JP4064424B2 - Transplantation and gene therapy for inflammatory or thrombotic conditions - Google Patents

Description

Detailed Description of the Invention

The present invention provides improvements in the fields of gene therapy and tissue and organ transplantation. In a broad aspect, the present invention relates to genetic modification of endothelial cells that reduces the sensitivity of the endothelial cells to inflammation or other activation stimuli.

  In particular, the present invention relates to genetic modification of endothelial cells undergoing platelet-mediated activation stimulation, and the endothelial cells inhibit platelet aggregation by expressing functional ATP diphosphohydrolase activity under endothelial cell activation or inflammatory conditions. It is something that can be done.

  In a preferred embodiment, the present invention is directed to a novel use of a polypeptide or class of polypeptides already identified as B cell activation markers, CD39. At present, CD39 is a cell surface glycoprotein associated with B lymphocytes, activated NK cells, certain T cells and endothelial cells, and so far no cell-specific functions have been assigned, It has been found to exert ATP- and ADP-degrading activity, ie ATP-diphosphohydrolase activity. Therefore, the novel use of CD39 contemplated by the present invention involves the suppression or inhibition of ADP-induced platelet aggregation and thrombus formation, particularly under cell activation conditions or with tissue inflammation. Accordingly, the present invention, in its further aspects and embodiments, genetically modifies mammalian cells and tissues or organs containing the cells so that such cells, organs or tissues can express CD39 protein and the expressed proteins Is maintained at a sufficient level under cell activation conditions, thereby inhibiting or inhibiting platelet aggregation (ie thrombus formation) on the cell surface.

  The present invention also encompasses the use of the CD39 protein or the gene encoding it in connection with further embodiments generally disclosed herein for ATP diphosphohydrolase active protein.

BACKGROUND OF THE INVENTION Thromboembolism can be attributed to various atherosclerotic and thrombotic conditions such as acute myocardial infarction, chronic unstable angina, transient cerebral ischemic attack, carotid endarterectomy, peripheral vascular disease, restenosis, And / or included in many vascular diseases including thrombosis after angioplasty or after anastomosis of a cardiovascular device such as a catheter or shunt. Other relevant are preeclampsia and various forms of vasculitis, such as Takayasu and rheumatic vasculitis. Importantly, in the field of allogeneic or xenotransplantation, thrombus formation in the vascular system of the graft is a serious problem affecting the viability of the transplanted tissues and organs.

  A recognized predisposition to the body's complex physiological thrombogenic mechanisms is the succession of events that cause platelet activation (also called platelet “adhesion” and “aggregation”). In summary, the endothelium (also known as “vascular endothelium”) consists of a layer of cells that line the heart and inside the lumen of blood vessels and lymph vessels. The “activation” process of endothelial cells by platelet and leukocyte-mediated damage and inflammation associated with the release of activators such as the cytokine TNFα has been described in the literature. See F. Bach et al., Immunological Reviews 141 (1994) 5-30 and Pober and Cotran, Transplantation 52 (1991) 1037-1042. A phenomenon associated with this process is the retraction of the endothelial surface and the expression of components of the subendothelial matrix such as collagen and von Willebrand factor (vWF).

  Accompanying endothelium “activation”, platelets circulating freely in the blood are usually “activated” by constituents of the exposed subendothelial matrix, as well as by thrombus and activated complement components. become. In this activated state, platelet glycoprotein (GP) IIb / IIIa and P-selectin expression is increased, increasing affinity for endothelium and subendothelial components. In addition, platelets begin to secrete biologically active components, particularly adenine nucleotides, ATP and ADP. ADP is paramount to a continuous platelet activation response and leads to further platelet recruitment. ATP also stimulates neutrophils through its P2y receptor, resulting in increased release of reactive oxygen intermediates. As each event continues to interrelate, platelet "aggregation" begins with the binding of ADP and agonists such as thrombin, epinephrine, ADP, collagen, and thromboxane A2 to the platelet membrane receptor. As a result of agonistic stimulation, latent fibrinogen receptor appears on the platelet surface and eventually fibrinogen binds to the platelet GPIIb / IIIa receptor complex, which is responsible for platelet aggregation and thrombus formation in vivo. It is considered to be a cause.

