, however, for injection into the body, since on account of their stage of differentiation as endothelial cells and the strong adhesion to the cell culture vessel associated therewith detachment without damage and conversion into an individual suspension would only be possible with great difficulty.
Instead of this, mononuclear cells freshly isolated from the blood were injected, of which it is known, however, that they can differentiate to give different cells in vivo, according to the local conditions in the particular organ in which they settle, and only partly (Asahara et al., 1997, see above) into endothelial cells.
The process claimed in this invention, of the marking of cells up to differentiation, is to be differentiated from the process of the dedifferentiation of cells known from the literature. This dedifferentiation is carried out by insertion of a gene into a cell which leads to an immortalization of this cell. Genes of this type are, for example:
- IE 84 of CMV
{Speir et al., Science 265, 391 (1994}) - E 1 A of AV
(Nevins, Science 258, 424 (1992)) 3) Detailed description of the invention
The process claimed in this invention and the cells prepared by this process are novel. The prior art is to carry out the differentiation in vitro by addition of growth factors in cell culture and to use the differentiated cells after mechanical or proteolytic detachment from the cell culture vessel.
For example, peripheral mononuclear cells, in particular CD34-positive cells, from human blood were differentiated into endothelial cell-like cells in cell culture by Asahara et al., Science 275, 964 (1997) by addition of serum and brain extract of cattle and used for the investigation of their properties.
These cells were not used, however, for injection into the body, since on account of their stage of differentiation as endothelial cells and the strong adhesion to the cell culture vessel associated therewith detachment without damage and conversion into an individual suspension would only be possible with great difficulty.
Instead of this, mononuclear cells freshly isolated from the blood were injected, of which it is known, however, that they can differentiate to give different cells in vivo, according to the local conditions in the particular organ in which they settle, and only partly (Asahara et al., 1997, see above) into endothelial cells.
The process claimed in this invention, of the marking of cells up to differentiation, is to be differentiated from the process of the dedifferentiation of cells known from the literature. This dedifferentiation is carried out by insertion of a gene into a cell which leads to an immortalization of this cell. Genes of this type are, for example:
- IE 84 of CMV
{Speir et al., Science 265, 391 (1994}) - E 1 A of AV
(Nevins, Science 258, 424 (1992)) 3) Detailed description of the invention
4 The choice of the starting cells, the promoters and the nucleotide sequences coding for receptors and/or growth factors and differentiation factors is carried out according to the intended use demanded for these cells, i.e. the state of differentiation of the cells demanded in the body and the therapeutic aim demanded with these cells.
3.1 ) Demanded state of differentiation: endothelial cells a) Intended use:
- substitution of endothelial cells in endothelial cell defects (e.g. after vasodilatation) or for the promotion of angiogenesis - use as a cellular vector for gene therapy as presented in detail in the Patent Applications EP A 0 893 493 and EP A 0 926 236 b) Choice of the starting cells:
- mononuclear cells obtained * from the blood, e.g. 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 (emerging, for example, at the surface of a supe~cially, e.g. mechanically, damaged epidermis) Erythrocytes, granulocytes and other cell components are separated from these body fluids by density gradient centrifugation and platelets are separated by differential centrifugation according to the methods known to the person skilled in the art - mononuclear cells having surface markers CD34, CD11, CD13, CD14, CD64 and/or CD68 obtained from organs, body cavities or the blood The isolation of these cells is carried out using the methods known to the person skilled in the art, for example by immunoadsorption on supports which are coated with monoclonal antibodies speck for the respective surface antigen.
- endothelial cells
3.1 ) Demanded state of differentiation: endothelial cells a) Intended use:
- substitution of endothelial cells in endothelial cell defects (e.g. after vasodilatation) or for the promotion of angiogenesis - use as a cellular vector for gene therapy as presented in detail in the Patent Applications EP A 0 893 493 and EP A 0 926 236 b) Choice of the starting cells:
- mononuclear cells obtained * from the blood, e.g. 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 (emerging, for example, at the surface of a supe~cially, e.g. mechanically, damaged epidermis) Erythrocytes, granulocytes and other cell components are separated from these body fluids by density gradient centrifugation and platelets are separated by differential centrifugation according to the methods known to the person skilled in the art - mononuclear cells having surface markers CD34, CD11, CD13, CD14, CD64 and/or CD68 obtained from organs, body cavities or the blood The isolation of these cells is carried out using the methods known to the person skilled in the art, for example by immunoadsorption on supports which are coated with monoclonal antibodies speck for the respective surface antigen.
- endothelial cells
5 Endothelial cells can be obtained using the methods known to the person skilled in the art, for example from fatty tissue, by scraping out veins or by detachment of the umbilical cord endothelium.
c) Selection of the genes for growth and differentiation factors and for their receptors Ail growth factors and their receptors are suitable which contribute to the proliferation and to the 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 (FGFa., FGF~}
epidermal growth factor (EGF) * insulin-like growth factor {IGF-1, IGF-2) * ~-endothelial cell growth factor (ECGF) * endothelial cell attachment factor (ECAF) * interteukin-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}
* interteron y (IFNY) oncostatin M
* B61 ' platelet derived endothelial cell growth factor (PDEGF) * stem cell factor (SCF) * transforming growth factor ~ (TGF-ji) ' angiogenin
c) Selection of the genes for growth and differentiation factors and for their receptors Ail growth factors and their receptors are suitable which contribute to the proliferation and to the 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 (FGFa., FGF~}
epidermal growth factor (EGF) * insulin-like growth factor {IGF-1, IGF-2) * ~-endothelial cell growth factor (ECGF) * endothelial cell attachment factor (ECAF) * interteukin-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}
* interteron y (IFNY) oncostatin M
* B61 ' platelet derived endothelial cell growth factor (PDEGF) * stem cell factor (SCF) * transforming growth factor ~ (TGF-ji) ' angiogenin
6 * pleiotrophin Flt-3 ligand (FL) - stem cell growth factor (Scg7) * Tie-2 ligands such as angiopoietin-1 * stromal derived factor-1 (SDF-1 ) and * TNFa - receptors * the VEGF receptor I (Flt) * 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 TGF{i receptor * theTie-2 receptor * the SDF-1 receptor * the pleiotrophin receptor * the EGF receptor * the TNFa receptor and/or * the SDF-1 receptor * the PDGF receptor (a and (i) d) Selection of the promoters - unrestrictedly activatable, endothelial cell-specifically, metabolically activatable, self potentiating and/or pharmacologically controllable and ~mbinations thereof (see section 4) 3.2) Demanded state of differentiation: osteoblasts a) Intended use - promotion of bone healing (e.g, on local or systemic adminisVation after bone fractures)
* 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 TGF{i receptor * theTie-2 receptor * the SDF-1 receptor * the pleiotrophin receptor * the EGF receptor * the TNFa receptor and/or * the SDF-1 receptor * the PDGF receptor (a and (i) d) Selection of the promoters - unrestrictedly activatable, endothelial cell-specifically, metabolically activatable, self potentiating and/or pharmacologically controllable and ~mbinations thereof (see section 4) 3.2) Demanded state of differentiation: osteoblasts a) Intended use - promotion of bone healing (e.g, on local or systemic adminisVation after bone fractures)
7 b) Selection of the cells:
- mononuclear cells obtained as described in 3.1.
- mononuclear cells having the surface markers CD34, CD11, CD13, CD14 and/or CD68 obtained as described in 3.1.
- fibrobiasts obtained, for example, from the hypodermis using the methods known to the person skilled in the art c) Selection of the genes for receptors of growth and differentiation factors and for growth factors and differentiation factors All growth and differentiation factors are suitable which contribute to the proliferation and to the differentiation of osteoblasts.
- Growth and differentiation factors These include, in particular, the bone morphogenic proteins (BMP), 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), CSzkaynak et al., EMBO J. 9, 2085 (1990), t5zkaynak et al., J. Biol. Chem. 267, 25220 (1992), Hino et al., Biochem.
