WO1997030083A1 - Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions - Google Patents

Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
cells
protein
bcl
cell
polypeptide
Prior art date
Application number
PCT/EP1997/000676
Other languages
French (fr)
Inventor
Fritz H. Bach
Christiane Ferran
Original Assignee
Novartis Ag
New England Deaconess Hospital Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Ag, New England Deaconess Hospital Corporation filed Critical Novartis Ag
Priority to AU18730/97A priority Critical patent/AU1873097A/en
Priority to JP09528990A priority patent/JP2000510326A/en
Priority to EP97905019A priority patent/EP0886650A1/en
Publication of WO1997030083A1 publication Critical patent/WO1997030083A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027New or modified breeds of vertebrates
    • A01K67/0271Chimeric vertebrates, e.g. comprising exogenous cells
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/20Animal model comprising regulated expression system
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/105Murine
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/108Swine
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/02Animal zootechnically ameliorated
    • A01K2267/025Animal producing cells or organs for transplantation
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/03Animal model, e.g. for test or diseases
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/03Animal model, e.g. for test or diseases
    • A01K2267/035Animal model for multifactorial diseases
    • A01K2267/0368Animal model for inflammation
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00Uses of viruses
    • C12N2799/02Uses of viruses as vector
    • C12N2799/021Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022Uses 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Environmental Sciences (AREA)
  • Cell Biology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A method of genetically modifying mammalian, especially endothelial cells to render them less susceptible to an inflammatory or other immunological activation stimulus is described, which comprises inserting in that cell or a progenitor thereof DNA encoding an anti-apoptotic polypeptide capable of inhibiting NF-λB and expressing the protein, whereby NF-λB in the cell is substantially inhibited in the presence of a cellular activating stimulus. Suitable polypeptides are selected from those having activity of a mammalian A20, BCL-2, BCL-XL (MCL-1) or A1 protein, including homologs and truncated forms of the native proteins. The BCL-2, BCL-XL or A1 active polypeptides can also be employed as homodimers or as heterodimers with another anti-apoptotic polypeptide of the BCL family. The method, which can be carried out in vivo or ex vivo or in vitro, is particularly useful in connection with allogeneic or, especially, xenogeneic transplantation, as well as to treat systemic or local inflammatory conditions. Transgenic or somatic recombinant non-human mammals can be prepared expressing such a polypeptide on a regulable basis by the endothelial cells thereof, and tissues or organs comprising such cells can be obtained for grafting into a mammalian recipient.