  Counteracting the platelet aggregation process is primarily a potent antithrombotic mechanism localized in the endothelium, such as (i) release of prostacyclins, (ii) production of nitric oxide and (iii) ADP-degradation There are various activities such as enzyme activity and fibrinolysis mechanism. However, these mechanisms are ineffective, failing to prevent many inflammatory vascular abnormalities or failing to maintain graft survival, resulting in very uncontrolled platelet activation and aggregation, ultimately It is obvious that vascular occlusion and platelet thrombosis may occur.

  Graft damage and loss associated with graft preservation-induced endothelial damage and in the case of allograft and xenograft rejection are thrombosis under conditions where endothelial tissue is activated This is an example of being easily affected by the disease. For example, after graft vasculature anastomosis, recipient platelets begin to interact with the endothelium and subendothelial cells of the graft. Activation of the graft endothelium in an inflammatory environment can initiate the platelet aggregation cascade, resulting in platelet adhesion and aggregation on the graft endothelium, making the graft more susceptible to thrombosis, Ultimately, graft dysfunction occurs.

  Considerable efforts have been made by researchers in the field to determine drug components that can control platelet aggregation. However, the antiplatelet agents currently in clinical use have side effects and are disadvantageous in lack of selectivity. Relatively new GPIIb / IIIa antagonists such as peptides, peptidomimetics, and antibodies are more selective and potent but do not provide a preventive function in the early stages of inflammation or injury. Certain purinergic P2T receptor antagonists and to some extent PAF antagonists have similar disadvantages. Thus, there is a great need for methods to prevent or minimally inhibit platelet aggregation that occurs in connection with endothelial cell activation. In particular, there is a need to prolong transplant organ survival while minimizing the toxicity and other adverse effects associated with available platelet activation inhibitors.

Summary of the Invention It has now been found that the regulation and inhibition of platelet aggregation under cell activation conditions is highly dependent on the maintenance of ecto ATP-diphosphohydrolase activity by endothelial cells. More specifically, activation of endothelial cells (hereinafter “EC”) in response to immunity or inflammatory stimuli leads to a reduction or loss of ADP-hydrolysis activity on the surface of the cells, and this ADP -It has been found that the reduction or loss of hydrolytic activity results in platelet adhesion and platelet aggregation to the endothelial cell surface, ultimately leading to thrombus formation.
In particular, the ability of EC to express cell-bound ATP-diphosphohydrolase activity capable of inhibiting platelet activation without an activator and conditions that enhance EC activation (eg, exposure to TNFα / complement) And xenograft hyperacute rejection / reperfusion injury / oxidative invasion) observed to have reduced or lost ecto-ATP-diphosphohydrolase activity resulting in a cellular environment with increased sensitivity to platelet aggregation It was done.

  Furthermore, the activity of native mammalian / pig ATP diphosphohydrolase was found to be susceptible to oxidation and, when oxidized, the protein loses its ability to inhibit platelet activation. At present, this phenomenon is believed to play a critical role in many pathogenic conditions including platelet aggregation and thrombus formation seen in graft rejection. Many medical conditions or disease states that require treatment aimed at inhibiting platelet aggregation are associated with high levels of toxic oxygen radicals and other reactive oxygen intermediates. One example of such a pathology is graft preservation injury and ischemia-reperfusion. The resulting disease states are reperfusion injury associated with myocardial infarction, disseminated intravascular coagulation associated with sepsis, alveolar fibrosis associated with adult respiratory syndrome and noncardiac pulmonary edema. Furthermore, injury to the endothelium is associated with the influx of activated monocytes, polymorphonuclear leukocytes, etc., which can also create toxic oxygen species.

  So far, we have recognized the general association with endothelial cell damage, inflammation and thrombosis, but the present invention first shows the reduced ability of the enzyme ATP diphosphohydrolase to prevent platelet aggregation under oxidatively invasive conditions. It was confirmed. This new feature is very important in the treatment of many pathological conditions that require restoration of cellular platelet activation-suppression function or antithrombotic function.

