WO1997030083A1 - Therapie genique de cellules endotheliales avec des proteines anti-apoptose pour les pathologies liees aux greffes et aux etats inflammatoires - Google Patents

Therapie genique de cellules endotheliales avec des proteines anti-apoptose pour les pathologies liees aux greffes et aux etats inflammatoires Download PDF

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WO1997030083A1
WO1997030083A1 PCT/EP1997/000676 EP9700676W WO9730083A1 WO 1997030083 A1 WO1997030083 A1 WO 1997030083A1 EP 9700676 W EP9700676 W EP 9700676W WO 9730083 A1 WO9730083 A1 WO 9730083A1
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cells
protein
bcl
cell
polypeptide
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PCT/EP1997/000676
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Fritz H. Bach
Christiane Ferran
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Novartis Ag
New England Deaconess Hospital Corporation
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Priority to JP09528990A priority Critical patent/JP2000510326A/ja
Priority to EP97905019A priority patent/EP0886650A1/fr
Priority to AU18730/97A priority patent/AU1873097A/en
Publication of WO1997030083A1 publication Critical patent/WO1997030083A1/fr

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    • C12N15/09—Recombinant DNA-technology
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/10—Mammal
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the invention relates to the field of anti-apoptotic gene therapy for transplantation and inflammatory conditions. It provides improvements in the field of gene therapy and tissue and organ transplantation. In its broad aspect, it relates to methods of treating cellular activation processes. In particular, it is concerned with genetic modification of endothelial cells to render them less susceptible to an inflammatory, immunological, or other activating stimulus.
  • the invention is specifically directed to genetic modification of a cell, in particular an endothelial cell, to render it capable of expressing a polypeptide capable of inhibiting cellular apoptosis. and to recombinant vectors therefor.
  • polypeptides capable of inhibiting apoptosis in mammalian cells include polypeptides having activity of a mammalian A20 protein, as well as. more generally, polypeptides having anti-apoptotic activity, in particular certain proteins of the BCL family.
  • the invention also concerns the resultant genetically modified cells, or tissues or organs comprising these cells; and non-human iransgenic or somatic recombinant animals so modified.
  • the invention is most particularly directed to transplantation of genetically modified cells, or graftable tissues or organs comprising such cells, into a mammalian recipient.
  • the mammalian recipient may be allogeneic or xenogeneic as to the cells.
  • va ⁇ ous means including the use of immune suppressants, as well as donor organs that express factors which inhibit the complement system of the recipient (Dalmasso, A.P., Immunopharmacologv 24 (2) [1992] 149-160).
  • endothelial cell activation refers to a continuum of changes characterizing endothelial ceils which are subjected to a stimulus such as a cytotoxic cytokine
  • TNF tumor necrosis factor
  • the endothelium (also referred to as the ' ascular endothelium”) consists of a layer of cells that line the cavities of the heart and of the blood and lymph vessels
  • the initial cellular response of such cells to an activating stimulus typically involves changes in the cell phe ⁇ otype, such as retraction of cells from one another, hemorrhage and edema, and trans-migration of leukocytes across the endo
  • a still further phase of cellular activation involves transc ⁇ ptional up-regulation of va ⁇ ous genes encoding interleukins. adhesion molecules, and procoagulant, prothrombotic components of the coagulation system
  • E-selectin is a tissue specific molecule which is expressed exclusively by endothelial cells (EC) upon activation, and therefore is a generally accepted indicator of Type II EC activation (Pober, J.S. and Cotran, R.S., Transplantation $Q 1 19901 537-544)
  • Apoptosis can be considered as preprogrammed cell death seen in the process of development, differentiation, or turnover of tissues (Wyllie, A. H. et al., Int. Rev. Cvtol.
  • apoptosis occurs when a cell activates an internally encoded suicide program as a result of either extrinsic or intrinsic signals. Morpho ⁇ logically, apoptosis is characterized by loss of contact with neighboring cells, concentration of cytoplasm, endonuclease activity-associated chromatin condensation and pyknosis, and segmentation of the nucleus, among others. Disappearance of microvilli from the cell surface and vesicle formation on the cell surface (membrane blebbing) are also observed. The remaining fragments of apoptotic body cells are ultimately phagocytosed by neighboring cells (Duvall, E. and Wyllie, A.
  • NF-i B An identified transcription factor for many of the genes susceptible to transc ⁇ ptional up-regulation in response to an activation stimulus such as TNF ⁇ , is "Nuclear Factor B". i.e. NF-i B (M. Grilli et al.. International Review of Cytology 143 [ 1993] 1 -61 ) NF- B exists as a preformed transcription factor in the cytoplasm of cells, which is inactivated by its association with a protein inhibitor of the IKB family. On exposure to cellular activating stimuli such as lipopolysaccharide (LPS), TNF, or oxygen radicals, the I B protein is rapidly phosphorylated and then degraded, thereby liberating the preformed NF- B and allowing its transmigration to the nucleus.
  • LPS lipopolysaccharide
  • TNF oxygen radicals
  • NF- ⁇ B In the nucleus, the binding of NF- ⁇ B to certain NF- B binding sites (also referred to as "KB elements") in promoter regions of the nuclear DNA initiates transcription of genes directly or indirectly under the control of said promoters.
  • Genes subject to up-regulation by NF- B upon stimulation of the cell with TNF include E-selectin, IL-8, and tissue factor, among others (F.H. Bach et al., Immunological Reviews Hi [1994] 1 -30; T. Collins, Lab. Invest. 6j£ [ 1993] 499-508; M.A. Read et al., J. Exp. Med. 122 [1994] 503-512).
  • the A20 gene is found to be inducible by TNF or other cellular activating factors (A.W.Opipari et al., J. Biol. Chem. 265 [ 1990] 14705-14708; C.D.Laherty et al.. J.Biol.Chem. 26£ [1993] 5032-5039).
  • TNF TNF or other cellular activating factors
  • A20 belongs to a sub-set of TNF-inducible genes which assist in ultimately conferring resistance to TNF-induced apoptosis (M. Tewari et al., J. Immunol. 154 [1995] 1699-1706; A.W. Opipari et al., J. Biol. Chem.
  • BCL-2 proteins of the BCL family of proteins also exert an anti-apoptotic effect.
  • BCL-2 proteins of the BCL family of proteins
  • BCL-X L proteins of the BCL
  • MCL-1 proteins of the BCL family of proteins
  • Al proteins of the BCL
  • NF- ⁇ B regulation of gene transcription is related to expression of an apoptosis inhibiting (i.e. "anti-apoptotic") protein. More particularly, it has been found that such a protein can exert a negative feedback control on NF- B-mediated gene transcription, namely, the anti-apoptotic protein functions as an inhibitor of the NF- ⁇ B transcription factor. This observed negative feedback effect may perhaps in certain cases be exerted via an anti-oxidative mechanism that directly or indirectly protects the NF- ⁇ B-I ⁇ B complex from dissociating, apparently by acting upstream of I ⁇ B degradation.
  • Such inhibitory function may normally assist in preventing apoptotic cell death.
  • expression of the anti-apoptotic protein in a cell may be at insufficient levels, or delayed relative to the rapid activation of NF-v B in the cell, so that inhibition of NF- ⁇ B is rendered ineffective to prevent cellular activation and apoptosis.
  • the method and other aspects of the invention may be used to treat inflammation or disease states associated with inflammation, e.g., septic shock, chronic rejection, xenograft rejection, atherosclerosis (restenosis), vasculitis, cardiac failure, or autoimmune diseases.
  • inflammation or disease states associated with inflammation e.g., septic shock, chronic rejection, xenograft rejection, atherosclerosis (restenosis), vasculitis, cardiac failure, or autoimmune diseases.
  • the invention relies on gene therapy techniques, utilizing an anti-apoptotic gene and its expressed product to inhibit NF- ⁇ B activation in mammalian cells susceptible to an activating stimulus.
  • the invention provides a mammalian cell (in particular, an endothelial cell) which is genetically modified to express an anti-apoptotic protein which is capable of substantially inhibiting NF- ⁇ B activation in the presence of a cellular activating stimulus.
  • a mammalian cell in particular, an endothelial cell
  • an anti-apoptotic protein which is capable of substantially inhibiting NF- ⁇ B activation in the presence of a cellular activating stimulus.
  • a "cellular activating stimulus” is tumor necrosis factor. TNF (i.e. TNF ⁇ ).
  • NF- ⁇ B activation is meant NF- B-mediated up-regulation of genes which are directly or indirectly under the control of an NF- ⁇ B binding site, such as, e.g., E-selectin in endothelial cells.
  • NF- ⁇ B activation constitutes the binding of NF- ⁇ B to KB regulatory sequences in the DNA of a cell in a manner sufficient (whether alone or in combination with other factors) to initiate transcription of a gene in operative association with said sequences.
  • NF- "B inhibition” is meant that NF- ⁇ B binding to NF- ⁇ B binding sites in the nuclear DNA is prevented.
  • NF-KB is considered “substantially inhibited” when, for evampic.