Description

GENE THERAPY OF ENTOTHELIAL CELLS WITH ANΗ-APOPTOπC PROTEINS FOR TRANS¬ PLANTATION AND INFLAMMATORY CONDITIONS
Field of the invention
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. Examples of 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. Rackpround of the invention
The well-characteπzed problem of "hyperacute rejection" accompanying transplantation of organs between discordant species, involving an immediate immunological response of recipient antibodies and complement system against the transplanted organ, has been addressed by vaπous 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).
However, a further condition associated with grafted tissue or organs, and with cells subjected to inflammatory processes in general, is the process known as ' activation" In particular, endothelial cell "activation" refers to a continuum of changes characterizing endothelial ceils which are subjected to a stimulus such as a cytotoxic cytokine |e g , tumor necrosis factor (TNF)], an inflammatory or infectious condition, reperfusion injury, atherosclerosis, vascu tis or graft rejection 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 (often referred to as "Type I" activation) typically involves changes in the cell pheπotype, such as retraction of cells from one another, hemorrhage and edema, and trans-migration of leukocytes across the endothelium. A still further phase of cellular activation ("Type II" activation), involves transcπptional up-regulation of vaπous genes encoding interleukins. adhesion molecules, and procoagulant, prothrombotic components of the coagulation system For example, 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)
A recognized phenomenon associated with continuous overexpression of such activation proteins, at the expense of normal cell functioning, is the tendency of the cell to undergo a process of active cellular suicide known as "apoptosis" (G. T. Williams and C A. Smith. £ej] 7_4 f I 993| 777-779, D.L. Vaux et al., Cell 26 [1994] 777-779). 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. 68 [ 1980] 251 -306) Cell death by 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. H., Immunology Today 7(4) [1986] 1 15-1 19; Trauth, B.C. et al., Science 245 [1989] 301-305). Apoptotic cell death is of fundamental importance in inflammation, embryogeπesis and lymphocyte selection. Avoidance of cell activation and apoptotic cell death accompanying inflammation in general, and particularly in connection with organ transplantation, has become a major goal for workers in the art. Graft injury and loss occurring in connection with graft preservation techniques, as well as accompanying graft rejection, exemplify the vulnerability of endothelial cells to such processes.
An identified transcription factor for many of the genes susceptible to transcπptional up-regulation in response to an activation stimulus such as TNFα, 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. In the nucleus, the binding of NF-κB 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).
For example, 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). There is evidence that 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. 267 [1992] 12424-12427; A.W. Opipari et al., J. Biol. Chem. 265 [1990] 14705-14708; Dixit et al. [1989], sjjpxa). A. Krikos and co-workers (J. Biol. Chem. 267 [1992] 17971-17976) demonstrated that induction of the A20 gene by TNFα is also mediated by NF-κB binding sites in the A20 promoter (see also CD. Laherty et al., J. Biol. Chem. 2δ£ [1993] 5032-5039).
Besides the A20 protein, certain proteins of the BCL (also referred to as BCL-2) family of proteins also exert an anti-apoptotic effect. Such proteins include BCL-2, BCL-XL, MCL-1 , and Al . However, the precise mechanisms by which the A20 protein or BCL proteins exert an anti-apoptotic effect have not been completely elucidated.
Summary of the invention
An important means of suppressing NF-κB-mediated activation of a cell has now been found. Unexpectedly, it was found that NF-κB 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-κB 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-κB-IκB complex from dissociating, apparently by acting upstream of IκB degradation. Such inhibitory function may normally assist in preventing apoptotic cell death. However, under conditions of severe cellular challenge, such as occurring in connection with transplantation, and particularly xenotransplantation, 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-κB is rendered ineffective to prevent cellular activation and apoptosis.
This finding has now been used to devise a method to treat endothelial or other cells susceptible to an inflammatory or other activating stimulus, and in particular to treat cells, tissues or organs which are subject to transplantation rejection. 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.
The invention relies on gene therapy techniques, utilizing an anti-apoptotic gene and its expressed product to inhibit NF-κB activation in mammalian cells susceptible to an activating stimulus.
Accordingly, in a first aspect 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-κB activation in the presence of a cellular activating stimulus. An example of a "cellular activating stimulus" is tumor necrosis factor. TNF (i.e. TNFα).
By "NF-κB activation" is meant NF- B-mediated up-regulation of genes which are directly or indirectly under the control of an NF-κB binding site, such as, e.g., E-selectin in endothelial cells. In functional terms, NF-κB activation constitutes the binding of NF-κB 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.
Bv "NF- "B inhibition" is meant that NF-κB binding to NF-κB 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 TNFα 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 TNFα.
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-κB and expressing the protein, whereby NF- B in the cell is substantially inhibited in the presence of a cellular activating stimulus. It was found that inhibition of NF-KB-initiated transcription by the anti-apoptotic protein, such as, e.g., an A20 protein, in a genetically modified cell is unexpectedly potent, even at moderate levels of transfection in vitro with the corresponding A20 gene (e.g., 0.5 μg plasmid DNA per approximately 5 x 105 cells), leading to effective suppression of induction of cytokine-inducible genes such as tissue factor, E-selectin and IicBα, all of which are associated with inflammation.
It will be apparent that 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.
Thus in a further aspect, 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
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-κB activ ation in the cells is substantially inhibited in the presence of a cellular activating stimulus.
The "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.
According to a further aspeci of the invention, there are provided 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. In its additional aspects, 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. Also within the scope of the invention are vectors for genetically modifying cells by insertion of anti-apoptotic protein-encoding polynucleotides, such as for example retroviral vectors, and especially, adenoviral vectors.
nescriotion of the drawings
Figure 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 TNFα. Also analyzed for comparison is HUVEC which is either non-stimulated ("NS") or stimulated with TNFα ("TNF").
Figure 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 TNFα ("TNF") or lipopolysaccharide ("LPS").
Figures 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 IκBα promoter ("IκBα Reporter") (FIG. 3B); and (c) porcine tissue factor (TF) promoter ("Tissue Factor Reporter") (FIG. 3C): and then stimulated with TNFα or LPS or maintained as a control.
Figure 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 TNFα or LPS or maintained as a control.
Figure 5A: Luciferase levels in BAEC co-transfected with either A20 or pAC and an RSV-LTR driven luciferase vector ("RSV-LUC Reporter").
Figure 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-XL, together with either the E-selectin reporter (FIG. 6a), the IκBα 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.
Figure 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, β-gal ("rAd.β-Gal"), using a B binding oligonucleotide derived from the human immuπoglobulin (lg) K promoter and, for comparison, a cold wild-type NFκB-specific probe ("sp-comp.") and a non-specific competitor ("nsp. comp.")(AP-I ).
Figure 9: Western blot of rAd.Bcl-2- (or, as a control, rAd.β-gal-) infected PAEC taken prior to ("0") , or ten minutes (" 10' ") or one hundred-twenty minutes ("120' ") following stimulation with TNF, with Iicβα as shown.
Figure 10: EMSA of nuclear extracts from rAd.Bcl-2- (or, as a control, rAd.β-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.").