  Peptide sequences with significant (eg 95% or more, typically 98% or more, eg 99% or more, or even 100%) homology corresponding to type I and type II ecto-ATP diphosphohydrolase For example, Christoforidis et al., Eur. J. Biochem. 234 (1) (November 15, 1995) 66-74, and CD39 lymphocyte activation markers [CRMaliszewski et al., J. Immunol. 153 (1994) 3574-3583]. It has not previously been recognized in the art that the CD39 protein or class of proteins encodes an ATP hydrolyzing function, in particular ecto-ATP diphosphohydrolase.

  Thus, the term “ATP diphosphohydrolase” or “ecto-ATP diphosphohydrolase” refers to and includes the native CD39 protein, particularly the native human CD39 protein.

  Accordingly, the present invention relates in its broader aspects to a method of genetically modifying mammalian cells, such as endothelial cells, to reduce their susceptibility to inflammation or immunological stimulation and platelet adhesion, the method comprising: The ability to stably express a polypeptide having ATP diphosphohydrolase activity under cell activation conditions, i.e., ATP diphosphohydrolase at a level sufficient to inhibit or inhibit platelet adhesion or aggregation on the cell surface. Providing such cells with the ability to express.

  “Stable” expression means that transcription and expression of the ATP diphosphohydrolase protein or analog thereof by the cell is maintained in an effective amount of antithrombosis (ie, platelet plug / thrombosis suppression). . The concentration of such a protein may be the same as expressed by the cell under hemostatic conditions, or may be higher or even lower, however, such a concentration of ATP diphosphohydrolase protein. “Stable” expression is not modified according to the present invention, ie, in the vascular system of the local microenvironment of the cell as compared to activation conditions similar to cells containing no inserted gene / protein, ie It is sufficient to cause reduction or suppression of platelet aggregation and platelet thrombus on the modified cell surface.

  “Cell activation conditions” refers to type I EC activation (representing initial post-stimulation events, including continuous intrusion of EC and bleeding and edema); and / or type II EC activation (over time) (Representing late events that occur and depend on transcriptional regulation and protein synthesis) (see Bach et al., Supra). A generally accepted indicator of type I EC activation is high levels of PAF and / or P-selectin in the cellular environment. A generally accepted indicator of type II EC activation is high levels of E-selectin in the cellular environment or cell membrane.

  Inhibition or inhibition of platelet adhesion or aggregation on the surface of cells modified according to the present invention is described, for example, in Marcus et al., J. Clin. Investig. 88 (1988) 1690-1696 and Born, Nature 194 (1962) 927-930. It can be measured by known methods as described [reviewed in Peerschke, Semin. Hematol. 22 (1985) 241]. A reduction of more than 50% and preferably more than 65% of platelet aggregate formation on the cell surface represents the platelet inhibition or suppression that is the object of the present invention.

  The stable or high level of ADP-hydrolysis activity provided by the present invention is achieved by using a vector construct comprising a polypeptide having ATP-diphosphohydrolase activity, in particular a DNA encoding an ATP diphosphohydrolase protein, It can be obtained under the control of a promoter capable of initiating transcription of DNA under activated or oxidatively invasive conditions, thus replacing the normally present activity of ATP diphosphohydrolase. Examples of such promoters include “constitutive” or “inducible” promoters.

  “Constitutive” means that protein expression is substantially independent of cell activators and is substantially continuous over the life of the cell.

  “Inducible” is controlled by the administration of an exogenous factor where protein expression is either typically absent from the cellular environment or either disappears or diminishes from the cellular environment under activated conditions. It can be done. Such exogenous factors include cytokines or growth factors.

  It is also within the scope of the present invention to achieve “stable” ATP-diphosphohydrolase activity by providing peptides having ADP-hydrolysis activity under oxidizing conditions. Thus, the present invention provides peptide analogs that have the activity of a natural ATP-diphosphohydrolase such as CD39, preferably human CD39 protein, and are substantially oxidation resistant.

  Also concomitant with the co-administration of an antioxidant to diseased cells, tissues or organs, concomitant with expression of ecto-ATP diphosphohydrolase.