  • transcription of the E-selectin gene by an endothelial cell genetically modified according to the invention and stimulated with TNF ⁇ is reduced by 60% or greater, and preferably 8 c /r or greater, and even 90% or greater, e.g., 95% and even 99% or greater, relative to an unmodifed cell (i.e. a cell not subject to genetic manipulation according to the invention ) which is also stimulated by TNF ⁇ .
  • the invention in its broader aspects also concerns a method of genetically modifying mammalian (e.g.. endothelial) cells to render them less susceptible to an inflammatory or other immunological activation stimulus by inserting in these cells, or progenitors thereof.
  • DNA encoding an anti-apoptotic protein capable of inhibiting NF- ⁇ B and expressing the protein, whereby NF- B in the cell is substantially inhibited in the presence of a cellular activating stimulus.
  • NF-KB-initiated transcription by the anti-apoptotic protein such as, e.g., an A20 protein
  • the anti-apoptotic protein such as, e.g., an A20 protein
  • a genetically modified cell is unexpectedly potent, even at moderate levels of transfection in vitro with the corresponding A20 gene (e.g., 0.5 ⁇ g plasmid DNA per approximately 5 x 10 5 cells), leading to effective suppression of induction of cytokine-inducible genes such as tissue factor, E-selectin and IicB ⁇ , all of which are associated with inflammation.
  • Such a therapy will be useful in general to treat patients afflicted with conditions which may benefit from inhibition of NF-KB activation, such as inflammation. Such a therapy will also be useful to moderate complications occurring in connection with organ transplantation, especially where the graft recipient is human, and most particularly where the graft is xenogeneic as to the recipient.
  • the invention comprises a method of transplanting donor endothelial or other mammalian cells (e.g., bone marrow stem cells as precursors of monocytcs. NK cells, or lymphocytes; or islet cells), or graftable tissues or organs comprising such cells, to a mammalian recipient in whose blood or plasma these cells, tissues or organs arc subject to activation, which comprises: la I gcncticallv modifying the donor cells, or progenitor cells thereof, by inserting therein
  • donor endothelial or other mammalian cells e.g., bone marrow stem cells as precursors of monocytcs. NK cells, or lymphocytes; or islet cells
  • graftable tissues or organs comprising such cells
  • DNA encoding an anii-apoptotic protein capable of inhibiting NF- B and (b ) transplanting the resultant modified donor cells, or tissues or organs comprising these cells, into the recipient, and expressing in the cells the anti-apoptotic protein, whereby
  • NF- ⁇ B activ ation in the cells is substantially inhibited in the presence of a cellular activating stimulus.
  • modified donor cells of step (b) will be understood to refer to cells which themselves arc subjected to genetic modification in step (a), as well as to progeny thereof.
  • donor endothelial cells, and tissues and organs comprising such cells, wherein the ceils are genetically modified to rcgulably or constitutively express an anti-apoptotic protein in a graft recipient, whereby NF-t B is substantially inhibited, for transplantation into a recipient species.
  • the graft recipient may be allogeneic or xenogeneic as to the donor cells, tissues or organs.
  • the invention provides a non-human transgenic or somatic recombinant mammal comprising DNA encoding an anti-apoptotic protein of a different species; and a method of preparing such non-human iransgenic or somatic recombinant mammal.
  • vectors for genetically modifying cells by insertion of anti-apoptotic protein-encoding polynucleotides such as for example retroviral vectors, and especially, adenoviral vectors.
  • FIG. 1 Analysis of antibody affinity purified protein extracted from: BAEC transfected with A20 vector (“A20”), BAEC transfected with empty pAC vector (“PAC”), or non-transfected BAEC ("NT") following stimulation with TNF ⁇ . Also analyzed for comparison is HUVEC which is either non-stimulated (“NS”) or stimulated with TNF ⁇ (“TNF”).
  • FIG 2 Luciferase levels in relative light units (RLU) in BAEC co-transfected with A20 and/or pAC vector ("pAC") together with the porcine E-selectin promoter region cloned into a luciferase expressing vector ("porcine E-selectin Reporter”); BAEC are either non-stimulated (“NS” or “control”) or stimulated with TNF ⁇ (“TNF”) or lipopolysaccharide (“LPS”).
  • RLU relative light units
  • FIG. 3A-3C Luciferase levels in BAEC co-transfected with either A20 or pAC and one of the following promoters cloned into a luciferase vector: (a) human IL-8 promoter ("IL-8 Reporter") (FIG. 3A); (b) porcine I ⁇ B ⁇ promoter ("I ⁇ B ⁇ Reporter”) (FIG. 3B); and (c) porcine tissue factor (TF) promoter (“Tissue Factor Reporter”) (FIG. 3C): and then stimulated with TNF ⁇ or LPS or maintained as a control.
  • IL-8 Reporter human IL-8 promoter
  • I ⁇ B ⁇ Reporter porcine I ⁇ B ⁇ Reporter
  • TF tissue factor
  • FIG. 4 Luciferase levels in BAEC co-transfected with either A20 or pAC and KB elements derived from the porcine E-selectin promoter cloned into a luciferase vector ("NFKB Reporter"), and then stimulated with TNF ⁇ or LPS or maintained as a control.
  • NFKB Reporter a luciferase vector
  • FIG. 5A Luciferase levels in BAEC co-transfected with either A20 or pAC and an RSV-LTR driven luciferase vector ("RSV-LUC Reporter").
  • FIG. 5B "C-labeled chloramphenicol levels, in counts per minute (CPM), in BAEC co-transfected with A20 and/or pAC and an HIV LTR-driven CAT vector ("H1V-CAT Reporter"). Cells are stimulated with the viral c-Tat protein ("C-Tat") or maintained as a control.
  • Figures 6A. 6B. 6C Luciferase levels in BAEC co-transfected with pAC and either Bcl-2 or Bcl-X L , together with either the E-selectin reporter (FIG. 6a), the I ⁇ B ⁇ reporter (FIG. 6B), or the NF KB reporter (FIG. 6C) cloned into a luciferase vector, and then stimulated with TNF or LPS or maintained as a non-stimulated control.
  • FIG. 7 Luciferase levels in BAEC co-transfected with pAC, full length A20, or truncated A20 clones #3 ["tA20(3)”] or #7 ["tA20(7)”] ( together with the E-selectin reporter cloned into a luciferase vector, and then stimulated with TNF or LPS or maintained as a non-stimulated ("NT") control.
  • Figure 8 EMSA of nuclear extracts from TNF-stimulated (+) or non-stimulated (-) PAEC infected with adenoviral Bcl-2 ("rAd.BcI-2") or, as a control, ⁇ -gal (“rAd. ⁇ -Gal”), using a B binding oligonucleotide derived from the human immu ⁇ oglobulin (lg) K promoter and, for comparison, a cold wild-type NF ⁇ B-specific probe ("sp-comp.”) and a non-specific competitor (“nsp. comp.”)(AP-I ).
  • sp-comp. cold wild-type NF ⁇ B-specific probe
  • nsp. comp. non-specific competitor
  • Figure 9 Western blot of rAd.Bcl-2- (or, as a control, rAd. ⁇ -gal-) infected PAEC taken prior to ("0") , or ten minutes (" 10' ”) or one hundred-twenty minutes (“120' ”) following stimulation with TNF, with Iic ⁇ as shown.
  • Figure 10 EMSA of nuclear extracts from rAd.Bcl-2- (or, as a control, rAd. ⁇ -gal-) infected PAEC prior to ("-") or two hours following (+) TNF stimulation, using the transcription factor cAMP responsive element ("CRE") as a probe and, for comparison, a cold wild-type CRE-specific probe ("sp-comp.”) and a non-specific competitor ("nsp. comp.”).
  • CRE transcription factor cAMP responsive element
  • Figure 1 1 Luciferase levels in BAEC co-transfected with either Al or pAC and a luciferase vector comprising 0.7 ⁇ g of either the (A) E-selectin or (B) NFKB reporter. Cells are stimulated with TNF or LPS or non-stimulated (control).
  • Figure 12 Nothern blot TNF-stimulated (+) or non-stimulated (-) HUVEC infected with adenoviral I ⁇ B ⁇ ("rAd.I B" ⁇ ) or A20 (“rAd.A20”) or, as a control. rAd. ⁇ -gal . Definitions
  • “Graft,” “transplant” or “implant” are used interchangeably to refer to biological material derived from a donor for transplantation into a recipient, and to the act of placing such biological material in the recipient.
  • “Host or "recipient” refers to the body of the patient in whom donor biologicai material is grafted.
  • Allogeneic refers to the donor and recipient being of the same species (also “allograft”). As a subset thereof, “syngeneic” refers to the condition wherein donor and recipient are genetically identical. “Autologous” refers to donor and recipient being the same individual. “Xenogeneic” (and “xenograft”) refer to the condition where the graft donor and recipient are of different species.
  • A20 refers to a natural mammalian A20 gene (including the cDNA thereof) or protein, including derivatives thereof having variations in DNA (or amino acid) sequence (such as silent mutations or deletions of up to 5 amino acids) which do not prejudice the capability of the natural protein to block NF-KB activation.