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 the promoters - unrestrictedly activatable promoters of the genes for the BMPs, metabolically activatabie, self-potentiating and/or pharmacologically controllable and combinations thereof (see section 4) 3.3) Demanded state of differentiation: glia cells a) Intended use:
- promotion of the healing of damage to the CNS
- mononuclear cells obtained as described in 3.1.
- mononuclear cells having the surface markers CD34, CD11, CD13, CD14 and/or CD68 obtained as described in 3.1.
- fibrobiasts obtained, for example, from the hypodermis using the methods known to the person skilled in the art c) Selection of the genes for receptors of growth and differentiation factors and for growth factors and differentiation factors All growth and differentiation factors are suitable which contribute to the proliferation and to the differentiation of osteoblasts.
- Growth and differentiation factors These include, in particular, the bone morphogenic proteins (BMP), 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), CSzkaynak et al., EMBO J. 9, 2085 (1990), t5zkaynak et al., J. Biol. Chem. 267, 25220 (1992), Hino et al., Biochem.
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 the promoters - unrestrictedly activatable promoters of the genes for the BMPs, metabolically activatabie, self-potentiating and/or pharmacologically controllable and combinations thereof (see section 4) 3.3) Demanded state of differentiation: glia cells a) Intended use:
- promotion of the healing of damage to the CNS
8 - cellular vector for gene therapy of disorders and/or damage to the CNS, such as, for example, presented 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 having the surface markers CD34, CD11, CD13, CD14 and/or CD68, obtained as described in 3.1 ) c) Selection of the genes for growth and differentiat'ron factors and for the associated receptors Growth factors and differentiation factors All growth factors which contribute to the growth and to the differentiation of glia cells are suitable within the meaning of the invention.
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 cel! line derived neurotrophic factor (GDNF) - the nerve growth factor (NGF; trk-A) Receptors All receptors for growth factors which contribute to the proliferation and to the differentiation of glia cells are suitable within the meaning of the invention.
These include, for example:
- the GGF receptor - the NGF receptor - the CNF receptor - the BdNF receptor
- mononuclear cells obtained as described in 3.1 ) - mononuclear cells having the surface markers CD34, CD11, CD13, CD14 and/or CD68, obtained as described in 3.1 ) c) Selection of the genes for growth and differentiat'ron factors and for the associated receptors Growth factors and differentiation factors All growth factors which contribute to the growth and to the differentiation of glia cells are suitable within the meaning of the invention.
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 cel! line derived neurotrophic factor (GDNF) - the nerve growth factor (NGF; trk-A) Receptors All receptors for growth factors which contribute to the proliferation and to the differentiation of glia cells are suitable within the meaning of the invention.
These include, for example:
- the GGF receptor - the NGF receptor - the CNF receptor - the BdNF receptor
9 - the NT-4 receptor - the GDNF receptor d) Selection of the promoters - unrestrictedly activatable, glia cell-specifically activatable, metabolically activatable, self-potentiating andlor pharmacologically controllable and combinations thereof (see section 4).
3.4) Demanded state of diffensntiation: synovial cells a) Intended use:
- prophylaxis and therapy of joint damage b) Cells - mononuclear cells isolated according to section 3.1 ) - blood cells having surface markers corresponding to section 3.1 ) - fibroblasts, obtained according to a method known to the person skilled in the art 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, for example, cytokines, cytokine inhibitors, enzyme inhibitors, antiadhesion molecules, antagonists of oxygen radicals and growth factors for cartilaginous cells which lead to an antiinflammatory reaction and differentiation of synovial cells.
These include, for example:
- transforming growth factor (TGF) ~-1 and -2 - interfeukin 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) adhesion molecules such as, for example, of CD18, /CAM-1, CD44 5 - receptors These include all those receptors whose activation leads to an antiinflammatory reaction and differentiation of synovial cells.
3.4) Demanded state of diffensntiation: synovial cells a) Intended use:
- prophylaxis and therapy of joint damage b) Cells - mononuclear cells isolated according to section 3.1 ) - blood cells having surface markers corresponding to section 3.1 ) - fibroblasts, obtained according to a method known to the person skilled in the art 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, for example, cytokines, cytokine inhibitors, enzyme inhibitors, antiadhesion molecules, antagonists of oxygen radicals and growth factors for cartilaginous cells which lead to an antiinflammatory reaction and differentiation of synovial cells.
These include, for example:
- transforming growth factor (TGF) ~-1 and -2 - interfeukin 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) adhesion molecules such as, for example, of CD18, /CAM-1, CD44 5 - receptors These include all those receptors whose activation leads to an antiinflammatory reaction and differentiation of synovial cells.
10 These include, for example:
TGF~ receptors IL-10 receptors * IGF-1 receptors * IL-4 receptors * bFGF receptors d) Promoters - nonspec~c, lymphocyte- and/or macrophage-specific and/or synovial cell-specific (see section 4) 3.5) Demanded state of differen~ation: antiinflammatory cells a) Intended use:
- prophylaxis and therapy of inflammations, autoimmune disorders and organ rejections b) Cells:
- mononuclear blood cells isolated according to section 3.1 ) - cells having cell surface markers isolated according to section 3.1 - fibrobiasts c) Genes for growth factors and differentiation factors andlor their receptors All cytokines and their receptors which are antiallergic or inhibit the antibody reaction or the cellular immune reaction are suitable within the meaning of the invention.
These include, for example:
TGF~ receptors IL-10 receptors * IGF-1 receptors * IL-4 receptors * bFGF receptors d) Promoters - nonspec~c, lymphocyte- and/or macrophage-specific and/or synovial cell-specific (see section 4) 3.5) Demanded state of differen~ation: antiinflammatory cells a) Intended use:
- prophylaxis and therapy of inflammations, autoimmune disorders and organ rejections b) Cells:
- mononuclear blood cells isolated according to section 3.1 ) - cells having cell surface markers isolated according to section 3.1 - fibrobiasts c) Genes for growth factors and differentiation factors andlor their receptors All cytokines and their receptors which are antiallergic or inhibit the antibody reaction or the cellular immune reaction are suitable within the meaning of the invention.
These include, for example:
11 - CyiOkineS
* interferons {IFNa, IFN~, IFNy) interleukin-4 (IL-4) * interleukin-6 {IL-fi) * interleukin-9 (IL-9) * interleukin-13 (IL-13) * LIF
* oncostatin * interleukin-10 (IL-10) * interleukin-12 (IL-12) * TGF(i tumor necrosis factor a (TNFa) * TNF(3 * interleukin-1 receptor antagonist (IL-1 RA) - receptors * receptors for IFNa, -~, -y * soluble iL-4 receptor * IL-4 receptor * IL-6 receptor * soluble IL-6 receptor soluble IL-2 receptor * IL-10 receptor * IL-12 receptor * 1 L-13 receptor TNFa receptor * TNF~ receptor * TGF(i receptor d) Selection of the promoters - unrestrictedly activatable lymphocytes and/or macrophage specfically activatable, metabolically activatable, self-potentiating andlor pharmacologically controllable and combinations thereof (see section 4).
3.6) Demanded state of differentiation: cells involved in inflammation a) Intended use:
* interferons {IFNa, IFN~, IFNy) interleukin-4 (IL-4) * interleukin-6 {IL-fi) * interleukin-9 (IL-9) * interleukin-13 (IL-13) * LIF
* oncostatin * interleukin-10 (IL-10) * interleukin-12 (IL-12) * TGF(i tumor necrosis factor a (TNFa) * TNF(3 * interleukin-1 receptor antagonist (IL-1 RA) - receptors * receptors for IFNa, -~, -y * soluble iL-4 receptor * IL-4 receptor * IL-6 receptor * soluble IL-6 receptor soluble IL-2 receptor * IL-10 receptor * IL-12 receptor * 1 L-13 receptor TNFa receptor * TNF~ receptor * TGF(i receptor d) Selection of the promoters - unrestrictedly activatable lymphocytes and/or macrophage specfically activatable, metabolically activatable, self-potentiating andlor pharmacologically controllable and combinations thereof (see section 4).