Figure 1 1: Luciferase levels in BAEC co-transfected with either Al or pAC and a luciferase vector comprising 0.7μg 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 IκBα ("rAd.I B"α) or A20 ("rAd.A20") or, as a control. rAd.β-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-κB 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). In a preferred embodiment, 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). In a further aspect, 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-κB 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-κB 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). In a preferred embodiment of the invention, 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).
Similarlv . "Bcl-v, " refers to a natural mammalian Bcl-xL 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-xL 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-XL protein" or "BCL-XL 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-Xt protein (for example, SEQ. ID. NO. 4 hereof). In a preferred embodiment of the invention, the BCL-XL 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).
"A I " refers to a natural mammalian Al 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 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). In a preferred embodiment of the invention, 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).
Detailed description
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-myπstate 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.
A cDNA sequence of the human A20 gene obtained from HUVEC, and the deduced amino acid sequence, are published by Opipaπ et al. [1990], supra, as indicated hereinabove. TNF-induction of A20 has been indicated to be mediated through NF-κB 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. At the protein level, the deduced sequence of 790 ammo acids (SEQ. ID. NO. 1 ) contains within its carboxy 1 terminal half 7 Cys /Cys, zinc finger repeats: six with the configuration Cys-X -Cys-Xπ-Cys-X:-Cys and one with the configuration Cys-X^-Cys-Xn-Cys-Xj-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. | I992|. supra)
In one embodiment of this invention, 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.
It has been found that the A20 protein acts with specificity to inhibit NFKB. For example, 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. In vitro, 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-XL (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.
In general, suitable deletion mutants of the BCL family may comprise, for example, at least one of the BH I , BH2. BH3 and BH4 regions of the native protein, for example, for each protein, one or more of the following peptide sequences (a.a. = amino acid position no.): BCL-2: about a.a. 10 to about a.a. 30; about a.a. 93 to about a.a. 107; about a.a. 135 to about a.a.155; about a.a. 187 to about a.a. 202, of SEQ. ID. NO. 3; BCL-X, : about a.a. 5 to about a.a. 24; about a.a. 86 to about a.a. 100; about a.a. 129 to about a.a.148; about a.a. 180 to about a.a. 195, of SEQ. ID. NO. 4; A l : about a.a. 27 to about a.a. 45; about a.a. 66 to about a.a. 99; about a.a. 133 to about a.a. 145. of SEQ. I D. NO. 5. Still other BCL family apoptosis-regulating polypeptides useful in the invention may compπse 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)
These anti -apoptotic polypeptides may exist in vivo in the form of homodimers or heterodimers with another anti-apoptotic polypeptide of the BCL family. Such anti- apoptotic polypeptides may also be found in heterodimer combinations with antagonist polypeptides of the BCL family such as BCL-XS (the alternatively spliced short form of
BCL-X), BAX and BAD
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-κB activation in said cell is substantially inhibited.
In a particular aspect, 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-κB and expressing that protein, whereby NF-κB is substantially inhibited in the cells in the presence of a cellular activating stimulus In a further aspect, 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-XL 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.
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. Alternatively, 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.
Preferably the protein encoding region is under the control of a constitutive or regulable promoter. By "constitutive" is meant substantially continuous transcription of the gene and expression of the protein over the life of the cell. By "regulable" 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. An example of 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. Alternatively, an example of a regulable promoter system in which transcπption is initiated by the withdrawal of tetracycline is described in M. Gossen and H. Bujard, EHΔS fiS [1992] 5547-5551.
Preferably, 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-κB activation. For example, cells of a donor mammal (e.g., porcine) may be genetically modified according to the invention by insertion of an anti-apoptotic gene (e.g., porcine or human) under the control of 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 matuπty 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. In such case, 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 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-κB activation Alternatively, 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.
In another embodiment, expression may be provided to occur as a result of w ithholding iciracycline from the cellular environment. Thus, 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>clιne. and which is induced in the absence of tetracycline. In such case, 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. 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 appropπate levels of expression. Preferably, the inserted DNA sequences are incorporated into the genome of the cell. Alternatively, 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.
Thus 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-κB activation is substantially inhibited. For example, the blood vessels of an organ (e.g., a kidney) can be temporarily clamped off from the blood circulation of the patient and the vessels perfused with 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.
In another aspect, 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. For example, 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.
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 duπng 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.
Alternative means of targeted gene delivery comprise DNA-protem conjugates, liposomes, etc
In yei another embodiment, 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
(a) modifying the donor cells by introducing therein DNA encoding an anti-apoptotic protein, and
(b) transplanting the resultant donor cells, or tissue or organs comprising such cells, into the recipient and expressing the protein, whereby NF-KB activation of the cells is substantially inhibited
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. For human recipients, it is envisaged that human (i.e. allogeneic) as well as pig (i.e. xenogeneic) donors will be suitable, but any other mammalian species (e.g., bovine or non-human primate) may also be suitable as donor
For example, porcine aortic endothelial cells (PAEC), or the progenitor cells thereof, can be genetically modified to express porcine or human anti-apoptotic, e.g. A20 protein at effective levels, for grafting into a human recipient. 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. In another aspect 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. In one embodiment, the gene is inserted in a particular locus, e.g., the thrombomodulin locus. Subsequently, 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.
Methods of preparing transgenic pigs are disclosed in e.g. Pinckert et al., Xeno 2. No. 1 [ I 994| 10-15.
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. For this purpose, 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.
Although any mammalian cell can be targeted for insertion of 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. In an alternative embodiment of the invention, the anti-apoptotic polypeptide, in a pharmaceutically acceptable carrier, may be applied directly to cells, tissues or organs in vivo.
It will be appreciated that 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. Alternatively 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.
Thus in a further aspect 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.
The following Examples are intended to be illustrative only and not limitative of the invention. Cultured BAEC are transfected with reporter constructs consisting of promoters of genes known to be upregulated upon EC activation, i.e. E-selectin. IκBα . IL-8 and tissue factor.
EXAMPLES
Materials and methods:
The following vectors are utilized in the Examples:
"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-xL expression plasmids: murine bcl-2 and bcl-xL 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. The 700 bp full-length Bcl-xL cDNA was also flag-tagged and cloned into a Clal/BamHl sites of the PAW neo-3 expression vector (PAW neo-3 is a 7kb expression plasmid containing ampicillin and neomycin resistance sites and a SFFV-LTR promoter before the polylinker cloning site) (SFFV = spleen focus forming virus).
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-κB reporter: 4 copies of NF-κB 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. 22Ω [1995] 3849-3857.
Porcine IκBα (also referred to as "ECI-6") reporter 600 bp fragment of the porcine ECI-6 IkBα promoter hgated into p-UBT-luc, with the creation of an additional Hind III site, as described by R. de Martin et al., EMBO J. 1 [1993] 2773-2779.
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 β-gal reporter: E. coll β-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.
Cell extracts are assayed for luciferase (or CAT) and galactosidase levels. a) Luαtcrase lev els (E-selectin. NF-κB. IL-8. TF and IκBα [ECI-6] promoters):
10 μ l of cellular extract are added to 90 μl of a solution containing 24 mM glvcv lghcine ipH 7.8). 2 mM ATP (pH 7.5) and 10 mM MgSO4. Samples are read on a Microlumai LB 96P luminometer (EG+G Berthold) using an injection mix consisting of 24 mM glycyl lycine and 0.1 mM lucifenn (Boehπnger, Mannheim, Germany). Luciferase activity is normalized for β-galactosidase using the following formula: (luciferase aciivity/β-gal activity) x 1000. Luciferase activity is also corrected for protein bv dividing the luciferase activity by protein concentration. Normalized luciferase activity is given in relative light units (RLU). CAT levels (HIV LTR activity):
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 β-galactosidase using the following formula: (cpm/β-gal activity) x 1000. Significance is determined by Student's t-test. c) β-palactosidase levels:
The RSV β-gal reporter serves as a control for transfection efficiency. The Tropix, Inc. Galacto-Light protocol (Tropix Inc., Bedford, MA, USA) is employed to measure β-galactosidase levels.
Rxample 1 : Transfected BAEC express human A20 protein
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. approximately 1 x 10* cells) is transfected with 0.5 μg of the A20 vector ("A20"); a second group is transfected with 0.5 μg of the pAC vector ("PAC"): and a third group is maintained as a non-transfected ("NT") control. All transfcciions arc done with 16 μg lipofectamine. Non-transfected, non-stimulated HUVEC ( "NS" ) or non-transfected. TNFα-stimulated HUVEC ("TNF") also serves as controls.
Cells arc washed twice with cysteine and methionine-free medium (ICN, Lisle, IL, USA), and then placed in the same medium supplemented with 100 μCi/ml Tran "S labelled cysicinc and mcthionine (ICN). After four hours, cells are harvested. Immuno- prccipitation w ith polyclonal rabbit anti-human A20 polyclonal serum on a polyacrylamidc SDS gel. as shown in FIG. 1, reveals the presence of a 3,S-labelled 80 kD A20 protein in the "A20" extract, but not the "PAC", "NT" or "NS" extracts. This protein is comparable to that seen in the TNF mulated HUVEC extract ("TNF"). Example. 2-4; Central Procedure
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. When the cells reach 50%-70% confluency, a total of 1.6 μg of DNA (comprising test plasmids, reporter constructs and the β-gal reporter) and 8 μg of lipofectamine are used to transfect the cells in each well. After incubation of the cells for 5 hours, FCS is added to the cells to make a final concentration of 107c. After incubation for 48 hours, the cells are stimulated by adding to triplicate wells 100 U/ml of TNFα 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 10s cells; in the bar graphs, the bars represent the mean of triplicate values; standard error is represented by a bracket).
Exam le 2: E-selectin reporter (A20 expression in BAEC inhibits E-selectin induction in a dose-dependent manner)
BAEC (bovine aortic endothelial cells) are cotransfected with 0.7 μg of the porcine E-.select reporter construct, together with the A20 expression plasmid or the pAC control plasmid or both. The header poπion of FIG. 2 indicates the amount of A20 plasmid provided to each cell population, as follows: lanes I . 5. 9: 0 μg A20; lanes 2. 6. 10: 0.125 μg A20; lanes 3,7, 1 1 : 0.5 μg A20; lanes 4.8.12: 0.7 μg A20. pAC is titrated with the A20 plasmid where necessary to bring the total concentration of A20 and pAC vector to 0.7 μg 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).
It is also apparent that A20 expression inhibits E-selectin induction in a dose-dependent manner: when 0.125 μg 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 μg 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 μg and higher.
Since maximal inhibition is obtained by transfecting with 0.5 to 0.7 μg A20 vector, the concentration of A20 plasmid used to transfect groups of cells in Examples 3, 4 and 5 is selected to be 0.5 μg.
Example X- IL-S. IKBP fECI-6) and TF reporter constructs
BAEC are cotran fected as described in the General Procedure above with 0.5 μg of either the A20 expression plasmid or the pAC control plasmid, and 0.7 μg of one of the above-indicated reporter constructs, which are up-regulated during EC activation. 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. 5A, the presence ("+") or absence ("-") of A20 or pAC is indicated in the header): a) 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 TNFα 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 TNFα 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). Further¬ more, A20 overexpression decreases the basal luciferase activity of the IL-8 reporter by 3-fold (FIG. 3 A, lane 1 v. 2). b) I Bα reporter: The results of the co-transfections performed using the porcine IκBα (ECI-6) reporter construct are similar to those seen with IL-8. Induction with TNFα and LPS reaches 1.6 and 3.6-fold, respectively. Inhibition is virtually complete when A20 is cotransfected with the IkBα reporter. TNFα- 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). Co-transfection with A20 is found to decrease by 5-fold the basal level of EC1-6 luciferase activity (FIG. 3B, lanes 1 v. 2). c) Tissue factor reporter: In a comparable manner, A20 expression inhibits the 3.5 and 4.5-fold induction of TF reporter activity after TNFα and LPS stimulation, respectively (FIG. 3C, lanes 3, 4, 5, 6). However, a decrease in basal TF reporter activity with A20 co-expression is not observed (FIG. 3C, lane 1 v. 2).
Example 4: NF-KB reporter
BAEC are cotransfected according to the General Procedure with 0.5 μg of either the A20 expression plasmid or the pAC control plasmid and 0.7 μg of the NF-κB 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 TNFα 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).
All the reporters listed above are known to be highly dependent on NF-κB. Activation of these reporters by either LPS or TNFα is found to be inhibited by expression of A20, demonstrating that the inhibitory effect of A20 on EC activation relates, at least in part and perhaps totally, to inhibition of NF-κB.
Example 5: RSV-LUC and H1V-CAT reporters
To test non-specific or toxic effects of A20 on the transcriptional machinery, ceils are transfected according to the General Procedure with a constitutive, non-inducible reporter. RSV-LUC, which is independent of NF-κB. Also tested is the HIV -CAT reporter, which is induced by the viral c-Tat protein through Spl rather than NF-κB binding (Zimmermann et al. [1991 ], supra). Cells are transfected with 0.5 μg 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 μg (HIV-CAT reporter) (as shown in the header of FIG. 5B). For the RSV-LUC reporter, cell groups are either non-stimulated ("Control") or TNF- or LPS-stimulated. For the HIV-CAT reporter, cells are either unstimulated ("Control") or stimulated with 0.2 μg 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).
The above demonstrates that expression of A20 prevents gene induction associated with endothelial cell activation. Reporter inhibition is seen when either TNF or LPS is used to stimulate the EC, pointing to the broad inhibitory effect of A20 on gene induction. The similar effect on LPS- and TNF-induced signaling also excludes any specific association of the action of A20 with the TNF response per se. The basal expression of the E-selectin, IL-8 and IκBα reporters is also significantly decreased in cells expressing A20. Inhibition is found to be dose-dependent.
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-κB activation.
Therefore in addition to its ability to protect cells from apoptosis, expression of A20 inhibits NF-κB activation, and thereby inhibits gene induction. This function places A20 in the category of genes that are dependent on NF-κB for their induction, but that subsequently inhibit NF-κB and thus, endothelial cell activation. Such genes presumably function in negative regulatory loops to regulate the extent and duration of endothelial cell activation.