  • the A20 gene (protein) may, for example, be porcine, bovine or human, or may be of a primate other than human, depending on the nature of the cells to be modified and the intended recipient species for transplantation.
  • a polypeptide having activity of an A20 protein or "A20 active protein” refers to a protein which is able to block or suppress NF- ⁇ B activation, and which is at least 70%, preferably at least 80%, and more preferably at least 90% (most preferably at least 95%) homologous to the protein sequence of a natural mammalian (e.g., human) A20 protein (for example, SEQ. ID. NO. 1 hereof).
  • the A20 protein of the invention is human and has the amino acid sequence corresponding to SEQ. ID. NO. 1 herein (as disclosed in A .Opipari et al. [ 1990], supra).
  • the A20 gene of the invention is at least 70%, and more preferably at least 80%, or at least 90% (e.g., at least 95%) homologous to, or corresponds to, SEQ. ID. NO. 2 herein.
  • Bcl-2 refers to a natural mammalian Bcl-2 gene (including the cDNA thereof) or protein (denoted by capital letters), including derivatives thereof having variations in DNA (or amino acid) sequence (such as silent mutations or deletions of up to 5 amino acids) which do not prejudice the capability of the natural protein to block NF- ⁇ B activation.
  • the BcI-2 gene (protein) may, for example, be porcine, bovine or human, or may be of a primate other than human, depending on the nature of the cells to be modified and the intended recipient species for transplantation.
  • a polypeptide having activity of BCL-2 protein or "BCL-2 active protein” refers to a protein which is able to block or suppress NF- ⁇ B activation, and which is at least 70%, preferably at least 80%, and more preferably at least 90% (most preferably at least 95%) homologous to the protein sequence of a natural mammalian (e.g.. human) BCL-2 (for example, SEQ. ID. NO. 3 hereof).
  • the BCL-2 polypeptide of the invention is human and has the amino acid sequence corresponding to SEQ. ID. NO. 3 (as disclosed by Tsujimoto, Y. and Croce, CM., P ⁇ JAS & [1986] 5214-5218. and in WO 95/00642).
  • Bcl-v refers to a natural mammalian Bcl-x L gene (including the cDNA ihcrcof) or protein (denoted by capital letters), including derivatives thereof having v ariations in DNA (or amino acid) sequence (such as silent mutations or deletions of up to 5 amino acids) which do not prejudice the capability of the natural protein to block NF-KB activation.
  • the Bcl-x L gene (protein) may, for example, be porcine, bovine or human, or may be of a primate other than human, depending on the nature of the cells to be modified and the intended recipient species for transplantation.
  • a polypeptide having activity of BCL-X L protein or "BCL-X L active protein” refers to a protein which is able to block or suppress NF-KB activation, and which is at least 70%. preferably at least 80%, and more preferably at least 90% (most preferably at least 95%) homologous to the protein sequence of a natural mammalian ( e.g.. human) BCL-X t protein (for example, SEQ. ID. NO. 4 hereof).
  • the BCL-X L polypeptide of the invention is hu an and has the amino acid sequence corresponding to SEQ. ID. NO. 4 (as also disclosed in WO 95/00642).
  • Al gene (protein) employed in the invention may, for example, be porcine, bovine or human, or may be of a primate other than human, depending on the nature of the cells to be modified and the intended recipient species for transplantation.
  • a polypeptide having activity of Al protein or "A 1 -active protein” refers to a protein which is able to block or suppress NF-KB activation, and which is at least 70%, preferably at least 80%, and more preferably at least 90% (most preferably at least 95%) homologous to the protein sequence of a natural mammalian (e.g., human) Al (for example, SEQ. ID. NO. 5 hereof).
  • the A I polypeptide of the invention is human and has the amino acid sequence corresponding lo SEQ. ID. NO. 5 (as disclosed in A. Karsan et al.. Blood. 87. No. 8 [April 15. 1996] 3089-3096).
  • the human A20 gene was originally cloned as an immediate early response gene which is rapidly but transiently expressed following TNF treatment of human umbilical vein endothelial cells (HUVEC) (Opipari et al. [ 1990], sjupia). It is now known that a protein having A20 activity can also be induced by other stimuli such as IL-1 in HUVEC (Dixit et al [ 1989], supra): CD40 cross-linking in B cells (Tewari et al. [1995], supra); or phorbol 12-my ⁇ state 13-acetate (PMA) or HTLV-I Tax protein in Jurkat T cells (Laherty et al. [ 1993], supra). An A20 protein is also constitutively present in mature resting T cells.
  • HUVEC human umbilical vein endothelial cells
  • a cDNA sequence of the human A20 gene obtained from HUVEC, and the deduced amino acid sequence, are published by Opipa ⁇ et al. [1990], supra, as indicated hereinabove.
  • TNF-induction of A20 has been indicated to be mediated through NF- ⁇ B binding sites in the A20 promoter, extending from -45 to -54 (5'-GGAAATCCCC-3') and from -57 to -66 (5 - GGAAAGTCCC-3 ) of the gene.
  • the deduced sequence of 790 ammo acids SEQ. ID. NO.
  • Cys /Cys contains within its carboxy 1 terminal half 7 Cys /Cys, zinc finger repeats: six with the configuration Cys-X -Cys-X ⁇ -Cys-X : -Cys and one with the configuration Cys-X ⁇ -Cys-Xn-Cys-X j -Cys, wherein X is any amino acid and the subscripts represent numbers of ammo acids between each of the indicated cysteines.
  • a novel finger loop domain composed of 1 1 amino acid residues has also been identified (Krikos ci al.
  • the "protein having A20 activity” comprises ammo acid residues 386-790 of SEQ. ID. NO. 1 , comprising the zinc finger region of the native protein sequence (i.e having 7 zinc binding domains), or a region at least 80% homologous to said residues.
  • Another suitable truncated from of the native human protein consists essentially of residues 373-790 of SEQ. ID. NO. 1 hereof.
  • Other deletion mutants found to be capable of inhibiting NFKB comprise the N-terminus and 2 zinc-binding domains of the polypeptide. e.g., ammo acids 1-538 of SEQ. ID. NO. 1.
  • the A20 protein acts with specificity to inhibit NFKB.
  • expression of JunB another TNF or LPS-inducible protein, is not found to be inhibited by A20 expression under conditions in which NFKB IS so inhibited.
  • the bcl-2 gene was originally cloned from the breakpoint of a t(14;18) translocation present in many human B cell lymphomas.
  • BCL-2 protein has been shown to prevent apoptotic cell death selectively in certain cell lines, suggesting the existence of multiple independent intracellular mechanisms of apoptosis, some of which can be prevented by BCL-2 and others of which are apparently unaffected by the gene (WO 95/00642).
  • Native proteins of the BCL i.e.
  • BCL-2 family are characterized by three conserved regions, referred to as BCL-2 homology regions 1 , 2 and 3 (abbreviated as BH-1 , BH-2 and BH-3), that are required for regulation of apoptosis and protein-protein interaction.
  • Proteins of the BCL family include anti-apoptotic polypeptides such as BCL-2, BCL-X L (the long form of a splice variant of BCL-X), MCL-1 and BAG-1 .
  • Another member of the BCL family comprises the Al protein.
  • Human Al has been found to comprise the BH 1 and BH2 regions characteristic of the BCL family (A. Karsan et al.. Blood £2, No.8 [April 15, 1996] 3089-3096; A. Karsan et al., J. Biol. Chem. 271 (44) [November 1 , 1996] 27201-27204).
  • Suitable anti-apoptotic polypeptides for use in the invention may comprise or consist essentially of regions BH1 and BH2 of native (e.g., human) Al protein, or an amino acid sequence which in the aggregate is at least 80%, preferably at least 90%, and more preferably at least 95%, homologous to the aggregate of the BH1 and BH2 regions of the native Al protein.
  • suitable deletion mutants of the BCL family may comprise, for example, at least one of the BH I , BH2.
  • BCL family apoptosis-regulating polypeptides useful in the invention may comp ⁇ se CDN-1 and CDN-2 (WO 95/15084); MCL-1 (Yang et al., J Cell Phvs 166 [ 1996] 523-536, particularly a polypeptide comprising one or more of amino acid residues 6-25, 209-223, 252-272, and 304-319 thereof; and BAG-1 (or homo- or heterodimers thereof with BCL-2 or other BCL family members) (Takayama et al., Cell, SQ [ 1995] 279-284)
  • anti -apoptotic polypeptides may exist in vivo in the form of homodimers or heterodimers with another anti-apoptotic polypeptide of the BCL family.
  • anti- apoptotic polypeptides may also be found in heterodimer combinations with antagonist polypeptides of the BCL family such as BCL-X S (the alternatively spliced short form of
  • the present invention also comprises a method of treating the dysfunctional or activation response of a cell to an inflammatory or other activation stimulus, comprising modifying said cell by inserting therein DNA encoding an anti-apoptotic protein, in operative association with a suitable promoter, and expressing said anti-apoptotic protein at effective levels whereby NF- ⁇ B activation in said cell is substantially inhibited.