3.6) Demanded state of differentiation: cells involved in inflammation a) Intended use:
12 - support of inflammatory and rejection reactions, for example in the course of infections or oncoses.
b) Cells:
- mononuclear blood cells isolated according to section 3.1 ) - cells having surface markers isolated according to section 3.1 ) - 1'<broblasts c) Genes for growth and differentiation factors and/or their receptors All cytokines and/or their receptors which promote an antibody-mediated or a cellular immune reaction are suitable within the meaning of the invention.
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
* IFNa, -/3, ~y - receptors * IL-1 receptor * IL-2 receptor * IFNa, -~, y receptor * IL-3 receptor * IL-5 receptor * IL-6 receptor * GM-CSF receptor * M-CSF receptor * intregrin beta 2 proteins
b) Cells:
- mononuclear blood cells isolated according to section 3.1 ) - cells having surface markers isolated according to section 3.1 ) - 1'<broblasts c) Genes for growth and differentiation factors and/or their receptors All cytokines and/or their receptors which promote an antibody-mediated or a cellular immune reaction are suitable within the meaning of the invention.
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
* IFNa, -/3, ~y - receptors * IL-1 receptor * IL-2 receptor * IFNa, -~, y receptor * IL-3 receptor * IL-5 receptor * IL-6 receptor * GM-CSF receptor * M-CSF receptor * intregrin beta 2 proteins
13 d) Promoters - unresfictedly activatable, lymphocyte and/or macrophage-specifically activatable, metabolically activatable, self potentiating and/or pharmacologically controllable and combinations thereof (see section 4).
4) Choice of the promoters Within the meaning of the invention, promoter sequences to be used are nucleotide sequences which, after binding of transcription factors, activate the transcription of a transgene located adjacent to the 3' end, such as, for example, of a gene for a receptor of a growth factor or differentiation factor or of a gene for a growth factor or differentiation factor. Within the meaning of the invention, at least one promoter sequence is inserted into the cell according to the invention. This promoter sequence can be combined with at least one further promoter sequence. The choice of the promoter sequence to be combined with the promoter sequence depends on the disorder to be treated. Thus the promoter sequence can be inducible unrestrictedly, endothelial cell-specifically, under certain metabolic conditions, such as, for example, by hypoxia or inducible by a pharmacon, virus-specifically and/or cell cycle-specifically activatable. Promoters of this type have already been mentioned 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. The promoter sequences to be selected inGude, for example:
4.1 ) Unrestrictively activatable promoters and activator sequences such as, for example - the promoter of RNA polymerise III
- the promoter of RNA polymerise II
- the CMV promoter and enhancer - the SV40 promoter 4.2) Metabolically activatable promoter and enhancer sequences such as, for example, the enhancer inducible by hypoxia (Semenza et al., PNAS
88: 5680 (1991 ), McBumey et a., Nucl. Acids Res. 19: 5755 (1991 )).
4) Choice of the promoters Within the meaning of the invention, promoter sequences to be used are nucleotide sequences which, after binding of transcription factors, activate the transcription of a transgene located adjacent to the 3' end, such as, for example, of a gene for a receptor of a growth factor or differentiation factor or of a gene for a growth factor or differentiation factor. Within the meaning of the invention, at least one promoter sequence is inserted into the cell according to the invention. This promoter sequence can be combined with at least one further promoter sequence. The choice of the promoter sequence to be combined with the promoter sequence depends on the disorder to be treated. Thus the promoter sequence can be inducible unrestrictedly, endothelial cell-specifically, under certain metabolic conditions, such as, for example, by hypoxia or inducible by a pharmacon, virus-specifically and/or cell cycle-specifically activatable. Promoters of this type have already been mentioned 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. The promoter sequences to be selected inGude, for example:
4.1 ) Unrestrictively activatable promoters and activator sequences such as, for example - the promoter of RNA polymerise III
- the promoter of RNA polymerise II
- the CMV promoter and enhancer - the SV40 promoter 4.2) Metabolically activatable promoter and enhancer sequences such as, for example, the enhancer inducible by hypoxia (Semenza et al., PNAS
88: 5680 (1991 ), McBumey et a., Nucl. Acids Res. 19: 5755 (1991 )).
14 4.3) Cell cycle-specifically activatable promoters These are, for example, the promoter of the cdc25B gene, of the cdc25C
gene, of the cyclin A gene, of the cdc2 gene, of the B-myb gene, of the DHFR gene, of the E2F-1 gene or else binding sequences for transcription factors occurring or activated during cell proliferation. These binding sequences include, for example, binding sequences for c-myc proteins.
Among these binding sequences are counted monomers or multimers of the nucleotide sequence designated as Myc E-box (5'-GGAAGCAGACCACGTGGTCTGCTTCC-3' (SEQ ID NO.: 1 ); Blackwood and Eisenmann, Science 251, 1211 (1991).
4.4) Self-potentiating andlor pharmacologically controllable promoters In the simplest case, a promoter can be inducible in the combination of identical or different promoters, for example in the form of a promoter which can be activated or switched off by tetracycline in the form of the tetracycline operator in combination with an appropriate repressor.
According to the invention, the promoter, however, can also be self potentiating with or also without a pharmacologically controllable promoter unit.
Self-potentiating andlor pharmacologically controllable promoters of this type have already been described in the Patent Application EP A 0 848 061, to which reference is expressly made.
4.5) Endothelial cell-specifically activatable promoters These include promoters or activator sequences from promoters or enhancers of those genes which code for proteins preferably formed in endothelial cells.
Within the meaning of the invention, promoters of the genes for the following proteins, for example, are to be used:
- brain-specific, endothelial glucose-1 transporter - endogiin - VEGF receptor-1 (flt-1 ) - VEGF receptor-2 (flt-1, KDR) - VEGF receptor-3 (flt-3) - tie-1 or tie-2 - B61 receptor (Eck receptor) 5 - endothelia, especially endothelia B or endothelia-1 - endothelia receptors, in particular the endothelia B receptor - mannose-6 phosphate receptors - von Willebrand factor - IL-1a, IL-1(i 10 - 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 alternatives to natural endothelial cell-specific promoters, synthetic activator sequences can also be used which consist of oligomerized binding sites for transcription factors which are preferentially or selectively active in endothelial cells. An example of this is the transcription factor GATA-2, whose binding site in the endothelia-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) Glia cell-spec~cally activatable promoters Promoters and activator sequences which are activated in glia cells are, for example, gene-regulatory sequences from genes which code, for example, for the following proteins: the Schwann cell-specific protein periaxin, glutamine synthetase, the glia cell-specific protein (glial fibrillary acid protein = GFAP), the glia cell protein S100, IL-6, CNTF, 5-HAT receptors, TNFa, IL-10, insulin-like growth factor receptor I and il or VEGF.
4.7) Synovtal cell-specifically activatable promoters Promoter and activator sequences which are activated in synovial cells are, for example, the promoter sequences of genes coding for matrix metalloproteinases (MMP), such as, for example, MMP-1 (interstitial collagenase), MMP-3 (stromelysinltransin) ar tissue inhibitors of metalloproteinases (TIMP), such as TIMP-1, -2, -3.
4.8 Lymphocyte- and/or macrophage-specifically activatable promoters Promoters and activator sequences which are activated in lymphocytes andlor macrophages are, for example, the promoter and activator sequences of the genes coding for cytokines, cytokine receptors and adhesion molecules and receptors for the Fc fragments of antibodies such as, for example, IL-receptor, IL-1a, IL-1~, IL-2, IL-2 receptor, IL-3, IL-3 receptor (a-subunit), IL-3 receptor (~i-subunit), IL-4, IL-4 receptor, IL-5, IL-6, IL-6 receptor, interferon regulatory factor 1 (IRF-1 ), the promoter of IRF-1 being activated to the same extent by IL-6 as by IFNY or IFN~i, IFNy responsive promoter IL-7, IL-8, IL-10, IL-11, IFNY, GM-CSF, GM-CSF
receptor (a-chain), IL-13, LIF, macrophage colony stirnulating factor (M-CSF) receptor, type I and type II macrophage scavenger receptors, MAC-1 (leukocyte function antigen), LFA-1a (leukocyte function antigen) or p150,95 (leukocyte function antigen).