While not intending to be bound thereby, it is proposed that an alternative mechanism exists by which 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. There is evidence that antioxidants such as PDTC can prevent gene induction associated with EC activation, by inhibition of NF-κB (E.B. Cunningham, Biochem.Biophvs.Res.Commun. 215 [1995] 212-218) and also to prevent TNF-mediated apoptosis (T.M. Buttke and P.A. Sandstrom. Immunol. Today 15 [1994] 7-10). These findings correlate with the fact that signaling via the TNF receptor results in a rapid rise in the levels of intracellular reactive oxygen intermediates that cause apoptosis via oxidative damage (Buttke and Sandstrom [1994], supra).
E am le *>• Adenoviral-mediated transfer of A20 to porcine aortic endothelial cells
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 IκBα. The percentage of inhibition is directly correlated to mRNA levels of A20 detected in infected cells. In accordance with Northern blot analysis, 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 β-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-xL expression nlas ids along with reporter constructs
Approximately 3 x 10 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 μg/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. In all experiments, BAEC are co-transfected with 0.5 μg of Bcl-2, Bcl-xL or pAC, and 0.7 μg of the E-selectin, ECI-6 (IκBα) or NF-κB - luciferase (luc) reporters, as well as 0.3 μg of the β-galactosidase (b-gal) reporter. After 5 hours incubation, 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
The effect of BCL-2 and BCL-XL expression upon EC activation is first studied using an endothelial cell-specific marker, E-selectin. BAEC (3x l 0-, to 5x10s cells) are co¬ transfected w ith the porcine E-selectin reporter construct (0.7 μg) as well as the bcl-2, the bcl-x, expression plasmids (0.5 μg) or the pAC control (0.5 μg) plasmid in conjunction with the RSV β-gal plasmid (0 3 μg)
The results, depicted in FIG. 6A. show that BCL-2 and BCL-XL overexpression leads to a significant decrease in the luciferase activity of the E-selectin reporter after both TNF and LPS stimulation In the pAC control, 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. 8) Inhibition is seen to be complete when BCL-2 is expressed in the cells No induction of the E-selectin reporter is seen upon TNF and LPS .stimulation (lanes 4 and 7 v lanes 6 and 9) The basal level of luciferase activity of the E-selectin reporter is not affected by BCL-2 or BCL-XL expression.
The results of the co-transfections performed using the porcine IκBα (ECI-6) reporter construct (FIG. 6B) are similar to those seen with E-selectin. Induction with TNF and LPS reaches 2 5 fold (lanes 1 v 4 and 6). BCL-XL and BCL-2 expression completely abolishes TNF- and LPS-induced luciferase activity following TNF and LPS stimulation (lanes 4 and 7 v. 5, 6 and 7, 8). The basal level of luciferase activity of the IκBα reporter is not affected by BCL-2 or BCL-XL expression.
BAEC are co-transfected with an NF-κB reporter construct that is solely dependent upon NF-κB, and either bcl-xL, bcl-2 or the empty vector, pAC (FIG. 6C). BCL-XL 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. In contrast with BCL-XL, BCL-2 expression totally abrogates the TNF and LPS inducibility of the NF-κB 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-XL, BCL-2 and pAC (lanes 1 v. 2 and 3).
Therefore the demonstrated EC inhibition is shown to be related to inhibition of the transcriptional factor NF-κB.
Example 8: A2Q mutant?
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 10s 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 μg/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 μg of the β-gal reporter is used, with 0.5 μg of: A20, or truncated A20 (tA20), or the control plasmid pAC, and 0.7 μg 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. Cell extracts are prepared 7 hours after stimulation and assayed for β-galactosidase and luciferase expression, as above. Two clones expressing the truncated form of the A20 are tested: clone #3 and #7. 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-κB 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. For example 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. When both plasmids are present in a cell, the tTA protein drives high level transcription of the anti-apoptotic gene of the invention. In the presence of tetracycline there is no expression of the anti-apoptotic transgene. 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). b) 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 tTAΛTA 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.
Example 10: Generation of transgenic nigs
A transgenic pig expressing a human anti-apoptotic gene (e.g., A20, bcl-2, bcl-xL, 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.β-gal-infected PAEC before, and two hours following, treatment with TNF (lOOU/ml). NF-κB 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-κB, whereas rAd.β-gal - infected cells demonstrate strong induction of NF-κB 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 IκBα 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.β-gal - infected PAEC. Protein concentration of the cytoplasmic extracts is quantitated by the Bradford method. IκBα expression is evaluated by Western blot. lκBα 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 IκBα 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. cAMP reponsive element (CRE)
To determine whether BCL-2 expression affects nuclear binding to a CRE probe, nuclear extracts are prepared from rAd.Bcl-2- or rAd.β-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 β-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-κB 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-κB:
To evaluate whether functional NF-κB activity is needed for the induction of Al , it is investigated whether A I continues to be inducible following TNF stimulation in HUVEC even in the presence of an overexpressed inhibitor of NF-κB (i.e. IκBα or A20). HUVEC are infected with the rAd.IκBα, rAd.A20 or the control rAd.β-gal at an MOI of 100. Northern blot reveals high levels of IκBα 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 IκBα or of A20 inhibits the induction of Al messenger RNA as seen in the control rAd.β-gal-infected cells. Similarly, induction of IκBα (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).
SEQUENCE LISTING
(1 ) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Novartis AG
(B) STREET: Schwarzwaldallee 215
(C) CITY: Basle
(E) COUNTRY: Switzerland
(F) POSTAL CODE (ZIP): CH-4058
(G) TELEPHONE: 61 -324 5269 (H) TELEFAX: 61 -322 7366
(ii) TITLE OF INVENTION: ANTI-APOPTOTIC GENE THERAPY FOR
TRANSPLANTATION AND INFLAMMATORY CONDITIONS
(iii ) NUMBER OF SEQUENCES: 5
(iv ) COMPUTER READABLE FORM:
( A ) MEDIUM TYPE: Floppy disk
(B ) COMPUTER: IBM PC compatible
(C ) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: WO PCT/EP97/....
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/601515
(B) FILING DATE: I4-FEB- I996
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/634995
(B) FILING DATE: I9-APR-I996 (2) INFORMATION FOR SEQ ID NO. 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 790 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
Met Ala Glu Gin Val Leu Pro Gin Ala Leu Tyr Leu Ser Asn Met Arg
1 5 10 15
Lys Ala Val Lys lie Arg Glu Arg Thr Pro Glu Asp lie Phe Lys Pro 20 25 30
Thr Asn Gly lie lie His His Phe Lys Thr Met His Arg Tyr Thr Leu 35 40 45
Glu Met Phe Arg Thr Cys Gin Phe Cys Pro Gin Phe Arg Glu lie lie 50 55 60
His Lys Ala Leu lie Asp Arg Asn lie Gin Ala Thr Leu Glu Ser Gin 65 70 75 80
Lys Lys Leu Asn Trp Cys Arg Glu Val Arg Lys Leu Val Ala Leu Lys 85 90 95
Thr Asn Gly Asp Gly Asn Cys Leu Met His Ala Thr Ser Gin Tyr Met 100 105 110
Trp Gly Val Gin Asp Thr Asp Leu Val Leu Arg Lys Ala Leu Phe Ser 115 120 125
Thr Leu Lys Glu Thr Asp Thr Arg Asn Phe Lys Phe Arg Trp Gin Leu 130 135 140
Glu Ser Leu Lys Ser Gin Glu Phe Val Glu Thr Gly Leu Cys Tyr Asp 145 150 155 160
Thr Arg Asn Trp Asn Asp Glu Trp Asp Asn Leu lie Lys Met Ala Ser
165 170 175
Thr Asp Thr Pro Met Ala Arg Ser Gly Leu Gin Tyr Asn Ser Leu Glu 180 185 190
Glu lie His lie Phe Val Leu Cys Asn lie Leu Arg Arg Pro lie lie 195 200 205 Val lie Ser Asp Lys Met Leu Arg Ser Leu Glu Ser Gly Ser Asn Phe 210 215 220
Ala Pro Leu Lys Val Gly Gly lie Tyr Leu Pro Leu His Trp Pro Ala 225 230 235 240
Gin Glu Cys Tyr Arg Tyr Pro lie Val Leu Gly Tyr Asp Ser His His 245 250 255
Phe Val Pro Leu Val Thr Leu Lys Asp Ser Gly Pro Glu lie Arg Ala 260 265 270
Val Pro Leu Val Asn Arg Asp Arg Gly Arg Phe Glu Asp Leu Lys Val 275 280 285
His Phe Leu Thr Asp Pro Glu Asn Glu Met Lys Glu Lys Leu Leu Lys 290 295 300