  • the invention comprises a method of treating the dysfunctional or activation response of a cell to an inflammatory or other activation stimulus, comprising modifying the cell by inserting therein DNA encoding a polypeptide having anti-apoptotic activity of an A20 protein in operative association with a suitable promoter, and expressing the polypeptide at effective levels whereby activation in the cell is substantially inhibited
  • It further comprises a method of inhibiting cellular activation in a mammalian subject susceptible to an inflammatory or immunological stimulus which comprises genetically modifying endothelial cells of the subject, by insertion of DNA encoding an anti-apoptotic protein capable of inhibiting NF- ⁇ B and expressing that protein, whereby NF- ⁇ B is substantially inhibited in the cells in the presence of a cellular activating stimulus
  • it comprises a method of treating the activation response of a cell to an inflammatory or other stimulus, comprising modifying that cell by inserting therein DNA encoding a polypeptide having anti-apoptotic activity of a BCL protein (such as BCL-2 and BCL-X L proteins), a homodimer of such a polypeptide, or a heterodimer of such a polypeptide with another anti-apoptotic protein of the BCL family, and expressing the polypeptide or di er at effective levels whereby activation in the cell is substantially inhibited.
  • a BCL protein such
  • the invention also includes the cells so modified, and corresponding tissues or organs comprising such cells.
  • the protein-encoding region and/or the promoter region of the inserted DNA may be heterologous. i.e. non-native to the cell.
  • one or both of the protein encoding regions and the promoter region may be native to the cell, provided that the promoter is other than the promoter which normally controls anti-apoptotic (e.g., A20) expression in the cell.
  • the protein coding sequence may be under the control of an appropriate signal sequence, e.g., a nucleus specific signal sequence.
  • the protein encoding region is under the control of a constitutive or regulable promoter.
  • constitutive is meant substantially continuous transcription of the gene and expression of the protein over the life of the cell.
  • regulatory is meant that transcription of the gene and expression of the protein is related to the presence, or absence, of a given substance.
  • An embodiment of “regulable” expression comprises “inducible” expression, i.e. whereby transcription (and thus protein expression) occurs on demand in response to a stimulus.
  • the stimulus may comprise endothelial cell activating stimuli or a predetermined external stimulus.
  • the endothelial cell activating stimuli may be any of the stimuli which give rise to changes in the endothelium of donor tissue or organs which stimulate coagulation.
  • the predetermined external stimulus may be a drug, cytokine or other agent.
  • an advantage of employing an inducible promoter for transplantation purposes is that the desired high level expression of the (e.g., A20) active protein can be obtained on demand in response to a predetermined stimulus, such as e.g., the presence of tetracycline in the cellular environment.
  • a predetermined stimulus such as e.g., the presence of tetracycline in the cellular environment.
  • a tetracycline-inducible promoter which is suitable for use in the invention is disclosed in P.A. Furth et al., PNAS 9J. [1994] 9302-9306.
  • an example of a regulable promoter system in which transc ⁇ ption is initiated by the withdrawal of tetracycline is described in M. Gossen and H. Bujard, EH ⁇ S fiS [1992] 5547-5551.
  • expression of the (e.g., A20) active protein is induced in response to a predetermined external stimulus, and the stimulus is applied beginning immediately prior to subjecting the cells to an activating stimulus, so that expression is already at effective levels to block NF- ⁇ B activation.
  • a donor mammal e.g., porcine
  • an anti-apoptotic gene e.g., porcine or human
  • a promoter which is inducible by a drug such as tetracycline.
  • the animal whether somatic recombinant or iransgenic, may be raised up to the desired level of matu ⁇ ty under tetracycline-free conditions, until such time as the cells, or tissue or organs comprising the cells, are to be surgically removed for transplantation purposes.
  • the donor animal prior to surgical removal of the organ, the donor animal may be administered tetracycline in order to begin inducing high levels of expression of the anti-apoptotic (e.g., A20) protein.
  • the anti-apoptotic e.g., A20
  • the organ can then be transplanted into a recipient (e.g., human), and tetracycline may continue to be administered to the recipient for a sufficient time to maintain the protein at the desired levels in the transplanted cells to inhibit NF- ⁇ B activation
  • a recipient e.g., human
  • tetracycline may continue to be administered to the recipient for a sufficient time to maintain the protein at the desired levels in the transplanted cells to inhibit NF- ⁇ B activation
  • the organ after being surgically removed from the donor, the organ can be maintained ex vivo in a tetracyc ne-containing medium until such time as grafting into a recipient is appropriate.
  • expression may be provided to occur as a result of w ithholding iciracycline from the cellular environment.
  • cells of a donor animal mav be genetically modified according to the invention by insertion of a gene encoding an anti-apoptotic (e.g.. A20) protein under the control of a promoter which is blocked by tctrac>cl ⁇ ne. and which is induced in the absence of tetracycline.
  • the animal mav be raised up to the desired level of maturity while being administered tetracycline, until such time as the cells, tissues of organs of the animal are to be harvested.
  • the donor animal Prior to surgical removal, the donor animal may be deprived of tetracycline in order to begin inductng expression of the protein, and the patient in whom the cells, tissue or organs are transplanted mav thereafter also be maintained tetracycline-free for a sufficient time to maintain approp ⁇ ate levels of expression.
  • the inserted DNA sequences are incorporated into the genome of the cell.
  • the inserted sequences may be maintained in the cell extrachromosomally, either stably or for a limited period.
  • the modification of endothelial or other mammalian cells according to the invention may be earned out in vivo or ex vivo.
  • the invention also comprises a method for inhibiting the dysfunctional or activation response of endothelial cells to an inflammatory or other activation stimulus in vivo in a patient in need of such therapy, comprising modifying such cells of the patient by inserting in the cells DNA encoding an anti-apoptotic protein in operative association with a constitutive or inducible promoter and expressing the protein at effective levels whereby NF- ⁇ B activation is substantially inhibited.
  • the blood vessels of an organ e.g., a kidney
  • a solution comprising a transmissible vector construct containing the anti-apoptotic (e.g., A20) gene, for a time sufficient for at least some cells of the organ to be genetically modified by insertion therein of the vector construct, and on removal of the clamps, blood flow can then be restored to the organ and its normal functioning resumed.
  • a transmissible vector construct containing the anti-apoptotic (e.g., A20) gene for a time sufficient for at least some cells of the organ to be genetically modified by insertion therein of the vector construct, and on removal of the clamps, blood flow can then be restored to the organ and its normal functioning resumed.
  • cell populations can be removed from the patient or a donor animal, gcncticallv modified ex vivo by insertion of vector DNA, and then re-implanted into the patient or transplanted into another recipient.
  • an organ can be removed from a patient or donor, subjected ex vivo to the perfusion step described above, and the organ can be re - raited into the patient or implanted into a different recipient of the same or different species.
  • retroviral vectors for gene delivery , retroviral vectors, and in particular replication-defective retroviral vectors lacking one or more of the gag, pol, and env sequences required for retroviral replication, are well-known in the art and may be used to transform endothelial or other mammalian cells.
  • PA I 7 or other producer cell lines producing helper-free viral vectors are well -described in the literature (A.D.Miller and C.Buttimore, Mol..Cell. Biology 6. 1 19861 2895-2902).
  • a representative retroviral construct comprises at least one v iral long terminal repeat and promoter sequences upstream of the nucleotide sequence of the therapeutic substance and at least one viral long terminal repeat and polyadenylation signal downstream of the nucleotide sequence.
  • Vectors derived from adenoviruses i.e. viruses causing upper respiratory tract disease and also present in latent infections in primates, are also known in the art.
  • the ability of adenoviruses to attach to cells at low ambient temperatures is an advantage in the transplant setting which can facilitate gene transfer du ⁇ ng cold preservation of tissue or organs.
  • Adenoviral-mediated gene transfer into vessels or organs by means of transduction perfusion as described hereinabove is also a means of genetically modifying cells in vivo or ex vivo.
  • the invention comprises a method for suppressing the activation response of donor cells, or tissue or organs comprising such cells, upon transplantation into a mammalian recipient in whom the cells are susceptible to activation, which comprises
  • the donor species may be any mammalian species which is the same or different from the recipient species, and which is able to provide the appropriate cells, tissue or organs for transplantation into the recipient species.
  • the donor may be of a species which is allogeneic or xenogeneic to that of the recipient
  • the recipient is a mammal, e.g., a primate, and is preferably human.
  • human i.e. allogeneic
  • pig i.e. xenogeneic
  • any other mammalian species e.g., bovine or non-human primate
  • porcine aortic endothelial cells can be genetically modified to express porcine or human anti-apoptotic, e.g. A20 protein at effective levels, for grafting into a human recipient.
  • porcine or human anti-apoptotic e.g. A20 protein at effective levels
  • Heterologous DNA encoding the A20 or other anti-apoptotic protein can be inserted into the animal or an ancestor of the animal at the single-cell or early morula stage.