4.9) Combination of identical or different promoters The combination of identical promoters is effected, for example, by linkage of a number of promoters one after the other in the reading direction from 5' to 3' of the nucleotide sequence.
For the combination of identical or different promoters, however, technologies are preferentially employed which have already been described in detail in the Patent Applications WO 96/06943;
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. Reference is expressly made to these patent applications in the context of this invention. Examples of technologies of this type are:
4.10) Chimeric promoters A chimeric promoter is the combination of a cell-specifically, metabolically or virus-specifically activatable activator sequence located upstream with a promoter module located downstream, which contains the nucleotide sequence CDE-CHR or E2FBS-CHR to which suppressive proteins bind, which as a result can inhibit the activation of the activator sequence located upstream in the Gp and G~ phase of the cell cycle (Vl/0 96/06943, Lucibello et al., EMBO J. 12 (1994)).
Continuing investigations on the functioning, in particular of the promoter element CDE-CHR, showed that the cell cycle-dependent regulation by the CDE-CHR element of an activator sequence located upstream is largely dependent on whether the activation sequence of transcription factors is activated with glutamine-rich activation domains (Zwicker et al., Nucl. Acids Res. 3822 (1995)).
Transcription factors of this type include, for example, Sp1 and NF-Y.
This consequently restricts the use of the promoter element CDE-CHR for chimeric promoters. The same is to be assumed for the promoter element E2F-8S-CHB of the B-myb gene (Zwicker et al., Nucl. Acids Res. 3822 (1995)).
4.11 ) Hybrid promoters The hybrid promoters have already been described in the Patent Application EP A 0 848 063. For the combination of the endothelial cell-specific promoter with at least ane further promoter, a gene construct, for example, is selected which contains all the following components:
The nucleotide sequence of the endothelial cell-specific promoter in a form in which at least one binding site for a transcription factor is mutated. By means of this mutation, the initiation of the transcription of the effector gene is blocked.
A transgene which codes as an effecior gene for an active compound.
At least one promoter or enhancer sequence which is activatable non-specifically, cell-specifically, virus-specifically, by tetracycline andlor cell cycle-specifically, which activates the transcription of at least one gene for at least one transcription factor which is mutated such that it can bind to the mutated binding sites) in the endothelial cell-specific promoter and activates this.
In an exemplary embodiment of this invention, the mutation in the promoter sequence can be, for example, a mutation of the TATA box of the cdc25B
promoter.
The mutation of the TATA can be, for example, TGTATAA. By means of this mutation, the DNA binding site of the normal TATA box-binding protein (TBP) is no longer recognized and the effector gene is no longer efficiently transcribed. Accordingly, the nucleic acid sequence which codes for the TBP must have a comutation. By means of this comutation, the TBP binds to the mutated TATA box (e.g. to TGTATAA) and thus leads to the efficient transcription of the effector gene. Comutations of the TBP gene of this type have been described, for example, by Strvbin and Stnuhl (Cell, 721 (1992) and by Heard et al. (EMBO J., 3519 (1993)).
4.12) Multiple promoters in combination with a nuclear retention signal and a nuclear export factor This technology has already been described in detail in the Patent Application EP A 0 805 209. Reference is made to this patent application.
According to the invention, a promoter of this type contains the following components:
- A first endothelial cell-specific, activatable promoter or enhancer sequence, which activates the basal transcription of a transgene.
- A transgene which codes as an effector gene for an active compound.
A nuclear retention signal (NRS) whose cDNA is linked indirectly or directly at the 5' end to the 3' end of the structural gene (b).
- Preferentially, the transcription product of the nuclear retention signal has a binding structure for a nuclear export factor.
- A further nonspecific, cell-specific, virus-specific, metabolically and/or cell cycle-specifically activatable promoter or enhancer sequence which activates the basal transcription of a nuclear export factor.
- A nucleic acid coding for a nuGear export factor (NEF) which binds to the transcription product of the nuclear retention signal and thereby mediates the transport of the transcription product of the transgene from the cell nucleus.
Preferably, the gene coding for the nuclear retention signal is selected from the group comprising the Rev responsive element (RRE) of HIV-1 or HIV-2, the RRE-equivalent retention signal of retrovirusss or the RRE-equivalent retention signal of the HBV.
The nuclear export factor is preferentially a gene selected from the group comprising the Rev gene of the viruses HIV-1, HIV-2, Visna-Maidi virus, caprine arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, of retroviruses, of HTLV or the gene of the hnRNP-A1 protein or the gene of the transcription factor TFIII-A.
4.13) Activator-responsive promoter unit Activator-responsive promoter units have already been described in detail in the Patent Application EP A 0 805 209. Reference is made to this patent application.
An activator-responsive promoter unit consists of the following components:
- one or more identical or different promoter or enhancer sequences which is or are activatable, for example, cell cycle-specifically, cell proliferation-dependently, metabolically, endothelial cell-spec~cally or virus-specifically or both cell cycle-specifically and metabolically, endothelial cell-specfically or virus-specifically (so-called chimeric promoters) - one or more identical or different activator subunits which are in each case located downstream of the promoter or enhancer sequences and is or are activated by this in their basal transcription - an activator responsive promoter which is activated by the expression products of one or more activator subunits.
In a preferred embodiment, activator-responsive promoter units according to the invention can be binding sequences for chimeric transcription factors from DNA-binding domains, protein-protein interaction domains and transactivation domains. All transcription factor binding sites mentioned in the application can be present singly (monomers) or in a number of copies (multimers, for example up to 10 copies).
An example of an activator-responsive promoter activated by two activator subunits is the LexA operator in combination with the SV40 promoter. The first activator subunit includes the cDNA for the LexA-DNA binding protein coding for the amino acids 1-81 or 1-202, whose 3' end is linked to the 5' end of the cDNA for the Ga180 protein (amino acids 1-435).
The second activator subunit comprises the cDNA of the Ga180 binding domain of the Gal4 protein coding for the amino acids 851-881, whose 3' end is linked to the 5' end of the cDNA of the SV40 large T antigen coding for the amino acids 126-132, whose 3' end is linked to the 5' end of the 5 cDNA of the transactivation domain of the VP16 of HSV-1 coding for the amino acids 406-488.
A further example of an activator-responsive promoter activated by two activator subunits is the binding sequence for the Gal4 protein in 10 combination with the SV40 promoter.
The first activation unit comprises the cDNA for the DNA binding domains of the Gal4 protein (amino acids 1-147), whose 3' end is linked to the 5' end of the cDNA for the Ga180 protein (amino acids 1-435).
The second activation subunit comprises the cDNA for the Ga180 binding domain of Gal4 (amino acids 851-881), whose 3' end is linked to the 5' end of the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 126-132), whose 3' end is linked to the 5' end of the cDNA for the transactivation domain of the VP16 of HSV-1 coding for the amino acids 406-488.
A further example of two activator subunits which activate the activator-responsive promoter consisting of the binding sequence for the Gal4 protein and the SV40 promoter is - a first activating unit which comprises the cDNA for the cytoplasmic domain of the CD4 T-cell antigen (amino acids 397-435), whose 5' end is linked to the 3' end of the cDNA for the transactivation domain of the VP16 from HSV-1 (amino acids 406-488), whose 5' end is in tum linked to the 3' end of the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 16-132) and - the second activation unit comprising the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 126-132), the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), whose 3' end is linked to the 5' end of the cDNA for the CD4 binding sequence of the p56 Ick protein (amino acids 1-71 ).
5) Examples for illustration of the subject of the invention The following examples describe how the genetic marking of CD14' cells for endothelial cells could appear.
a) Isolation of D14' cells Peripheral, mononuclear blood cells PBMC are isolated from healthy donors with the aid of Ficoll density gradient centrifugation according to the manufacturer (Ficoll-Paque, Pharmacia).