  Accordingly, the present invention, in its more specific aspects, genetically modifies mammalian cells, such as endothelial cells and monocytes, NK cells, lymphocytes, erythrocytes and islet cells, which inhibit platelet aggregation. A method comprising the steps of: a functional ecto-ATP diphosphohydrolase protein or an oxidation resistant analogue thereof, which encodes a polypeptide having ATP diphosphohydrolase activity in the cell or their progenitor cells Operably linked to an inducible promoter, and expressing such a polypeptide, particularly ecto-ATP diphosphohydrolase, from the cells at an effective level of inhibiting platelet aggregation under cell activation conditions Consists of.

  “Functional” means that the expressed ATP-diphosphohydrolase of such cells hydrolyzes platelet secreted ADP to AMP and monophosphate.

  The present invention also includes a method of controlling platelet aggregation, thereby preventing or reducing a thrombotic condition in a mammalian subject in need of treatment, wherein the subject's cells are sensitive to platelet-mediated activation. Preferably, endothelial cells are genetically introduced by inserting DNA encoding a polypeptide having ATP diphosphohydrolase activity or their oxidation resistant analogs, particularly in operative association with a suitable promoter. Modifying and expressing the polypeptide from such cells at an effective level of inhibiting platelet aggregation. Preferably, the cells are modified in vivo, i.e. while in the subject's body.

  In other embodiments, the cell population can be removed from the patient, genetically modified ex vivo by insertion of vector DNA, and then reimplanted into the subject. The subject is preferably a human.

  In a further aspect, the present invention relates to donor allogeneic or xenogeneic cells, preferably endothelial cells or transplantable tissues or organs containing such cells, to which these cells or tissues or organs are responsive to activation stimuli in their blood or plasma. Including transplanting to a susceptible mammalian recipient, the method comprising:

  (A) By inserting such donor cells or their progenitor cells into them, operably linked to a promoter, a DNA encoding a polypeptide having the activity of ATP diphosphohydrolase or their oxidation-resistant analog Genetically modified;

(B) The obtained modified donor cell, tissue, or organ is transplanted into a recipient, and a polypeptide having ATP diphosphohydrolase activity is expressed from the obtained modified cell, tissue, or organ at an effective level for inhibiting platelet aggregation. Consists of.

  “Modified donor cells” in step (b) refer to the cells that are themselves the subject of the genetic modification in step (a), as well as their progenitor cells. These also form part of the present invention.

  Steps (a) and (b) can be performed in any order; that is, the donor allogeneic or xenogeneic cell, tissue, or organ is modified or genetically manipulated (eg, before or after transplantation into the recipient) By transfection, transduction or transformation).

  For example, endothelial cells from porcine tissues or organs can be genetically modified in vivo by inserting DNA encoding human ATP-diphosphohydrolase proteins or their oxidation resistant analogs under the control of a promoter, The modified cell or tissue is then used to transplant into a human recipient. Once transplanted, the transgenic cells or tissues or organs express functional human ecto-ATP-diphosphohydrolase or their oxidation-resistant analogs, even in the presence of other down-regulation factors and in an inflammatory environment.

  For example, since porcine or bovine ATP-diphosphohydrolase factors have cross-species activity, porcine or bovine protein expression transgenic (or somatic cell recombinant) animals can be used for transplantation into humans. It can be usefully employed for cell, tissue and organ replacement. However, preferably, human proteins or analogs are used in appropriate vectors to modify porcine donor cells or organs and make them transgenic (or somatic recombination) for transplantation purposes.

  A somatic recombination or transgenic donor animal can modify the animal's cells, or earlier, eg, at the embryonic stage, by well-known techniques to produce an animal that expresses the desired protein. Can be obtained.

  Donor cells or tissues can also be genetically modified ex vivo, whereby cells, tissues or organs that have been extracted from the donor and maintained in culture are genetically modified as described above, and then When transplanted into a recipient, the graft can then express the desired functional protein.

  The genetic modification of the donor is preferably performed in vivo.