  • the preferred stage is the single-cell stage, although the process may be carried out between the two and eight cell stages.
  • a transgenic non-human animal can be thereby obtained which will pass the heterologous DNA on to offspring.
  • genes can be inserted into somatic/body cells of the donor animal to provide a somatic recombinant animal, from whom the DNA construct is not capable of being passed on to offspring (see, e.g., Miller, A.D. and Rosman, G.J., Biotechniques 2 [1989] 980-990).
  • Appropriate well-known methods of inserting foreign cells or DNA into animal tissue include micro-injection, embryonic stem cell manipulation, electroporation, cell gun. transduction. transfection, retroviral infection, adenoviruses, etc.
  • the gene is inserted in a particular locus, e.g., the thrombomodulin locus.
  • the construct is introduced into embryonic stem cells, and the resulting progeny express the construct in a tissue specific manner, paralleling the expression of thrombomodulin, i.e. in the vascular endothelium.
  • Genetically modified endothelial cells may be administered by intravenous or intra-arterial injection under defined conditions. Tissues or organs comprised thereof may also be removed from a donor and grafted into a recipient by well-known surgical procedures. Prior to implantation, the treated endothelial cells, tissue or organ may be screened for genetically modified cells containing and expressing the construct.
  • the vector construct can also be provided with a second nucleotide sequence encoding an expression product that confers resistance to a selectable marker substance. Suitable selection markers for screening include the neo gene, conferring resistance to neomycin or the neomycin analog, G418.
  • the anti-apoptotic gene such as monocytes, NK cells, lymphocytes, or islet cells
  • the preferred cells for manipulation are endothelial cells.
  • the recipient species will primarily be human, but other mammals, such as non-human primates, may be suitable recipients.
  • the anti-apoptotic polypeptide, in a pharmaceutically acceptable carrier may be applied directly to cells, tissues or organs in vivo.
  • the modified donor cells and tissues and organs defined above have a supplementary function in the prevention of xenotransplant rejection since complement-mediated events also participate in hyperacute rejection of such transplants (A.P. Dalmasso et al., Transplantation 5.2 [1991] 530-533). Therefore, the genetic material of the cells of the donor organ is typically also altered such that activation of the complement pathway in the recipient is prevented. This may be done by providing transgenic animals that express the complement inhibitory factors of the recipient species.
  • the endothelial cells of a donor organ obtained from such an animal can be modified by gene therapy techniques to provide the endothelial cells defined above.
  • a vector containing DNA encoding a protein having anti-apoptotic (e.g., A20) activity can be introduced into the transgenic animal at the single cell or early morula stage. In this way. the resulting transgenic animal will express the complement inhibitory factors and will have endothelial cells as defined above.
  • the invention also provides endothelial cells, tissue, donor organs and non-human transgenic or somatic recombinant animals as defined above which express one or more human complement inhibitory factors.
  • Cultured BAEC are transfected with reporter constructs consisting of promoters of genes known to be upregulated upon EC activation, i.e. E-selectin. I ⁇ B ⁇ . IL-8 and tissue factor.
  • pAC 8.8 kB plasmid vector containing a CMV promoter, a pUC19 polylinker site, and an SV40 splice/polyA site (J.Herz and R.D.Gerard. PNAS 90 [1993] 2812-2816).
  • A20 expression plasmid (“A20” in Figures): human A20 cDNA (Opipari et al. [ 1990], supra) (SEQ. ID. NO. 2), subcloned into the pAC expression vector at the XBal restriction site.
  • Bcl-2 and Bcl-x L expression plasmids murine bcl-2 and bcl-x L genes (W. Fang et al.. J. Immunol. 155 [ 1995] 66-75).
  • the 830 bp full-length bcl-2 cDNA was flag-tagged and cloned in the PAW neo-3 expression vector into a Clal/Xbal expression vector.
  • Porcine E-selectin reporter bp -1286 to +484 of the porcine E-selectin promoter cloned into the pMAMneo-luc plasmid vector by replacing the mm TV promoter (Clontech, Palo Alto, CA) (this includes the first complete intron and exon, as well as the beginning of the 2nd exon up to the ATG site).
  • Porcine NF- ⁇ B reporter 4 copies of NF- ⁇ B binding sites derived from the porcine E-selectin promoter inserted upstream of a TK minimal promoter driving the full length luciferase gene in a pT3/T7-luc vector (Clontech).
  • the vector backbone is a Bluescript KS+ plasmid (Stratagene, La Jolla CA, USA).
  • Human IL-8 reporter human IL-8 (hIL-8) promoter cloned into p-UBT luc.
  • Porcine TF reporter -4000 to +34 fragment of the porcine TF promoter cloned into p-UBT luc, a luciferase reporter gene vector (R. de Martin et al., Gene 124 [1993] 137-138), according to the method of T. Moll et al, J. Biol. Chem. 22 ⁇ [1995] 3849-3857.
  • HIV-CAT reporter -1 17 bp to the TATA box start of the HIV-wt LTR, cloned upstream of the CAT gene (CAT3N polylinker), prepared as described by . Zimmermann et al.. Virology J 2 [1991 ] 874-878.
  • RSV ⁇ -gal reporter E. coll ⁇ -gal gene inserted into the pRc/RSV vector (lnvitrogen. San Diego, CA. USA) at the Not 1 site.
  • RSV-LUC reporter full-length luciferase gene cloned into the pRc/RSV vector.
  • a Promega kit (Promega. Madison, WI, USA) is used to incubate cells in C-labeled chloramphenicol and n-butyryl coenz e A - containing medium (the CAT protein transfers the n-butyryl moiety of the coenzyme to chloramphenicol).
  • Cells are extracted into xylene, which is mixed with scintillation liquid and counted in a scintillation counter ( 1900 TR. Packard, Downes Grove, IL, USA).
  • Counts per minute (CPM) are normalized for ⁇ -galactosidase using the following formula: (cpm/ ⁇ -gal activity) x 1000. Significance is determined by Student's t-test.
  • the RSV ⁇ -gal reporter serves as a control for transfection efficiency.
  • the Tropix, Inc. Galacto-Light protocol (Tropix Inc., Bedford, MA, USA) is employed to measure ⁇ -galactosidase levels.
  • Bovine aortic endothelial cells (BAEC) are isolated and cultured in 10 cm plates in Dulbecco's Modified Eagle Medium (DMEM), supplemented with L-glutamine (2 mM). penicillin G (100 units/ml), and fetal calf serum (FCS) (10%). Cells are maintained at 37"C in a humidified incubator with a 5% CO, atmosphere. When the cells reach 70% confluencv. one group (i.e.
  • NT non-transfected
  • All transfcciions arc done with 16 ⁇ g lipofectamine.
  • Non-transfected, non-stimulated HUVEC ( “NS” ) or non-transfected. TNF ⁇ -stimulated HUVEC (“TNF”) also serves as controls.
  • Approximately 3 x 10 BAEC are plated per well in 6-well plates in 2 ml DMEM as supplemented and under the conditions described in Example 1.
  • a total of 1.6 ⁇ g of DNA (comprising test plasmids, reporter constructs and the ⁇ -gal reporter) and 8 ⁇ g of lipofectamine are used to transfect the cells in each well.
  • FCS is added to the cells to make a final concentration of 107c.
  • the cells are stimulated by adding to triplicate wells 100 U/ml of TNF ⁇ or 100 ng/ml of lipopolysaccharide (LPS) (Sigma E.Coli OB55).
  • Non-stimulated cells serve as control ("NS" or "control”). Seven hours after stimulation, the cells are harvested (in the following Examples all volume or weight amounts are on a per well basis; the expression “cell population” or “group of cells” refers to the cell population of a single well plate, i.e. estimated to be approximately 5 x 10 s cells; in the bar graphs, the bars represent the mean of triplicate values; standard error is represented by a bracket).
  • BAEC bovine aortic endothelial cells
  • BAEC bovine aortic endothelial cells
  • the header po ⁇ ion of FIG. 2 indicates the amount of A20 plasmid provided to each cell population, as follows: lanes I . 5. 9: 0 ⁇ g A20; lanes 2. 6. 10: 0.125 ⁇ g A20; lanes 3,7, 1 1 : 0.5 ⁇ g A20; lanes 4.8.12: 0.7 ⁇ g A20.
  • pAC is titrated with the A20 plasmid where necessary to bring the total concentration of A20 and pAC vector to 0.7 ⁇ g per well.
  • FIG. 2 is a bar graph representing the results of a luciferase assay of each group of cells. Induction of the luciferase gene under the control of the E-selectin promoter is correlatable to the amount in relative light units (RLU) detected in the assay.
  • FIG. 2 demonstrates that stimulation of the cells with TNF or LPS results in substantial increases in activity of the E-selectin reporter in the untreated control (lane 1 ); or in the stimulated cells co-transfected with only the pAC control (lanes 5 and 9), where there are 8 and 14-fold increases in E-seiectin activity. Stimulated cells transfected with the A20 construct show significant inhibition of induction of the E-selectin reporter (lanes 5 v. 8, 9 v. 12).