The PBMCs obtained are washed twice in cold PBS and incubated at 4°C
for 15 min with anti-CD14 antibody-coupled magnetic beads (CD14 Micro Beads, Miltneyi Biotec), then washed with cold PBS and suspended in PBS
which contains 0.002% EDTA and 1 % human serum albumin.
The detachment of the CD14' cells from the column is effected with the aid of the Vario MACS separation kit (Miltenyi Biotec) according to the instructions of the manufacturer. The purity of the CD14+ cells prepared in this way is in the range between 70% and 95%.
b) Construction of the plasmid The following DNA sequence is prepared in the reading direction from 5' to 3' using the method known to the person skilled in the art.
- the DNA binding sequence of 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 the 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 acid 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 Figure 1:
This nucleic acid sequence (Figure 1 ) is cloned into a pxP2 plasmid vector (Norden, BioTechniques 6, 454 (1988)).
The respective components of the nucleic acid constructs are connected to one another by means of suitable restriction sites which are introduced onto the ends of the different components with the aid of PCR amplification.
The combination of the components is effected with the aid of restriction-specific enzymes and with the aid of DNA ligases.
c) Transfection of CD14' cells The CD14' cells are adjusted to a concentration of 1 xl0~lml of culture medium [medium 199 with 20°~ fetal calf serum and penicillin/streptomycin/amphotericin (Gibco-BRL)j, inoculated into 60 mm culture dishes and incubated at 37°C for 10 min with a complex of the plasmid shown in b) (coding for VEGF receptor) and Supertect (Quiagen, Diisseldort).
The complex is prepared according to the details of the manufacturer of Supertect (Quiagen).
The success of the transaction is determined by detection of the VEGF
receptor II (DR) m-RNA with the aid of RT-PCR. The RT-PCR is carried out as described by Sewing et al., J. Cell Sci. 104, 545 (1993).
d) Culturing of the VEGF receptor II (KDR~-expressing CD14 cells in vitro VEGF receptor II {KDR)-expressing CD14 cells are adjusted to 1 x 106/ml and incubated for 7 days in the culture medium "EGM-2" (Biowhittaker), to which has been added 50 ng/mi of VEGF (Pepro Tech, London, England) and 10% fetal calf serum, 5% horse serum and 0.8 ~g/ml of hydrocortisone.
After 7 days, the cells are investigated immunocytochemically with the aid of specific antibodies (see Table 1 ) using the methodology of the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method known to the person skilled in the art. m-RNAs speck for CD14, CD31, CD34, CD36, CD144 and vWF are additionally detected with the aid of the RT-PCR
(Sewing et al., J. Cell Sci. 104, 545 (1993}).
The following results were achieved:
The majority of the cells present in the cell culture have already differentiated after 7 days into cells which exhibit the characteristic surtace markers of endothelial cells (see Table 2).
Table 1 PreferredCluster Antigen Antibody source cell desig-(company) specificitynation (CD) EC CD31 platelet endothelial DAKO
adhesion molecule (PECAM) CD34 sialomucin expressed Becton Dickinson on , hematopoietic progenitorDAKO
cells and vascular endothelium von Willebrand factor PharMingen, (vWF) DAKO
CD54 intercellular adhesion PharMingen molecule (ICAM-1 ) CD51/61 avl(i3 integrin complex PharMingen (vitronectin receptor}
CD62E E-selectin/ELAM-1 (endothelialPharMingen leukocyte adhesion molecule) CD105 endoglin PharMingen CD106 vascular cell adhesion PharMingen molecule (VCAM-1 ) CD144 vascular endothelial PharMingen (VE}-cadherin (cadherin 5) cells of the receptor for LPS-LPS-BP Becton Dickinson monocytic lineage HLA-DR MHC class VI molecule Immunotech CD36 receptor for thrombospondin PharMingen and collagen CD64 Fcy receptor DAKO
CD68 oxidi2ed LDL receptor DAKO
PreferredCluster Antigen Antibody source cell desig-(~mPany) specificitynation (CD) dendriticCD1a putative antigen presentingDAKO
cells molecule structurally related to MHC-class I
CD80 B7-1 PharMingen (T cell costimulatory molecule) CD83 40-45 KD giycoprotein Serotec CDBfi B7-2 PharMingen cell costimulatory molecule) leukocytesCD45 galectin-1 receptor Becton Dickinson CD13 metallproteinase DAKO
CD33 sialoadhesin (function DAKO
unknown) Table 2 Surface marker Day 0 Day 7 EC marker CD31 (PECAM-1 ) + +
CD34 (sialomucin) - {+) CD54 {ICAM-1 ) (+) +
CD36 (TSP receptor) + +
CD51/61 (av/~3 integrin)- +
CD105 (endoglin) (+) +
VWF _ EC-specific KDR _ (+) Tie-2 (AP receptor) - (+) CD144 (V-cadherin) - +
M~'d marker CD14 (LPS receptor) +
CD64 (Fcy receptor) + +
CD68 (OxLDL receptor)+ +
H l~1-D R {+) +
DC marker CD1a {IgSF) - _ CD86 (B7-2) + +
a~xcs zlariac l 1 ) 0a11iML IlfT0a~1TI011 s li) 11??LICJWT:
tA) ~_ ~~t Xarion Rourrsl Dsutschlaad GsDB
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tiv) t~) raDrmc ssas: rairx (9) O0~1p0?=Rs IaH PC Qospatibls tC1 OPER7ITIlfO axaTalls tC-D0a/lla-DOS
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gene, of the cyclin A gene, of the cdc2 gene, of the B-myb gene, of the DHFR gene, of the E2F-1 gene or else binding sequences for transcription factors occurring or activated during cell proliferation. These binding sequences include, for example, binding sequences for c-myc proteins.
Among these binding sequences are counted monomers or multimers of the nucleotide sequence designated as Myc E-box (5'-GGAAGCAGACCACGTGGTCTGCTTCC-3' (SEQ ID NO.: 1 ); Blackwood and Eisenmann, Science 251, 1211 (1991).
4.4) Self-potentiating andlor pharmacologically controllable promoters In the simplest case, a promoter can be inducible in the combination of identical or different promoters, for example in the form of a promoter which can be activated or switched off by tetracycline in the form of the tetracycline operator in combination with an appropriate repressor.
According to the invention, the promoter, however, can also be self potentiating with or also without a pharmacologically controllable promoter unit.
Self-potentiating andlor pharmacologically controllable promoters of this type have already been described in the Patent Application EP A 0 848 061, to which reference is expressly made.
4.5) Endothelial cell-specifically activatable promoters These include promoters or activator sequences from promoters or enhancers of those genes which code for proteins preferably formed in endothelial cells.
Within the meaning of the invention, promoters of the genes for the following proteins, for example, are to be used:
- brain-specific, endothelial glucose-1 transporter - endogiin - VEGF receptor-1 (flt-1 ) - VEGF receptor-2 (flt-1, KDR) - VEGF receptor-3 (flt-3) - tie-1 or tie-2 - B61 receptor (Eck receptor) 5 - endothelia, especially endothelia B or endothelia-1 - endothelia receptors, in particular the endothelia B receptor - mannose-6 phosphate receptors - von Willebrand factor - IL-1a, IL-1(i 10 - 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 alternatives to natural endothelial cell-specific promoters, synthetic activator sequences can also be used which consist of oligomerized binding sites for transcription factors which are preferentially or selectively active in endothelial cells. An example of this is the transcription factor GATA-2, whose binding site in the endothelia-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) Glia cell-spec~cally activatable promoters Promoters and activator sequences which are activated in glia cells are, for example, gene-regulatory sequences from genes which code, for example, for the following proteins: the Schwann cell-specific protein periaxin, glutamine synthetase, the glia cell-specific protein (glial fibrillary acid protein = GFAP), the glia cell protein S100, IL-6, CNTF, 5-HAT receptors, TNFa, IL-10, insulin-like growth factor receptor I and il or VEGF.
4.7) Synovtal cell-specifically activatable promoters Promoter and activator sequences which are activated in synovial cells are, for example, the promoter sequences of genes coding for matrix metalloproteinases (MMP), such as, for example, MMP-1 (interstitial collagenase), MMP-3 (stromelysinltransin) ar tissue inhibitors of metalloproteinases (TIMP), such as TIMP-1, -2, -3.