Glu Tyr Leu Met Val lie Glu lie Pro Val Gin Gly Trp Asp His Gly 305 310 315 320
Thr Thr His Leu lie Asn Ala Ala Lys Leu Asp Glu Ala Asn Leu Pro 325 330 335
Lys Glu lie Asn Leu Val Asp Asp Tyr Phe Glu Leu Val Gin His Glu 340 345 350
Tyr Lys Lys Trp Gin Glu Asn Ser Glu Gin Gly Arg Arg Glu Gly His 355 360 365
Ala Gin Asn Pro Met Glu Pro Ser Val Pro Gin Leu Ser Leu Met Asp 370 375 380
Val Lys Cys Glu Thr Pro Asn Cys Pro Phe Phe Met Ser Val Asn Thr 385 390 395 400
Gin Pro Leu Cys His Glu Cys Ser Glu Arg Arg Gin Lys Asn Gin Asn 405 410 415
Lys Leu Pro Lys Leu Asn Ser Lys Pro Gly Pro Glu Gly Leu Pro Gly 420 425 430
Met Ala Leu Gly Ala Ser Arg Gly Glu Ala Tyr Glu Pro Leu Ala Trp 435 440 445
Asn Pro Glu Glu Ser Thr Gly Gly Pro His Ser Ala Pro Pro Thr Ala 450 455 460
Pro Ser Pro Phe Leu Phe Ser Glu Thr Thr Ala Met Lys Cys Arg Ser 465 470 475 480
Pro Gly Cys Pro Phe Thr Leu Asn Val Gin His Asn Gly Phe Cys Glu 485 490 495
Arg Cys His Asn Ala Arg Gin Leu His Ala Ser His Ala Pro Asp His 500 505 510
Thr Arg His Leu Asp Pro Gly Lys Cys Gin Ala Cys Leu Gin Asp Val 515 520 525 Thr Arg Thr Phe Asn Gly lie Cys Ser Thr Cys Phe Lys Arg Thr Thr 530 535 540
Ala Glu Ala Ser Ser Ser Leu Ser Thr Ser Leu Pro Pro Ser Cys His 545 550 555 560
Gin Arg Ser Lys Ser Asp Pro Ser Arg Leu Val Arg Ser Pro Ser Pro 565 570 575
His Ser Cys His Arg Ala Gly Asn Asp Ala Pro Ala Gly Cys Leu Ser 580 585 590
Gin Ala Ala Arg Thr Pro Gly Asp Arg Thr Gly Thr Ser Lys Cys Arg 595 600 605
Lys Ala Gly Cys Val Tyr Phe Gly Thr Pro Glu Asn Lys Gly Phe Cys 610 615 620
Thr Leu Cys Phe lie Glu Tyr Arg Glu Asn Lys His Phe Ala Ala Ala 625 630 635 640
Ser Gly Lys Val Ser Pro Thr Ala Ser Arg Phe Gin Asn Thr lie Pro 645 650 655
Cys Leu Gly Arg Glu Cys Gly Thr Leu Gly Ser Thr Met Phe Glu Gly 660 665 670
Tyr Cys Gin Lys Cys Phe lie Glu Ala Gin Asn Gin Arg Phe His Glu 675 680 685
Ala Lys Arg Thr Glu Glu Gin Leu Arg Ser Ser Gin Arg Arg Asp Val 690 695 700
Pro Arg Thr Thr Gin Ser Thr Ser Arg Pro Lys Cys Ala Arg Ala Ser 705 710 715 720
Cys Lys Asn lie Leu Ala Cys Arg Ser Glu Glu Leu Cys Met Glu Cys 725 730 735
Gin His Pro Asn Gin Arg Met Gly Pro Gly Ala His Arg Gly Glu Pro 740 745 750
Ala Pro Glu Asp Pro Pro Lys Gin Arg Cys Arg Ala Pro Ala Cys Asp 755 760 765
His Phe Gly Asn Ala Lys Cys Asn Gly Tyr Cys Asn Glu Cys Phe Gin 770 775 780
Phe Lys Gin Met Tyr Gly 785 790 (2) INFORMATION FOR SEQ I I) NO. 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4440 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Figure imgf000042_0001
TGCCTTGACC AGGACTTGGG ACTTTGCGAA AGGATCGCGG GGCCCGGAGA GGTGTTGGAG AGCACAATGG CTGAACAAGT CCTTCCTCAG GCTTTGTATT TGAGCAATAT GCGGAAAGCT GTGAAGATAC GGGAGAGAAC TCCAGAAGAC ATTTTTAAAC CTACTAATGG GATCATTCAT CATTTTAAAA ^ATGCACCG ATACACACTG GAAATGTTCA GAACTTGCCA GTTTTGTCCT CAGTTTCGGG ATCATCCA CAAAGCCCTC ATCGACAGAA ACATCCAGGC CACCCTGGAA AGCCAGAAGA AACTCAACTG GTGTCGAGAA GTCCGGAAGC TTGTGGCGCT GAAAACGAAC GGTGACGGCA ATTGCCTCAT GCATGCCACT TCTCAGTACA TGTGGGGCGT TCAGGACACA GACTTGGTAC TGAGGAAGGC GCTGTTCAGC ACGCTCAAGG AAACAGACAC ACGCAACTTT AAATTCCGCT GGCAACTGGA GTCTCTCAAA TCTCAGGAAT TTGTTGAAAC GGGGCTTTGC
Figure imgf000043_0001
-Ifr- _.900/-.6d-IX3d e800C/-.6 OΛV TGCTTCAAAA GGACTACAGC AGAGGCCTCC TCCAGCCTCA GCACCAGCCT CCCTCCTTCC TGTCACCAGC GTTCCAAGTC AGATCCCTCG CGGCTCGTCC GGAGCCCCTC CCCGCATTCT TGCCACAGAG CTGGAAACGA CGCCCCTGCT GGCTGCCTGT CTCAAGCTGC ACGGACTCCT GGGGACAGGA CGGGGACGAG CAAGTGCAGA AAAGCCGGCT GCGTGTATTT TGGGACTCCA GAAAACAAGG GCTTTTGCAC ACTGTGTTTC ATCGAGTACA GAGAAAACAA ACATTTTGCT GCTGCCTCAG GGAAAGTCAG TCCCACAGCG TCCAGGTTCC AGAACACCAT TCCGTGCCTG GGGAGGGAAT GCGGCACCCT TGGAAGCACC ATGTTTGAAG GATACTGCCA GAAGTGTTTC ATTGAAGCTC AGAATCAGAG ATTTCATGAG GCCAAAAGGA CAGAAGAGCA ACTGAGATCG AGCCAGCGCA GAGATGTGCC TCGAACCACA CAAAGCACCT CAAGGCCCAA GTGCGCCCGG GCCTCCTGCA AGAACATCCT GGCCTGCCGC AGCGAGGAGC TCTGCATGGA GTGTCAGCAT CCCAACCAGA GGATGGGCCC TGGGGCCCAC CGGGGTGAGC CTGCCCCCGA AGACCCCCCC AAGCAGCGTT πrPGGGCCCC CGCCTGTGAT CATTTTGGCA ATGCCAAGTG CAACGGCTAC TGCAACGAAT TTTCAGTT CAAGCAGATG TATGGCTAAC CGGAAACAGG TGGGTCACCT CCTGCAAGAA GTGGGGCCTC GAGCTGTCAG TCATCATGGT GCTATCCTCT GAACCCCTCA GCTGCCACTG CAACAGTGGG CTTAAGGGTG TCTGAGCAGG AGAGGAAAGA TAAGCTCTTC GTGGTGCCCA CGATGCTCAG GTTTGGTAAC CCGGGAGTGT TCCCAGGTGG CCTTAGAAAG CAAAGCTTGT AACTGGCAAG GGATGATGTC AGATTCAGCC CAAGGTTCCT CCTCTCCTAC CAAGCAGGAG GCCAGGAACT TCTTTGGACT TGGAAGGTGT GCGGGGACTG GCCGAGGCCC CTGCACCCTG CGCATCAGGA CTGCTTCATC GTCTTGGCTG AGAAAGGGAA AAGACACACA
Figure imgf000044_0001
AGTCGCGTGG GTTGGAGAAG CCAGAGCCAT TCCACCTCCC CTCCCCCAGC ATCTCTCAGA GATGTGAAGC CAGATCCTCA TGGCAGCGAG GCCCTCTGCA AGAAGCTCAA GGAAGCTCAG GGAAAATGGA CGTATTCAGA GAGTGTTTGT AGTTCATGGT TTTTCCCTAC CTGCCCGGTT CCTTTCCTGA GGACCCGGCA GAAATGCAGA ACCATCCATG GACTGTGATT CTGAGGCTGC TGAGACTGAA CATGTTCACA TTGACAGAAA AACAAGCTGC TCTTTATAAT ATGCACCTTT TAAAAAATTA GAATATTTTA CTGGGAAGAC GTGTAACTCT TTGGGTTATT ACTGTCTTTA CTTCTAAAGA AGTTAGCTTG AACTGAGGAG TAAAAGTGTG TACATATATA ATATACCCTT ACATTATGTA TGAGGGATTT TTTTAAATTA TATTGAAATG CTGCCCTAGA AGTACAATAG GAAGGCTAAA TAATAATAAC CTGTTTTCTG GTTGTTGTTG GGGCATGAGC TTGTGTATAC ACTGCTTGCA TAAACTCAAC CAGCTGCCTT TTTAAAGGGA GCTCTAGTCC TTTTTGTGTA ATTCACTTTA TTTATTTTAT TACAAACTTC AAGATTATTT AAGTGAAGAT ATTTCTTCAG CTCTGGGGAA AATGCCACAG TGTTCTCCTG AGAGAACATC CTTGCTTTGA GTCAGGCTGT GGGCAAGTTC CTGACCACAG GGAGTAAATT GGCCTCTTTG ATACACTTTT GCTTGCCTCC CCAGGAAAGA AGGAATTGCA TCCAAGGTAT ACATACATAT TCATCGATGT TTCGTGCTTC TCCTTATGAA ACTCCAGCTA TGTAATAAAA AACTATACTC TGTGTTCTGT TAATGCCTCT GAGTGTCCTA CCTCCTTGGA GATGAGATAG GGAAGGAGCA GGGATGAGAC TGGCAATGGT CACAGGGAAA GATGTGGCCT TTTGTGATGG TTTTATTTTC TGTTAACACT GTGTCCTGGG GGGGCTGGGA AGTCCCCTGC ATCCCATGGT ACCCTGGTAT TGGGACAGCA AAAGCCAGTA ACCATGAGTA TGAGGAAATC TCTTTCTGTT GCTGGCTTAC AGTTTCTCTG TGTGCTTTGT
Figure imgf000045_0001
o o o o o o o o
CN OO ^f O VO CN CO j"
O O H CN CN ro ro ^J"
^t ^1 ^1 ^* ^Jt "^ ^3*
Figure imgf000046_0001
<2\ INFORMATION FOR SEQ ID NO. 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu lie Val Met
1 5 10 15
Lys Tyr lie His Tyr Lys Leu Ser Gin Arg Gly Tyr Glu Trp Asp Ala 20 25 30
Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly lie 35 40 45
Phe Ser Ser Gin Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp 50 55 60
Pro Val Ala Arg Thr Ser Pro Leu Gin Thr Pro Ala Ala Pro Gly Ala 65 70 75 80
Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Ala 85 90 95
Leu Arg Gin Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Gly Asp Phe 100 105 110
Ala Glu Met Ser Ser Gin Leu His Leu Thr Pro Phe Thr Ala Arg Gly 115 120 125
Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 130 135 140
Gly Arg lie Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu 145 150 155 160
Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn lie Ala Leu Trp 165 170 175
Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp lie Gin Asp Asn 180 185 190
Gly Gly Trp Val Gly Ala Ser Gly Asp Val Ser Leu Gly 195 200 205 (2) INFORMATION FOR SEQ ID NO. 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 233 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Ser Gin Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys
1 5 10 15
Leu Ser Gin Lys Gly Tyr Ser Trp Ser Gin Phe Ser Asp Val Glu Glu
20 25 30
Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro
35 40 45
Ser Ala lie Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala
50 55 60
Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp Ala Arg Glu Val 65 70 75 80 lie Pro Met Ala Ala Val Lys Gin Ala Leu Arg Glu Ala Gly Asp Glu
85 90 95
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gin Leu
100 105 110
His lie Thr Pro Gly Thr Ala Tyr Gin Ser Phe Glu Gin Val Val Asn
115 120 125
Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg lie Val Ala Phe Phe
130 135 140
Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gin 145 150 155 160
Val Leu Val Ser Arg lie Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp
165 170 175
His Leu Glu Pro Trp lie Gin Glu Asn Gly Gly Trp Asp Thr Phe Val
180 185 190
Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gin Glu
195 200 205
Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val
210 215 220
Leu Leu Gly Ser Leu Phe Ser Arg Lys 225 230 INFORMATION FOR SEQ ID NO. 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Thr Asp Cys Glu Phe Gly Tyr lie Tyr Arg Leu Ala Gin Asp Tyr 1 5 10 15
Leu Gin Cys Val Leu Gin lie Pro Gin Pro Gly Ser Gly Pro Ser Lys 20 25 30
Thr Ser Arg Val Leu Gin Asn Val Ala Phe Ser Val Gin Lys Glu Val 35 40 45
Glu Lys Asn Leu Lys Ser Cys Leu Asp Asn Val Asn Val Val Ser Val 50 55 60
Asp Thr Ala Arg Thr Leu Phe Asn Gin Val Met Glu Lys Glu Phe Glu 65 70 75 80
Asp Gly lie lie Asn Trp Gly Arg lie Val Thr lie Phe Ala Phe Glu 85 90 95
Giy lie Leu lie Lys Lys Leu Leu Arg Gin Gin lie Ala Pro Asp Val 100 105 110
Asp Thr Tyr Lys Glu lie Ser Tyr Phe Val Ala Glu Phe lie Met Asn 115 120 125
Asn Thr Gly Glu Trp lie Arg Gin Asn Gly Gly Trp Glu Asn Gly Phe 130 135 140
Val Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe Leu Glu Val 145 150 155 160
Thr Gly Lys He Cys Glu Met Leu Ser Leu Leu Lys Gin Tyr Cys 165 170 175