  • A20 expression inhibits E-selectin induction in a dose-dependent manner: when 0.125 ⁇ g of A20 are used, the inhibition reaches 53% for TNF-stimulated cells and 78% for LPS-stimulated cells (lane 5 v. 6, 9 v. 10). Virtually complete inhibition is achieved when the amount of A20 used is 0.5 ⁇ g and higher, as compared to the basal levels detected in the non-stimulated BAEC transfected with the empty vector (lane 1 v. lanes 7, 8, 1 1 and 12). In addition, A20 expression decreases the basal, unstimulated activity of the E-selectin reporter by 2-fold when used at 0.5 ⁇ g and higher.
  • the concentration of A20 plasmid used to transfect groups of cells in Examples 3, 4 and 5 is selected to be 0.5 ⁇ g.
  • FIGS. 3A-3C are bar graphs representing the results of a luciferase assay for each reporter transfection (in FIGS. 3A-3C, as well as FIG.4 and FIG.
  • IL-8 reporter When the IL-8 reporter is cotransfected with empty pAC vector, luciferase activity increases 2.5 and 2.7-fold after stimulation with TNF ⁇ and LPS, respectively (FIG. 3A, lanes I v. 3 and 5). However, when the IL-8 reporter is cotransfected with the A20 expression plasmid, luciferase levels after TNF ⁇ or LPS stimulation are reduced to below that seen with non-stimulated pAC-transfected cells (60% below the luciferase activity of unstimulated cells, lane 1 v. 4 and 6).
  • I B ⁇ reporter The results of the co-transfections performed using the porcine I ⁇ B ⁇ (ECI-6) reporter construct are similar to those seen with IL-8. Induction with TNF ⁇ and LPS reaches 1.6 and 3.6-fold, respectively. Inhibition is virtually complete when A20 is cotransfected with the IkB ⁇ reporter. TNF ⁇ - or LPS- induced luciferase activities are also lower than the basal levels noted with the empty vector (FIG. 3B, lane 1 v. lanes 4 and 6).
  • BAEC are cotransfected according to the General Procedure with 0.5 ⁇ g of either the A20 expression plasmid or the pAC control plasmid and 0.7 ⁇ g of the NF- ⁇ B reporter construct, and the results are shown in the bar graph comprising FIG. 4. Results demonstrate that A20 expression abrogates the 12 and 28-fold induction of reporter activity in response to TNF ⁇ and LPS. respectively (FIG. 4, lanes 3 v. 4, 5 v. 6). There is no apparent significant difference between the basal levels of luciferase activity between A20 and pAC transfected cells (FIG. 4. lane 1 v. 2).
  • ceils are transfected according to the General Procedure with a constitutive, non-inducible reporter.
  • RSV-LUC which is independent of NF- ⁇ B.
  • HIV -CAT reporter which is induced by the viral c-Tat protein through Spl rather than NF- ⁇ B binding (Zimmermann et al. [1991 ], supra).
  • Cells are transfected with 0.5 ⁇ g of either A20 or pAC (RSV-LUC reporter) (as shown in the header of FIG. 5A), or A20 titrated with pAC to make up a total of 0.5 ⁇ g (HIV-CAT reporter) (as shown in the header of FIG. 5B).
  • RSV-LUC reporter cell groups are either non-stimulated ("Control”) or TNF- or LPS-stimulated.
  • TNF- or LPS-stimulated cells are either unstimulated ("Control") or stimulated with 0.2 ⁇ g of the c-Tat protein. It is found that basal luciferase activities of the RSV-LUC reporter are comparable to that seen in the A20 and pAC transfected BAEC.
  • FIGS. 5A-5B are bar graphs representing the results of a luciferase assay. It is apparent that no significant induction is achieved upon TNF or LPS stimulation in either the pAC- or the A20-expressing cells; luciferase values remain comparable among the 2 groups (FIG. 5 A). With regard to HIV-CAT, the results demonstrate that A20 expression affects neither the basal levels nor the 10 to 15-fold induction of the reporter observed upon stimulation with c-Tat (FIG. 5B, lane I v. lanes 2, 3, 4 and lane 1 v. lanes 6, 7, 8).
  • Expression of A20 has no apparent effect on either the constitutive activity of the RSV-LUC reporter or the c-Tat stimulation of the HIV-CAT reporter, which also demonstrates a lack of effect of A20 on Spl , which illustrates the specificity of A20 in blocking NF- ⁇ B activation.
  • A20 inhibits NF- ⁇ B activation, and thereby inhibits gene induction.
  • This function places A20 in the category of genes that are dependent on NF- ⁇ B for their induction, but that subsequently inhibit NF- ⁇ B and thus, endothelial cell activation. Such genes presumably function in negative regulatory loops to regulate the extent and duration of endothelial cell activation.
  • A20 functions as an antioxidant.
  • the full-length human A20 cDNA encodes 7 Cys2/Cys2 repeats, which characterizes it as a Zn finger protein with a potentially high Zn binding capacity (Opipari et al. [1990], sjinja).
  • Zn can act as an antioxidant by two mechanisms: the protection of sulfhydryl groups against oxidation and the inhibition of the production of reactive oxygens by transition metals, mainly iron and copper.
  • antioxidants such as PDTC can prevent gene induction associated with EC activation, by inhibition of NF- ⁇ B (E.B.
  • a recombinant A20 adenovirus (rAd.A20) is constructed by homologous recombination between a transfer vector containing the human A20 cDNA, pAC.CMV.NLS-A20, and pJM17, a plasmid-bome form of the adenovirus 5 genome.
  • the encoded A20 protein is unmodified.
  • Homologous recombination is performed in 293 cells.
  • Clonal viruses are obtained by limiting dilution cloning in 96-well plates, and analyzed by Northern blotting for the presence of A20 mRNA. After identification of a positive recombinant A20 adenovirus, amplification is performed in 293 cells.
  • Cesium chloride purified adenovirus is used to infect porcine aortic endothelial cells (PAEC) at a multiplicity of infection (MOD of 500 to 2500/cell.
  • A20 infection is checked by Northern blot analysis of infected cells. 48 hours after infection, cells are stimulated with 100 U/ml of TNF or 100 ng/ml of LPS. mRNA is extracted 2-6 hours following EC stimulation.
  • Northern blot analysis shows that A20 adenovirus-infected cells abrogate by 60-90% the TNF- and LPS-mediated induction of E-selectin, IL-8, and I ⁇ B ⁇ . The percentage of inhibition is directly correlated to mRNA levels of A20 detected in infected cells.
  • A20 expression in PAEC inhibits by up to 90% the surface expression of E-selectin as assessed by ELISA. Mock-infected cells as well as PAEC infected with a ⁇ -galactosidase rAD are used as controls. These results further demonstrate that expression of A20 inhibits EC activation.
  • Example 7 Co-transfer of BAEC with Bcl-2 and Bcl-x L expression nlas ids along with reporter constructs
  • bovine aortic endothelial cells obtained from culture in 10 cm plates as described in Example 1, are plated per well in a 6- well plate in 2 ml of DMEM as supplemented and under the conditions described in Example 1. When the cells reach 50%-70% confluency, a total of 1.5-1.6 ⁇ g/well of DNA (test plasmids and reporter constructs) is added to 8 mg of lipofectamine per well and incubated at room temperature for 30 minutes before being added to the cells in triplicate.
  • DNA test plasmids and reporter constructs
  • BAEC are co-transfected with 0.5 ⁇ g of Bcl-2, Bcl-x L or pAC, and 0.7 ⁇ g of the E-selectin, ECI-6 (I ⁇ B ⁇ ) or NF- ⁇ B - luciferase (luc) reporters, as well as 0.3 ⁇ g of the ⁇ -galactosidase (b-gal) reporter.
  • FCS is added to the medium to achieve a final concentration of 10% 48 hours thereafter the cells are stimulated with either human recombinant TNF (lOOU/ml) or LPS (lOOng/ml), and are harvested 7 h after stimulation
  • BAEC endothelial cell-specific marker, E-selectin.
  • BAEC 3x l 0 -, to 5x10 s cells
  • the porcine E-selectin reporter construct 0.7 ⁇ g
  • the bcl-2, the bcl-x, expression plasmids 0.5 ⁇ g
  • the pAC control 0.5 ⁇ g
  • results, depicted in FIG. 6A. show that BCL-2 and BCL-X L overexpression leads to a significant decrease in the luciferase activity of the E-selectin reporter after both TNF and LPS stimulation
  • induction with either TNF or LPS leads to a 35- and 50-fold increase in the activity of the E-selectm reporter, respectively.
  • BCL-X, expression inhibits TNF- and LPS-mduced luciferase activity very significantly, this inhibition reaching respectively 95% and 90% of the control following TNF and LPS stimulation (lanes 4 and 7 v . 5.
  • BAEC are co-transfected with an NF- ⁇ B reporter construct that is solely dependent upon NF- ⁇ B, and either bcl-x L , bcl-2 or the empty vector, pAC (FIG. 6C).