4.8 Lymphocyte- and/or macrophage-specifically activatable promoters Promoters and activator sequences which are activated in lymphocytes andlor macrophages are, for example, the promoter and activator sequences of the genes coding for cytokines, cytokine receptors and adhesion molecules and receptors for the Fc fragments of antibodies such as, for example, IL-receptor, IL-1a, IL-1~, IL-2, IL-2 receptor, IL-3, IL-3 receptor (a-subunit), IL-3 receptor (~i-subunit), IL-4, IL-4 receptor, IL-5, IL-6, IL-6 receptor, interferon regulatory factor 1 (IRF-1 ), the promoter of IRF-1 being activated to the same extent by IL-6 as by IFNY or IFN~i, IFNy responsive promoter IL-7, IL-8, IL-10, IL-11, IFNY, GM-CSF, GM-CSF
receptor (a-chain), IL-13, LIF, macrophage colony stirnulating factor (M-CSF) receptor, type I and type II macrophage scavenger receptors, MAC-1 (leukocyte function antigen), LFA-1a (leukocyte function antigen) or p150,95 (leukocyte function antigen).
4.9) Combination of identical or different promoters The combination of identical promoters is effected, for example, by linkage of a number of promoters one after the other in the reading direction from 5' to 3' of the nucleotide sequence.
For the combination of identical or different promoters, however, technologies are preferentially employed which have already been described in detail in the Patent Applications WO 96/06943;
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. Reference is expressly made to these patent applications in the context of this invention. Examples of technologies of this type are:
4.10) Chimeric promoters A chimeric promoter is the combination of a cell-specifically, metabolically or virus-specifically activatable activator sequence located upstream with a promoter module located downstream, which contains the nucleotide sequence CDE-CHR or E2FBS-CHR to which suppressive proteins bind, which as a result can inhibit the activation of the activator sequence located upstream in the Gp and G~ phase of the cell cycle (Vl/0 96/06943, Lucibello et al., EMBO J. 12 (1994)).
Continuing investigations on the functioning, in particular of the promoter element CDE-CHR, showed that the cell cycle-dependent regulation by the CDE-CHR element of an activator sequence located upstream is largely dependent on whether the activation sequence of transcription factors is activated with glutamine-rich activation domains (Zwicker et al., Nucl. Acids Res. 3822 (1995)).
Transcription factors of this type include, for example, Sp1 and NF-Y.
This consequently restricts the use of the promoter element CDE-CHR for chimeric promoters. The same is to be assumed for the promoter element E2F-8S-CHB of the B-myb gene (Zwicker et al., Nucl. Acids Res. 3822 (1995)).
4.11 ) Hybrid promoters The hybrid promoters have already been described in the Patent Application EP A 0 848 063. For the combination of the endothelial cell-specific promoter with at least ane further promoter, a gene construct, for example, is selected which contains all the following components:
The nucleotide sequence of the endothelial cell-specific promoter in a form in which at least one binding site for a transcription factor is mutated. By means of this mutation, the initiation of the transcription of the effector gene is blocked.
A transgene which codes as an effecior gene for an active compound.
At least one promoter or enhancer sequence which is activatable non-specifically, cell-specifically, virus-specifically, by tetracycline andlor cell cycle-specifically, which activates the transcription of at least one gene for at least one transcription factor which is mutated such that it can bind to the mutated binding sites) in the endothelial cell-specific promoter and activates this.
In an exemplary embodiment of this invention, the mutation in the promoter sequence can be, for example, a mutation of the TATA box of the cdc25B
promoter.
The mutation of the TATA can be, for example, TGTATAA. By means of this mutation, the DNA binding site of the normal TATA box-binding protein (TBP) is no longer recognized and the effector gene is no longer efficiently transcribed. Accordingly, the nucleic acid sequence which codes for the TBP must have a comutation. By means of this comutation, the TBP binds to the mutated TATA box (e.g. to TGTATAA) and thus leads to the efficient transcription of the effector gene. Comutations of the TBP gene of this type have been described, for example, by Strvbin and Stnuhl (Cell, 721 (1992) and by Heard et al. (EMBO J., 3519 (1993)).
4.12) Multiple promoters in combination with a nuclear retention signal and a nuclear export factor This technology has already been described in detail in the Patent Application EP A 0 805 209. Reference is made to this patent application.
According to the invention, a promoter of this type contains the following components:
- A first endothelial cell-specific, activatable promoter or enhancer sequence, which activates the basal transcription of a transgene.
- A transgene which codes as an effector gene for an active compound.
A nuclear retention signal (NRS) whose cDNA is linked indirectly or directly at the 5' end to the 3' end of the structural gene (b).
- Preferentially, the transcription product of the nuclear retention signal has a binding structure for a nuclear export factor.
- A further nonspecific, cell-specific, virus-specific, metabolically and/or cell cycle-specifically activatable promoter or enhancer sequence which activates the basal transcription of a nuclear export factor.
- A nucleic acid coding for a nuGear export factor (NEF) which binds to the transcription product of the nuclear retention signal and thereby mediates the transport of the transcription product of the transgene from the cell nucleus.
Preferably, the gene coding for the nuclear retention signal is selected from the group comprising the Rev responsive element (RRE) of HIV-1 or HIV-2, the RRE-equivalent retention signal of retrovirusss or the RRE-equivalent retention signal of the HBV.
The nuclear export factor is preferentially a gene selected from the group comprising the Rev gene of the viruses HIV-1, HIV-2, Visna-Maidi virus, caprine arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, of retroviruses, of HTLV or the gene of the hnRNP-A1 protein or the gene of the transcription factor TFIII-A.
4.13) Activator-responsive promoter unit Activator-responsive promoter units have already been described in detail in the Patent Application EP A 0 805 209. Reference is made to this patent application.
An activator-responsive promoter unit consists of the following components:
- one or more identical or different promoter or enhancer sequences which is or are activatable, for example, cell cycle-specifically, cell proliferation-dependently, metabolically, endothelial cell-spec~cally or virus-specifically or both cell cycle-specifically and metabolically, endothelial cell-specfically or virus-specifically (so-called chimeric promoters) - one or more identical or different activator subunits which are in each case located downstream of the promoter or enhancer sequences and is or are activated by this in their basal transcription - an activator responsive promoter which is activated by the expression products of one or more activator subunits.
In a preferred embodiment, activator-responsive promoter units according to the invention can be binding sequences for chimeric transcription factors from DNA-binding domains, protein-protein interaction domains and transactivation domains. All transcription factor binding sites mentioned in the application can be present singly (monomers) or in a number of copies (multimers, for example up to 10 copies).
An example of an activator-responsive promoter activated by two activator subunits is the LexA operator in combination with the SV40 promoter. The first activator subunit includes the cDNA for the LexA-DNA binding protein coding for the amino acids 1-81 or 1-202, whose 3' end is linked to the 5' end of the cDNA for the Ga180 protein (amino acids 1-435).
The second activator subunit comprises the cDNA of the Ga180 binding domain of the Gal4 protein coding for the amino acids 851-881, whose 3' end is linked to the 5' end of the cDNA of the SV40 large T antigen coding for the amino acids 126-132, whose 3' end is linked to the 5' end of the 5 cDNA of the transactivation domain of the VP16 of HSV-1 coding for the amino acids 406-488.
A further example of an activator-responsive promoter activated by two activator subunits is the binding sequence for the Gal4 protein in 10 combination with the SV40 promoter.
The first activation unit comprises the cDNA for the DNA binding domains of the Gal4 protein (amino acids 1-147), whose 3' end is linked to the 5' end of the cDNA for the Ga180 protein (amino acids 1-435).
The second activation subunit comprises the cDNA for the Ga180 binding domain of Gal4 (amino acids 851-881), whose 3' end is linked to the 5' end of the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 126-132), whose 3' end is linked to the 5' end of the cDNA for the transactivation domain of the VP16 of HSV-1 coding for the amino acids 406-488.