Claims

Claims:
1. A mammalian endothelial cell which is genetically modified to express an anti-apoptotic protein which is capable of inhibiting NF-κB activation in the presence of a cellular activating stimulus.
2. A donor endothelial cell, or a tissue or organ comprising such a cell, wherein the cell is genetically modified to regulably or constitutively express an anti-apoptotic protein in a graft recipient, whereby NF-κB is substantially inhibited, for transplantation into a recipient species.
3. A method of genetically modifying a mammalian endothelial cell to render it less susceptible to an inflammatory or other immunological activation stimulus, which comprises inserting in that cell, or a progenitor thereof, DNA encoding an anti-apoptotic protein capable of inhibiting NF-κB and expressing the protein, whereby NF-κB activation in the cell is substantially inhibited in the presence of a cellular activating stimulus.
4. A method of inhibiting cellular activation in a mammalian subject susceptible to an inflammator) 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-κB and expressing that protein, whereby NF-κB is substantially inhibited in the cells in the presence of u cellular activating stimulus.
5. A method of transplanting donor endothelial or other mammalian cells, or graftable tissues or organs comprising such cells, to a mammalian recipient in whose blood or plasma these cells, tissues or organs are subject to activation, which comprises:
(a) genetically modifying the donor cells, or progenitor cells thereof, by inserting therein DNA encoding an anti-apoptotic protein capable of inhibiting NF-κB; and (b) transplanting the resultant modified donor cells, or tissues or organ comprising these cells, into the recipient, and expressing in the cells the anti-apoptotic protein, whereby NF-κB activation in the cells is substantially inhibited in the presence of a cellular activating stimulus.
6. A cell according to claim 1 or 2 or a method according to any one of claims 3 to 5 wherein the anti-apoptotic protein is
- a polypeptide having activity of an A20 protein; or
- a polypeptide having activity of BCL-2 protein, a homodimer of that polypeptide, or a heterodimer of that polypeptide and another anti-apoptotic polypeptide of the
BCL family; or
- a polypeptide having activity of BCL-XL protein, a homodimer of that polypeptide, or a heterodimer of that polypeptide and another anti-apoptotic polypeptide of the
BCL family; or
- a polypeptide having activity of A l protein, a homodimer of that polypeptide, or a heterodimer of that polypeptide and another anti-apoptotic polypeptide of the
BCL family.
7. A cell according to claim 1 or 2 which is porcine.
8. A cell according to claim 1 or 2 which is human.
9. A non-human transgenic or somatic recombinant mammal comprising DNA encoding an anti-apoptotic protein of a different species.
10. A mammal according to claim 9 which is porcine.
1 1. A mammal according to claim 10 wherein the anti-apoptotic protein is human.
PCT/EP1997/000676 1996-02-14 1997-02-13 Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions WO1997030083A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU18730/97A AU1873097A (en) 1996-02-14 1997-02-13 Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions
JP09528990A JP2000510326A (en) 1996-02-14 1997-02-13 Anti-apoptotic gene therapy for transplantation and inflammatory conditions
EP97905019A EP0886650A1 (en) 1996-02-14 1997-02-13 Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US60151596A 1996-02-14 1996-02-14
US08/601,515 1996-02-14
US63499596A 1996-04-19 1996-04-19
US08/634,995 1996-04-19

Publications (1)

Publication Number Publication Date
WO1997030083A1 true WO1997030083A1 (en) 1997-08-21

Family

ID=27083879

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/000676 WO1997030083A1 (en) 1996-02-14 1997-02-13 Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions

Country Status (5)

Country Link
EP (1) EP0886650A1 (en)
JP (1) JP2000510326A (en)
AU (1) AU1873097A (en)
CA (1) CA2245503A1 (en)
WO (1) WO1997030083A1 (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998019712A1 (en) * 1996-11-08 1998-05-14 St. Elizabeth's Medical Center Of Boston, Inc. Methods for regulating angiogenesis
WO1999013073A2 (en) * 1997-09-08 1999-03-18 Rpr Gencell Asia/Pacific Inc. Viral vector system capable of expressing an apoptosis-associated gene
WO1999055382A1 (en) * 1998-04-29 1999-11-04 The Uab Research Foundation Adenoviral vector encoding anti-apoptotic bcl-2 gene and uses thereof
WO2001011031A2 (en) * 1999-05-27 2001-02-15 University Of Pittsburgh Of The Commonwealth System Of Higher Education Gene transfer to pancreatic beta cells for prevention of islet dysfunction
WO2001067110A1 (en) * 2000-03-08 2001-09-13 Akzo Nobel N.V. Synergistic activation of regulatory elements by rel proteins and a steroid receptor
WO2001081387A1 (en) * 2000-04-25 2001-11-01 Jin Woo Kim Human cervical cancer suppressor protein, polynucleotide encoding the protein, cell transformed with the polynucleotide and method for suppressing proliferation of cancer cell using the expression vector
KR100434591B1 (en) * 2002-04-16 2004-06-04 김진우 Human cancer suppressor gene, protein encoded therein, expression vector containing same, and cell transformed by said vector
US7282488B2 (en) 2000-05-22 2007-10-16 The Johns Hopkins Univeristy Genetic engineering of vascular grafts to resist disease
US7297685B2 (en) 2000-01-21 2007-11-20 Beth Israel Deaconess Medical Center Use of pro-apoptotic factors in treatment of atherosclerosis
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

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007573A1 (en) * 1990-10-31 1992-05-14 Somatix Therapy Corporation Genetic modification of endothelial cells
WO1992020795A1 (en) * 1991-05-17 1992-11-26 Cetus Oncology Corporation INHIBITOR OF NF-λB TRANSCRIPTIONAL ACTIVATOR AND USES THEREOF
WO1993020219A1 (en) * 1992-04-06 1993-10-14 The Government Of The United States As Represented By The Secretary, Department Of Health And Human Services Control and/or prevention of binding of nf-kb/rel/dorsal (nrd) family proteins to dna
WO1993019605A1 (en) * 1992-04-06 1993-10-14 Ochoa Augusto C Evaluation and treatment of patients with progressive immunosuppression
WO1994010305A1 (en) * 1992-11-02 1994-05-11 Sandoz Ltd. Transformed endothelial cells
WO1995000642A1 (en) * 1993-06-22 1995-01-05 Arch Development Corporation Vertebrate apoptosis gene: compositions and methods

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007573A1 (en) * 1990-10-31 1992-05-14 Somatix Therapy Corporation Genetic modification of endothelial cells
WO1992020795A1 (en) * 1991-05-17 1992-11-26 Cetus Oncology Corporation INHIBITOR OF NF-λB TRANSCRIPTIONAL ACTIVATOR AND USES THEREOF
WO1993020219A1 (en) * 1992-04-06 1993-10-14 The Government Of The United States As Represented By The Secretary, Department Of Health And Human Services Control and/or prevention of binding of nf-kb/rel/dorsal (nrd) family proteins to dna
WO1993019605A1 (en) * 1992-04-06 1993-10-14 Ochoa Augusto C Evaluation and treatment of patients with progressive immunosuppression
WO1994010305A1 (en) * 1992-11-02 1994-05-11 Sandoz Ltd. Transformed endothelial cells
WO1995000642A1 (en) * 1993-06-22 1995-01-05 Arch Development Corporation Vertebrate apoptosis gene: compositions and methods