  • BCL-X L expression significantly decreases the 10- and 26-fold induction of reporter activity in response to TNF and LPS, respectively (lanes 4 and 7 v. 5 and 8). This inhibition reaches 50% and 70%, respectively.
  • BCL-2 expression totally abrogates the TNF and LPS inducibility of the NF- ⁇ B reporter (lanes 4 and 7 v. 6 and 9). There appears to be no significant difference in the basal levels of luciferase activity between BCL-X L , BCL-2 and pAC (lanes 1 v. 2 and 3).
  • a truncation of the A20 gene from bp 1 182 to 2450 and spanning the 7 Zn binding domains of the molecule is obtained by digestion of the 2.4 kB cDNA with Ncol. This fragment is expressed as a polypeptide of 417 amino acid residues (residues 373 to 790 of SEQ. ID. NO. 1 ).
  • the truncated A20 gene is cloned into pBac 4 (Promega) and then subcloned into the pAC expression vector to be used in co-transfection experiments in BAEC. In these experiments, 2 x 10 s BAEC are plated per well in a 6-well plate with 2 ml of medium as described above.
  • Cells are transfected once they reach 50-70% confluence. 1.5-1 .6 ⁇ g/well of DNA (test plasmids and reporter constructs) are added to 4 units of lipofectamine per well and incubated at room temperature for 30 minutes before being added to the cells in triplicate. In this experiment, 0.3 ⁇ g of the ⁇ -gal reporter is used, with 0.5 ⁇ g of: A20, or truncated A20 (tA20), or the control plasmid pAC, and 0.7 ⁇ g of the E-selectin-luc reporter. 48 hours after transfection, cells are challenged with either 100 U/ml of TNF or 100 ng/ml of LPS.
  • FIG. 7 shows that expression of the truncated form of A20, i.e. consisting essentially of the 7 Zn binding domains of the molecule, inhibits as efficiently as A20. the induction of the E-selectin reporter upon stimulation by TNF or LPS.
  • Example 9 Regulable gene expression in transgenic mice a) Inducible tetracycline expression system:
  • a system for temporal regulation of anti-apoptotic gene expression is highly desirable to inhibit NF- ⁇ B activation on a controllable basis.
  • An inducible expression system can be employed to regulate anti-apoptotic gene expression in vivo, in particular the binary plasmid system described by Gossen and Bujard, PNAS [ 1992], supra, which is inducible by the withdrawal of tetracycline; or the tetracycline-dependent system disclosed by Furth et al., PNAS [1994], supra.
  • the Gossen and Bujard system employs a first plasmid containing a bacterial, tetracycline-sensiiive DNA binding protein fused to the HSV-VP16 transcriptional activation domain (tTA) expressed from a constitutive CMV promoter.
  • a second plasmid contains 7 copies of the binding site for tTA, downstream of which the anti-apoptotic gene is cloned into the vector.
  • the tTA protein drives high level transcription of the anti-apoptotic gene of the invention.
  • tetracycline In the presence of tetracycline there is no expression of the anti-apoptotic transgene.
  • tetracycline In the absence of tetracycline, there is high level expression of the anti-apoptotic gene (in the Furth et al. system, the presence of tetracycline promotes expression of the anti-apoptotic gene, whereas in the absence of tetracycline there is no expression of the anti-apoptotic transgene).
  • transgenic mice For the generation of transgenic mice the anti-apoptotic gene is cloned into a suitable vector, for example, as described by Gossen and Bujard, PNAS [1992] supra. Two separate founder strains are generated for tTA and the anti-apoptotic gene. Transgenic mice of each strain are rendered homozygous by crossing heterozygous animals. Homozygous animals of each strain are bred as lines. Crossing tTA ⁇ TA mice with, e.g... bcl-2/bcl-2 mice results in double transgenic mice carrying both tTA and Bcl-2 transgenes. These crossings are carried out under cover of tetracycline to prevent anti- apoptotic transgene expression during embryogenesis. Mice carrying the tTA and anti- apoptotic transgene, respectively, are identified by Southern blotting to prevent expression of the anti-apoptotic gene during embryogenesis.
  • mice that express the anti-apoptotic gene in EC can be used as donors for xenotransplantation (heart and/or kidney) into rats for modelling purposes.
  • a transgenic pig expressing a human anti-apoptotic gene (e.g., A20, bcl-2, bcl-x L , A l ) is prepared by techniques disclosed in Pinckert et al. [1994], supra.
  • Example 11 Adenoviral-mediated BCL-2 expression inhibits NF-KB activation
  • Nuclear extracts are prepared from rAd.Bcl-2 or rAd. ⁇ -gal-infected PAEC before, and two hours following, treatment with TNF (lOOU/ml).
  • TNF lOOU/ml
  • NF- ⁇ B activation and binding to a KB binding oligonucleotide derived from the human Immunoglobulin (lg) K promoter is evaluated by electrophoretic mobility shift assay (EMSA) (FIG. 8).
  • Nuclear extracts from PAEC expressing BCL-2 reveal little constitutive, and no inducible. binding of NF- ⁇ B, whereas rAd. ⁇ -gal - infected cells demonstrate strong induction of NF- ⁇ B binding activity following TNF stimulation. Specificity of DNA binding is confirmed by the use of excess cold wild-type (specific competitor) or a non ⁇ specific competitor (AP-1 ) probe as controls (lanes 3 and 4).
  • Example 12 BCL-2 expression in PAEC inhibits I ⁇ B ⁇ degradation following TNF treatment
  • Cytoplasmic extracts are prepared prior to, as well as ten minutes or two hours following, TNF treatment of rAd.Bcl-2 - or rAd. ⁇ -gal - infected PAEC. Protein concentration of the cytoplasmic extracts is quantitated by the Bradford method. I ⁇ B ⁇ expression is evaluated by Western blot. l ⁇ B ⁇ is detected using anti-MAD-3 rabbit polyclonal IgG anti-serum (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) and peroxidase-conjugated goat anti-rabbit secondary antibody followed by enhanced chemiluminescence (ECL) detection (Amersham Corp.). Results show that BCL-2 expression in PAEC inhibits the usual I ⁇ B ⁇ degradation that occurs 10 minutes following TNF stimulation. Results shown are representative of 3 independent experiments (FIG. 9).
  • Example 13 BCL-2 expression in the EC does not affect binding of the transcription factor.
  • CRE cAMP reponsive element
  • nuclear extracts are prepared from rAd.Bcl-2- or rAd. ⁇ -gal - infected PAEC before, and two hours following, treatment with TNF (lOOU/ml) and assayed by EMSA (electrophoretic mobility shift assay) for their binding activity of a radio-labeled CRE oligonucleotide. No difference is observed between the Bcl-2- and the ⁇ -gal - infected cells (FIG. 10).
  • Example 14 Function of the Bel ene Al in endothelial cells a) A I expression in EC inhibits TNF- and LPS-induced activation through inhibition of NF-KB:
  • HUVEC when stimulated with TNF, express Al .
  • the maximum induction at the mRNA level occurs at approximately three hours following TNF stimulation.
  • Expression of A l in the EC inhibits activation following TNF and LPS treatment; this inhibitory effect relates to inhibition of NF- ⁇ B activation.
  • BAEC are co-transfected with an expression plasmid encoding for Al and reporter constructs comprising the promoter region of E-selectin linked to the luciferase gene and a reporter solely dependent upon NF-KB for its induction (FIG. 11).
  • b) Expression of Al is dependent on NF- ⁇ B:
  • HUVEC are infected with the rAd.I ⁇ B ⁇ , rAd.A20 or the control rAd. ⁇ -gal at an MOI of 100.
  • Northern blot reveals high levels of I ⁇ B ⁇ and of A20 mRNA in the cells. Forty-eight hours following infection, EC are stimulated with 100U of TNF for three hours. RNA is extracted. Expression of Al is analyzed by Northern blot analysis.
  • Results demonstrate that expression of I ⁇ B ⁇ or of A20 inhibits the induction of Al messenger RNA as seen in the control rAd. ⁇ -gal-infected cells. Similarly, induction of I ⁇ B ⁇ (another NF-B dependent gene) is inhibited in the A20-expressing cells as compared to controls, further confirming the ability of A20 to block up-regulation of NF-KB dependent genes (FIG. 12).