A further example of two activator subunits which activate the activator-responsive promoter consisting of the binding sequence for the Gal4 protein and the SV40 promoter is - a first activating unit which comprises the cDNA for the cytoplasmic domain of the CD4 T-cell antigen (amino acids 397-435), whose 5' end is linked to the 3' end of the cDNA for the transactivation domain of the VP16 from HSV-1 (amino acids 406-488), whose 5' end is in tum linked to the 3' end of the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 16-132) and - the second activation unit comprising the cDNA of the nuclear localization signal of SV40 (SV40 large T; amino acids 126-132), the cDNA for the DNA binding domain of the Gal4 protein (amino acids 1-147), whose 3' end is linked to the 5' end of the cDNA for the CD4 binding sequence of the p56 Ick protein (amino acids 1-71 ).
5) Examples for illustration of the subject of the invention The following examples describe how the genetic marking of CD14' cells for endothelial cells could appear.
a) Isolation of D14' cells Peripheral, mononuclear blood cells PBMC are isolated from healthy donors with the aid of Ficoll density gradient centrifugation according to the manufacturer (Ficoll-Paque, Pharmacia).
The PBMCs obtained are washed twice in cold PBS and incubated at 4°C
for 15 min with anti-CD14 antibody-coupled magnetic beads (CD14 Micro Beads, Miltneyi Biotec), then washed with cold PBS and suspended in PBS
which contains 0.002% EDTA and 1 % human serum albumin.
The detachment of the CD14' cells from the column is effected with the aid of the Vario MACS separation kit (Miltenyi Biotec) according to the instructions of the manufacturer. The purity of the CD14+ cells prepared in this way is in the range between 70% and 95%.
b) Construction of the plasmid The following DNA sequence is prepared in the reading direction from 5' to 3' using the method known to the person skilled in the art.
- the DNA binding sequence of 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 the 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 acid 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 Figure 1:
This nucleic acid sequence (Figure 1 ) is cloned into a pxP2 plasmid vector (Norden, BioTechniques 6, 454 (1988)).
The respective components of the nucleic acid constructs are connected to one another by means of suitable restriction sites which are introduced onto the ends of the different components with the aid of PCR amplification.
The combination of the components is effected with the aid of restriction-specific enzymes and with the aid of DNA ligases.
c) Transfection of CD14' cells The CD14' cells are adjusted to a concentration of 1 xl0~lml of culture medium [medium 199 with 20°~ fetal calf serum and penicillin/streptomycin/amphotericin (Gibco-BRL)j, inoculated into 60 mm culture dishes and incubated at 37°C for 10 min with a complex of the plasmid shown in b) (coding for VEGF receptor) and Supertect (Quiagen, Diisseldort).
The complex is prepared according to the details of the manufacturer of Supertect (Quiagen).
The success of the transaction is determined by detection of the VEGF
receptor II (DR) m-RNA with the aid of RT-PCR. The RT-PCR is carried out as described by Sewing et al., J. Cell Sci. 104, 545 (1993).
d) Culturing of the VEGF receptor II (KDR~-expressing CD14 cells in vitro VEGF receptor II {KDR)-expressing CD14 cells are adjusted to 1 x 106/ml and incubated for 7 days in the culture medium "EGM-2" (Biowhittaker), to which has been added 50 ng/mi of VEGF (Pepro Tech, London, England) and 10% fetal calf serum, 5% horse serum and 0.8 ~g/ml of hydrocortisone.
After 7 days, the cells are investigated immunocytochemically with the aid of specific antibodies (see Table 1 ) using the methodology of the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method known to the person skilled in the art. m-RNAs speck for CD14, CD31, CD34, CD36, CD144 and vWF are additionally detected with the aid of the RT-PCR
(Sewing et al., J. Cell Sci. 104, 545 (1993}).
The following results were achieved:
The majority of the cells present in the cell culture have already differentiated after 7 days into cells which exhibit the characteristic surtace markers of endothelial cells (see Table 2).
Table 1 PreferredCluster Antigen Antibody source cell desig-(company) specificitynation (CD) EC CD31 platelet endothelial DAKO
adhesion molecule (PECAM) CD34 sialomucin expressed Becton Dickinson on , hematopoietic progenitorDAKO
cells and vascular endothelium von Willebrand factor PharMingen, (vWF) DAKO
CD54 intercellular adhesion PharMingen molecule (ICAM-1 ) CD51/61 avl(i3 integrin complex PharMingen (vitronectin receptor}
CD62E E-selectin/ELAM-1 (endothelialPharMingen leukocyte adhesion molecule) CD105 endoglin PharMingen CD106 vascular cell adhesion PharMingen molecule (VCAM-1 ) CD144 vascular endothelial PharMingen (VE}-cadherin (cadherin 5) cells of the receptor for LPS-LPS-BP Becton Dickinson monocytic lineage HLA-DR MHC class VI molecule Immunotech CD36 receptor for thrombospondin PharMingen and collagen CD64 Fcy receptor DAKO
CD68 oxidi2ed LDL receptor DAKO
PreferredCluster Antigen Antibody source cell desig-(~mPany) specificitynation (CD) dendriticCD1a putative antigen presentingDAKO
cells molecule structurally related to MHC-class I
CD80 B7-1 PharMingen (T cell costimulatory molecule) CD83 40-45 KD giycoprotein Serotec CDBfi B7-2 PharMingen cell costimulatory molecule) leukocytesCD45 galectin-1 receptor Becton Dickinson CD13 metallproteinase DAKO
CD33 sialoadhesin (function DAKO
unknown) Table 2 Surface marker Day 0 Day 7 EC marker CD31 (PECAM-1 ) + +
CD34 (sialomucin) - {+) CD54 {ICAM-1 ) (+) +
CD36 (TSP receptor) + +
CD51/61 (av/~3 integrin)- +
CD105 (endoglin) (+) +
VWF _ EC-specific KDR _ (+) Tie-2 (AP receptor) - (+) CD144 (V-cadherin) - +
M~'d marker CD14 (LPS receptor) +
CD64 (Fcy receptor) + +
CD68 (OxLDL receptor)+ +
H l~1-D R {+) +
DC marker CD1a {IgSF) - _ CD86 (B7-2) + +
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Claims (16)
1. A 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.
(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 process as claimed in claim 1, wherein the cells from step (b) integrate into a tissue group after return to the human body.
3. The process as claimed in one of claims 1 and 2, wherein in step (b) the gene for a growth and/or differentiation factor and/or for a receptor of a growth and/or differentiation factor is transfected as part of a nucleic acid construct which, under the control of a promoter, can express both the gene for the growth and/or differentiation factor and/or for the receptor mentioned, and prophylactically or therapeutically active proteins or enzymes for the activation of pharmaceutical precursors.
4. The process as claimed in one of claims 1 to 2, wherein the promoter is activatabte cell type-specifically, cell cycle-specifically, metabolically or pharmacologically.
5. The process as claimed in one of claims 1 to 4, wherein the cells from step (a) are selected from the group comprising mononuclear cells from blood, veins, capillaries, arteries, the umbilical cord, the placenta, suspensions of cells from bone, spleen, lymph nodes, lymph and connective tissue fluid, peritoneal cell suspensions, pleural cell suspensions, endothelial cells and fibroblasts.
6. The process as claimed in claim 5, wherein the mononuclear cells contain surface markers selected from the group consisting of CD34, CD11, CD13, CD14 and CD68.
7. The process as claimed in claim 6, wherein the desired differentiation corresponds to that of an endothelial cell and the gene for the 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 (FGF.alpha., FGF.beta.), epidermal growth factor (EGF), insulin-like growth factor (IGF-1, IGF-2), .beta.-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 y (IFN.gamma.), oncostatin M, B61, platelet derived endothelial cell growth factor (PDEGF), stem cell factor (SCF), transforming growth factor .beta. (TGF-.beta.), angiogenin, pleiotrophin, Flt-3 ligand (FL), Tie-2 ligands (angiopoietin-1, angiopoietin-2), stromal derived factor-1 (SDF-1) and TNF.alpha.; and the gene for the receptor of the growth and/or differentiation factor is selected from the group comprising the 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, the SCF receptor, TGF.beta. receptor, Tie-2 receptor, SDF-1 receptor, pleiotrophin receptor, EGF
receptor, TNF.alpha. receptor and/or SDF-1 receptor and PDGF receptor (.alpha.
and .beta.).
receptor, TNF.alpha. receptor and/or SDF-1 receptor and PDGF receptor (.alpha.
and .beta.).