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
A. KARSAN ET AL.: "cloning of a human Bcl-2 homologue: inflammatory cytokines induce human A1 in cultured endothelial cells", BLOOD, vol. 87, no. 8, 15 April 1996 (1996-04-15), PHILADELPHIA, PA,US, pages 3089 - 3096, XP000197498 *
A. KRIKOS ET AL.: "Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappaB elements", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 25, 1992, BETHESDA,MD, US, pages 17971 - 17976, XP000673587 *
C. FERRAN ET AL.: "Adenovirus-mediated gene transfer of A20 renders endothelial cells resistant to activation: A means of evaluating the role od endothelial cell activation in xenograft rejection", TRANSPLANTATION PROCEEDINGS, vol. 29, no. 1-2, 1997, STAMFORD, CT,US, pages 879 - 880, XP000197598 *
C. FERRAN ET AL: "Inhibition of NF-kappa-B by pirrolidine dithiocarbamate blocks endothelial cell activation", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 214, no. 1, 1995, ORLANDO, FL, pages 212 - 223, XP000676429 *
J.T. COOPER ET AL.: "A20 blocks endothelial cell activation through a NF-kappa-B-dependent mechanism", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 30, 26 July 1996 (1996-07-26), BETHESDA,MD,US, pages 18068 - 18073, XP000677230 *
J.T. COOPER ET AL.: "A20 expression inhibits endothelial cell activation", TANSPLANTATION PROCEEDINGS, vol. 29, no. 1-2, 1997, STAMFORD, CT, US, pages 881, XP000197597 *
K-I. LIN ET AL.: "Bcl-2 suppresses apoptosis and activation of NF-kappa B induced by Sindbis virus infection", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 21, no. 2, 1995, BETHESDA,MD,US, pages 1068, XP000197612 *
M. JAATTELA ET AL.: "A20 zinc finger protein inhibits TNF and Il-1 signaling", JOURNAL OF IMMUNOLOGY, vol. 156, no. 3, 1 February 1996 (1996-02-01), BETHESDA, MD,US, pages 1166 - 1173, XP000676426 *
M. TEWARI ET AL.: "Lymphoid expression and regulation of A20, an inhibitor of programmed cell death", JOURNAL OF IMMUNOLOGY, vol. 154, 1995, BETHESDA, MD, US, pages 1699 - 1706, XP000673576 *
W. FANG ET AL.: "Bcl-XL rescues WEHI 231 B Lymphocytes from oxidant-mediated death following diverse apoptotic stimuli", JOURNAL OF IMMUNOLOGY, vol. 155, 1995, BETHESDA, MD, US, pages 66 - 75, XP000673577 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (en) * 1996-11-08 1998-05-14 St. Elizabeth's Medical Center Of Boston, Inc. Methods for regulating angiogenesis
US9095580B2 (en) 1996-11-08 2015-08-04 Genesys Research Institution Inc. Compositions and methods for regulating angiogenesis
EP1618898A3 (en) * 1996-11-08 2007-12-12 Caritas St. Elizabeth's Medical Center of Boston, Inc. Methods for regulating angiogenesis
WO1999013073A2 (en) * 1997-09-08 1999-03-18 Rpr Gencell Asia/Pacific Inc. Viral vector system capable of expressing an apoptosis-associated gene
WO1999013073A3 (en) * 1997-09-08 1999-06-10 Rpr Gencell Asia Pacific Inc Viral vector system capable of expressing an apoptosis-associated gene
WO1999055382A1 (en) * 1998-04-29 1999-11-04 The Uab Research Foundation Adenoviral vector encoding anti-apoptotic bcl-2 gene and uses thereof
WO2001011031A2 (en) * 1999-05-27 2001-02-15 University Of Pittsburgh Of The Commonwealth System Of Higher Education Gene transfer to pancreatic beta cells for prevention of islet dysfunction
WO2001011031A3 (en) * 1999-05-27 2001-08-23 Univ Pittsburgh Gene transfer to pancreatic beta cells for prevention of islet dysfunction
US7297685B2 (en) 2000-01-21 2007-11-20 Beth Israel Deaconess Medical Center Use of pro-apoptotic factors in treatment of atherosclerosis
WO2001067110A1 (en) * 2000-03-08 2001-09-13 Akzo Nobel N.V. Synergistic activation of regulatory elements by rel proteins and a steroid receptor
US7252934B2 (en) 2000-03-08 2007-08-07 N.V. Organon Synergistic activation of regulatory elements by Rel proteins and a steroid receptor
JP2003526346A (en) * 2000-03-08 2003-09-09 アクゾ・ノベル・エヌ・ベー Synergistic activity of regulatory molecules by Rel protein and steroid receptor
WO2001081387A1 (en) * 2000-04-25 2001-11-01 Jin Woo Kim Human cervical cancer suppressor protein, polynucleotide encoding the protein, cell transformed with the polynucleotide and method for suppressing proliferation of cancer cell using the expression vector
US7282488B2 (en) 2000-05-22 2007-10-16 The Johns Hopkins Univeristy Genetic engineering of vascular grafts to resist disease
KR100434591B1 (en) * 2002-04-16 2004-06-04 김진우 Human cancer suppressor gene, protein encoded therein, expression vector containing same, and cell transformed by said vector
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 (en) * 2004-10-04 2012-11-28 University of Washington Methods of inhibiting cell death or inflammation in a mammal
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

Also Published As

Publication number Publication date
JP2000510326A (en) 2000-08-15
EP0886650A1 (en) 1998-12-30
CA2245503A1 (en) 1997-08-21
AU1873097A (en) 1997-09-02

Similar Documents

Publication Publication Date Title
KR101629071B1 (en) Engineered cells expressing multiple immunomodulators and uses thereof
AU2018236775A1 (en) Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof
AU692196B2 (en) Translational enhancer DNA
US7053062B2 (en) Compositions and methods for inducing gene expression
US5972605A (en) Assays for regulators of mammalian telomerase expression
WO1997030083A1 (en) Gene therapy of entothelial cells with anti-apoptotic proteins for transplantation and inflammatory conditions
BG60624B1 (en) Endogenous modification of gene expression by regulatory element
WO1997016533A1 (en) Mammalian artificial chromosomes and methods of using same
JP2002507895A (en) Transcriptional activator with stepwise transactivation ability
US20060294610A1 (en) Alpha1-3 galactosyltransferase gene and promoter
US20110088106A1 (en) Compositions and methods for mediating cell cycle progression
US20040224389A1 (en) Viral vectors encoding apoptosis-inducing proteins and methods for making and using the same
KR100558288B1 (en) Transgenic animal model for degenerative diseases of cartilage
AU680636B2 (en) Transformed endothelial cells expressing proteins having IKB activity
JP2002543792A (en) Supply and integration of transposon sequences by vector
WO1996022370A9 (en) Human fas gene promoter region
WO1996022370A1 (en) Human fas gene promoter region
Roberts et al. Smooth muscle alternative splicing induced in fibroblasts by heterologous expression of a regulatory gene.
JP2000500341A (en) Generation of mammalian somatic mosaicism by recombinant substrates
WO1997012040A1 (en) GENE THERAPY WITH MODIFIED p65 PROTEINS
WO2001027256A9 (en) Chimeric transcriptional regulatory element and methods for prostate-targeted gene expression
JP2004526404A (en) Method of regulating angiogenesis by controlling the expression of pituitary tumor transforming gene (PTTG)
EP1373498A2 (en) Animal tissue with carbohydrate antigens compatible for human transplantation
AU2002242538A1 (en) Animal tissue with carbohydrate antigens compatible for human transplantation
WO2006113220A2 (en) Transgenic mammals expressing human preproinsulin

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1997905019

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2245503

Country of ref document: CA

Ref country code: CA

Ref document number: 2245503

Kind code of ref document: A

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997905019

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997905019

Country of ref document: EP