  • Lys Ala Val Lys lie Arg Glu Arg Thr Pro Glu Asp lie Phe Lys Pro 20 25 30
  • Lys Glu lie Asn Leu Val Asp Asp Tyr Phe Glu Leu Val Gin His Glu 340 345 350
  • Cys Lys Asn lie Leu Ala Cys Arg Ser Glu Glu Leu Cys Met Glu Cys 725 730 735
  • Lys Tyr lie His Tyr Lys Leu Ser Gin Arg Gly Tyr Glu Trp Asp Ala 20 25 30
  • Gly Arg lie Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu 145 150 155 160
  • Val Leu Val Ser Arg lie Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp
  • Asp Gly lie lie Asn Trp Gly Arg lie Val Thr lie Phe Ala Phe Glu 85 90 95

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Abstract

L'invention concerne une méthode permettant de modifier les cellules des mammifères, en particulier les cellules endothéliales, afin de les rendre moins sensibles à un stimulus inflammatoire ou à un stimulus d'activation immunologique. Selon cette méthode, on introduit dans une cellule de ce type ou dans une de ses progénitrices un ADN codant pour un polypeptide anti-apoptose capable d'inhiber NF-λB et d'exprimer la protéine, NF-λB dans la cellule étant sensiblement inhibé en présence d'un stimulus activateur cellulaire. On sélectionne les polypeptides convenables parmi ceux ayant l'activité d'une protéine A20, BCL-2, BCL-XL(MCL-1) ou A1 de mammifère, y compris les homologues et les formes tronquées des protéines natives. Les polypeptides actifs BCL-2, BCL-XL ou A1 peuvent également être employés comme homodimères ou hétérodimères avec un autre polypeptide anti-apoptose de la famille BCL. La méthode, qui peut être mise en ÷uvre in vivo, ou ex vivo, ou in vitro, est particulièrement utile en relation avec les greffes allogéniques ou, plus particulièrement, xénogéniques, ainsi que pour traiter les états inflammatoires généraux ou locaux. Il est possible de préparer des mammifères non humains transgéniques ou recombinants somatiques de sorte que les cellules endothéliales de ceux-ci expriment un polypeptide de ce type et que cette expression soit régulée, ainsi que des tissus ou organes contenant ces cellules et pouvant être greffés chez un mammifère receveur.
PCT/EP1997/000676 1996-02-14 1997-02-13 Therapie genique de cellules endotheliales avec des proteines anti-apoptose pour les pathologies liees aux greffes et aux etats inflammatoires WO1997030083A1 (fr)

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JP09528990A JP2000510326A (ja) 1996-02-14 1997-02-13 移植および炎症状態に対する抗アポトーシス性遺伝子療法
EP97905019A EP0886650A1 (fr) 1996-02-14 1997-02-13 Therapie genique de cellules endotheliales avec des proteines anti-apoptose pour les pathologies liees aux greffes et aux etats inflammatoires
AU18730/97A AU1873097A (en) 1996-02-14 1997-02-13 Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions

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WO1998019712A1 (fr) * 1996-11-08 1998-05-14 St. Elizabeth's Medical Center Of Boston, Inc. Procede de regulation de l'angiogenese
WO1999013073A2 (fr) * 1997-09-08 1999-03-18 Rpr Gencell Asia/Pacific Inc. Systeme de vecteurs viraux capables d'exprimer un gene associe a l'apoptose
WO1999055382A1 (fr) * 1998-04-29 1999-11-04 The Uab Research Foundation Vecteur adenoviral codant pour un gene bcl-2 anti-apoptotique et utilisation de ce vecteur
WO2001011031A2 (fr) * 1999-05-27 2001-02-15 University Of Pittsburgh Of The Commonwealth System Of Higher Education TRANSFERT DE GENES DANS DES CELLULES β PANCREATIQUES POUR PREVENIR LE DYSFONCTIONNEMENT DE CES CELLULES REGROUPEES EN ILOTS
WO2001067110A1 (fr) * 2000-03-08 2001-09-13 Akzo Nobel N.V. Activation synergique d'elements regulateurs par des proteines rel et un recepteur de steroide
WO2001081387A1 (fr) * 2000-04-25 2001-11-01 Jin Woo Kim Proteine suppressive du cancer du col de l'uterus chez la femme, polynucleotide codant pour elle, cellule transformee par le polynucleotide et methode de suppression de la proliferation de cellules cancereuses utilisant le vecteur d'expression
KR100434591B1 (ko) * 2002-04-16 2004-06-04 김진우 인간 암 억제 유전자, 이에 의해 코딩되는 단백질, 이를포함하는 발현 벡터 및 이 벡터로 형질전환된 세포
US7282488B2 (en) 2000-05-22 2007-10-16 The Johns Hopkins Univeristy Genetic engineering of vascular grafts to resist disease
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US7470538B2 (en) 2002-12-05 2008-12-30 Case Western Reserve University Cell-based therapies for ischemia
US7964565B2 (en) * 2004-10-04 2011-06-21 University of Washington Center for Commercialization, a Public Institution of Higher Education Method of inhibiting inflammation in a mammal by administering Bcl protein
US9095580B2 (en) 1996-11-08 2015-08-04 Genesys Research Institution Inc. Compositions and methods for regulating angiogenesis
US9505821B2 (en) * 2006-10-03 2016-11-29 Rutgers, The State University Of New Jersey ATAP peptides, nucleic acids encoding the same and associated methods of use

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US6569428B1 (en) 1996-11-08 2003-05-27 St. Elizabeth's Medical Center Of Boston, Inc. Pharmaceutical products comprising endothelial cell precursors
US5980887A (en) * 1996-11-08 1999-11-09 St. Elizabeth's Medical Center Of Boston Methods for enhancing angiogenesis with endothelial progenitor cells
WO1998019712A1 (fr) * 1996-11-08 1998-05-14 St. Elizabeth's Medical Center Of Boston, Inc. Procede de regulation de l'angiogenese
US9095580B2 (en) 1996-11-08 2015-08-04 Genesys Research Institution Inc. Compositions and methods for regulating angiogenesis
EP1618898A3 (fr) * 1996-11-08 2007-12-12 Caritas St. Elizabeth's Medical Center of Boston, Inc. Procédure de régulation de l' angiogenèse
WO1999013073A2 (fr) * 1997-09-08 1999-03-18 Rpr Gencell Asia/Pacific Inc. Systeme de vecteurs viraux capables d'exprimer un gene associe a l'apoptose
WO1999013073A3 (fr) * 1997-09-08 1999-06-10 Rpr Gencell Asia Pacific Inc Systeme de vecteurs viraux capables d'exprimer un gene associe a l'apoptose
WO1999055382A1 (fr) * 1998-04-29 1999-11-04 The Uab Research Foundation Vecteur adenoviral codant pour un gene bcl-2 anti-apoptotique et utilisation de ce vecteur
WO2001011031A2 (fr) * 1999-05-27 2001-02-15 University Of Pittsburgh Of The Commonwealth System Of Higher Education TRANSFERT DE GENES DANS DES CELLULES β PANCREATIQUES POUR PREVENIR LE DYSFONCTIONNEMENT DE CES CELLULES REGROUPEES EN ILOTS
WO2001011031A3 (fr) * 1999-05-27 2001-08-23 Univ Pittsburgh TRANSFERT DE GENES DANS DES CELLULES β PANCREATIQUES POUR PREVENIR LE DYSFONCTIONNEMENT DE CES CELLULES REGROUPEES EN ILOTS
US7297685B2 (en) 2000-01-21 2007-11-20 Beth Israel Deaconess Medical Center Use of pro-apoptotic factors in treatment of atherosclerosis
WO2001067110A1 (fr) * 2000-03-08 2001-09-13 Akzo Nobel N.V. Activation synergique d'elements regulateurs par des proteines rel et un recepteur de steroide
US7252934B2 (en) 2000-03-08 2007-08-07 N.V. Organon Synergistic activation of regulatory elements by Rel proteins and a steroid receptor
JP2003526346A (ja) * 2000-03-08 2003-09-09 アクゾ・ノベル・エヌ・ベー Ｒｅｌタンパク質とステロイド受容体による調節分子の相乗活性
WO2001081387A1 (fr) * 2000-04-25 2001-11-01 Jin Woo Kim Proteine suppressive du cancer du col de l'uterus chez la femme, polynucleotide codant pour elle, cellule transformee par le polynucleotide et methode de suppression de la proliferation de cellules cancereuses utilisant le vecteur d'expression
US7282488B2 (en) 2000-05-22 2007-10-16 The Johns Hopkins Univeristy Genetic engineering of vascular grafts to resist disease
KR100434591B1 (ko) * 2002-04-16 2004-06-04 김진우 인간 암 억제 유전자, 이에 의해 코딩되는 단백질, 이를포함하는 발현 벡터 및 이 벡터로 형질전환된 세포
US7470538B2 (en) 2002-12-05 2008-12-30 Case Western Reserve University Cell-based therapies for ischemia
US7964565B2 (en) * 2004-10-04 2011-06-21 University of Washington Center for Commercialization, a Public Institution of Higher Education Method of inhibiting inflammation in a mammal by administering Bcl protein
US8304389B2 (en) 2004-10-04 2012-11-06 University Of Washington Through Its Center For Commercialization, A Public Institution Of Higher Education Methods of inhibiting cell death or inflammation in a mammal by administering a BCL protein
EP2527837A1 (fr) * 2004-10-04 2012-11-28 University of Washington Procédés d'inhibition de mort cellulaire ou inflammation chez un mammifère
US9505821B2 (en) * 2006-10-03 2016-11-29 Rutgers, The State University Of New Jersey ATAP peptides, nucleic acids encoding the same and associated methods of use

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