8. The process as claimed in claim 6, wherein the desired differentiation corresponds to that of an osteoblast and the gene for a growth and/or differentiation factor is selected from a group comprising BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8, pleiotrophin and midkine; and the gene for the receptor for the growth and/or differentiation factor is selected from the group comprising the receptors for BMP 1-8, PTN and midkine.
9. The process as claimed in claim 6, wherein the desired differentiation corresponds to that of a glia cell and the gene for a growth and/or differentiation factor is selected from a group comprising the 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 comprising the GGF receptor, NGF receptor, CNF receptor, BdNF receptor, NT-4 receptor and the GDNF receptor.
10. The process as claimed in claim 6, wherein the desired differentiation corresponds to that of a synovial cell and the gene for a growth and/or differentiation factor is selected from a group comprising transforming growth factor (TGF) .beta.-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 the gene for the receptor of the growth and/or differentiation factor is selected from the group comprising TGF-.beta. receptors, IL-10 receptors, IGF-1 receptors, IL-4 receptors and bFGF receptors.
11. The process as claimed in claim 6, wherein the desired differentiation corresponds to that of an antiinflammatory cell and the gene for a growth and/or differentiation factor is selected from the group comprising cytokines, interferons (IFN.alpha., IFN.beta., IFN.gamma.), interleukin-4 (IL-4), interleukin-6 (IL-6), interieukin-9 (IL-9), interleukin-13 (IL-13), LIF, oncostatin, interleukin-10 (IL-10), interleukin-12 (IL-12), TGF.beta., tumor necrosis factor .alpha. (TNF.alpha., TNF.beta. and the interleukin-1 receptor antagonist (IL-1RA); and the gene for the receptor of the growth and/or differentiation factor is selected from the group comprising 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, TNF.alpha., TNF.beta. receptor, TGF.beta.
receptor and receptors for IFN.alpha., -.beta., -.gamma..
receptor and receptors for IFN.alpha., -.beta., -.gamma..
12. The process as claimed in claim 6, wherein the desired differentiation corresponds to that of a cell involved in inflammation and the gene for a growth and/or differentiation factor is selected from a group comprising interleukin-1, interleukin-2, interteukin-4, interleukin-5, interleukin-5, interleukin-6, LIF, interleukin-7, interieukin-8, interleukin-11, GM-CSF, M-CSF, G-CSF and IFN.alpha., -.beta., -.gamma.; and the gene for the receptor of the growth and/or differentiation factor is selected from the group comprising the IL-1 receptor, IL-2 receptor, IFN.alpha., -.beta., -.gamma.
receptor, IL-3 receptor, IL-5 receptor, IL-6 receptor, GM-CSF receptor, M-CSF receptor, integrin beta 2 proteins.
receptor, IL-3 receptor, IL-5 receptor, IL-6 receptor, GM-CSF receptor, M-CSF receptor, integrin beta 2 proteins.
13. The process as claimed in claim 6, wherein the cells from step (a) are CD14-positive, peripheral mononuclear blood cells (PBMC) which are transfected with a plasmid comprising the following elements in the 5'-3' direction:
- the DNA binding sequence of LexA
(nucleotide sequence 5'-TACTGTATGTACATACAGTA-3') - the coding sequence for the von Willebrand factor (vWF) promoter (nucleotide sequence -487 to +247) - DNA coding for the VEGF receptor II (KDR) - the vWF promoter - the coding sequence for the binding domain of LexA (amino acid 1-81);
- the coding sequence for the transactivation domain of HSV-1 VP16 (amino acid 406 to 488);
- the polyadenylation signal of SV40.
- the DNA binding sequence of LexA
(nucleotide sequence 5'-TACTGTATGTACATACAGTA-3') - the coding sequence for the von Willebrand factor (vWF) promoter (nucleotide sequence -487 to +247) - DNA coding for the VEGF receptor II (KDR) - the vWF promoter - the coding sequence for the binding domain of LexA (amino acid 1-81);
- the coding sequence for the transactivation domain of HSV-1 VP16 (amino acid 406 to 488);
- the polyadenylation signal of SV40.
14. A cell, obtainable by a process as claimed in one of claims 1 to 13.
15. The cell as claimed in claim 14 for use in gene therapy.
16. The use of a cell as claimed in claim 14 for the production of a therapeutic in endothelial cell defects, for the promotion of angiogenesis, for bone healing, for the promotion of the healing of damage to the CNS, for the prophylaxis and therapy of joint damage, inflammations, autoimmune disorders, organ rejections and for assisting inflammatory and rejection reactions in infections or oncoses.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19850986.3 | 1998-11-05 | ||
| DE19850986A DE19850986A1 (en) | 1998-11-05 | 1998-11-05 | The genetic engineering of cells and their use for the prophylaxis and therapy of diseases |
| PCT/EP1999/007902 WO2000028010A2 (en) | 1998-11-05 | 1999-10-19 | The genetic determination of genes and its use for the prophylaxis and therapy of diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2349497A1 true CA2349497A1 (en) | 2000-05-18 |
Family
ID=7886784
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002349497A Abandoned CA2349497A1 (en) | 1998-11-05 | 1999-10-19 | The genetic determination of genes and its use for the prophylaxis and therapy of diseases |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1127109A2 (en) |
| JP (1) | JP2002529080A (en) |
| AU (1) | AU1040700A (en) |
| CA (1) | CA2349497A1 (en) |
| DE (1) | DE19850986A1 (en) |
| WO (1) | WO2000028010A2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0015761D0 (en) * | 2000-06-27 | 2000-08-16 | Univ Bristol | Polypeptide |
| US6962982B2 (en) | 2001-06-22 | 2005-11-08 | Roche Diagnostics Corporation | Soluble complexes of target proteins and peptidyl prolyl isomerase chaperones and methods of making and using them |
| DE10158680B4 (en) * | 2001-11-30 | 2004-04-08 | Universitätsklinikum Hamburg-Eppendorf | Process for ex vivo expansion and differentiation of multipotent stem cells |
| FR3018819A1 (en) * | 2014-03-19 | 2015-09-25 | Univ Bourgogne | TREATMENT OF THE INFLAMMATORY AND DYSIMMUNITARY RESPONSE |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5980887A (en) * | 1996-11-08 | 1999-11-09 | St. Elizabeth's Medical Center Of Boston | Methods for enhancing angiogenesis with endothelial progenitor cells |
| WO1998039427A2 (en) * | 1997-03-06 | 1998-09-11 | University Of Massachusetts | Gene therapy using bone marrow transplants transfected with therapeutic genes under the control of tissue-specific promoters |
-
1998
- 1998-11-05 DE DE19850986A patent/DE19850986A1/en not_active Withdrawn
-
1999
- 1999-10-19 AU AU10407/00A patent/AU1040700A/en not_active Abandoned
- 1999-10-19 EP EP99953880A patent/EP1127109A2/en not_active Withdrawn
- 1999-10-19 JP JP2000581177A patent/JP2002529080A/en not_active Withdrawn
- 1999-10-19 CA CA002349497A patent/CA2349497A1/en not_active Abandoned
- 1999-10-19 WO PCT/EP1999/007902 patent/WO2000028010A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| DE19850986A1 (en) | 2000-05-25 |
| WO2000028010A3 (en) | 2000-07-27 |
| WO2000028010A2 (en) | 2000-05-18 |
| EP1127109A2 (en) | 2001-08-29 |
| AU1040700A (en) | 2000-05-29 |
| JP2002529080A (en) | 2002-09-10 |
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