  In accordance with a further aspect of the present invention, expression of a DNA encoding a polypeptide having ATP-diphosphohydrolase activity at a platelet-suppressing effective level under cell activation conditions in allogeneic or xenograft recipients as donors Provided is a cell of a donor mammalian species, in particular an endothelial cell or tissue or organ, modified as possible.

  The present invention further provides a non-human transgenic or somatic recombination comprising heterologous DNA encoding a polypeptide having ATP-diphosphohydrolase activity in the cell under cell activation conditions, particularly in the endothelial cell. Methods are provided for the production of mammals, and such cells, tissues and organs themselves, and such non-human transgenic or somatic recombinant mammals. Such non-human transgenic or somatic recombinant mammals are of particular porcine species, however, murine transgenics expressing human ATP diphosphohydrolase are also within the scope of the present invention.

  Methods of inhibiting platelet aggregation and thereby treating thrombotic abnormalities in a mammalian (eg, human) subject, and such polypeptides or pharmaceutically acceptable salts thereof, or they in a pharmaceutically acceptable carrier Also comprising a pharmaceutical composition comprising, preferably in soluble form, the method comprising subjecting a subject to a recombinant peptide having ATP-diphosphohydrolase activity or a pharmaceutically acceptable salt thereof, Or administering an effective amount of a platelet aggregation inhibitor of their oxidation resistant analogs.

  Further encompassed are artificial venous organ devices comprising synthetic biocompatible materials coated with recombinant ATP-diphosphohydrolase or their oxidation resistant analogs.

  Such treatment is useful for alleviating thrombotic conditions in a patient, and in particular, ameliorating thrombotic complications that occur with organ transplantation, particularly when the graft recipient is a human. The invention further relates to recombinant polypeptides having ATP diphosphohydrolase activity or their pharmaceutically acceptable salts, or their oxidation resistant analogues, particularly in the manufacture of a medicament for reducing platelet aggregation in thrombosis In particular, the use of human CD39 protein.

DESCRIPTION OF THE FIGURES FIG. 1: Regulation of ecto-ADPase: bar graph showing the inhibitory effect of human γTNFα on ecto-ATP diphosphohydrolase activity:
■ = TLC nmol ADP / million cells / min;
□ = LeBel / Fiske μmol phosphate / hour / mg cell protein [Example 1 (c)]. γTNFα = recombinant tumor necrosis factor α.
FIG. 2: LWB (RheinWeaver Berke) ectoADPase (double reciprocal plot of enzyme kinetics): This shows the kinetics of stationary phase and cytokine-mediated PAEC.
■ = Control; □ = TNF
[Example 1 (d)].
FIG. 3: Inhibition of ecto-ADPase activity by oxidative invasion and cell activation (HOOH 5 μM / ecto-ADPase): bar graph showing peroxide and cytokine-mediated loss of ecto-ATP diphosphohydrolase activity against PAEC [Example 2 (a)]. BME = β-mercaptoethanol.
FIG. 4: Protective effect of β-mercaptoethanol on ecto-ADPase activity: bar graph showing that β-mercaptoethanol (BME) protects the cytokine-mediated loss of ecto-ATP-diphosphohydrolase activity against PAEC. [Example 2 (b)]. BME = β-mercaptoethanol.
FIG. 5: Ecto-ADPase regulation kinetics: bar graph showing ecto-ATP diphosphohydrolase regulation kinetics by TNFα and oxidizing agents: (in order on the graph) ■ = control; □ = XO / X; ■ = HOOH; = TNF
[Example 2 (c)]. XO / X = xanthine oxidase / xanthine; HOOH = hydrogen peroxide.
FIG. 6: Modulation of ecto-ADPase activity by antioxidants: Plot of ecto-ATP diphosphohydrolase activity of activated PAECs treated with antioxidants [Example 3]. SOD = superoxide dismutase; Cat = catalase.
FIG. 7: Reperfusion injury: bar graph showing ecto-ATP diphosphohydrolase activity in purified rat glomeruli as a function of in vivo reperfusion time [Example 5]. Isch = ischemic time (minutes); Reperf = reperfusion time (minutes).
FIG. 8: Effect of CVF: bar graph showing the effect of pretreatment of rat glomeruli with ischemia followed by reperfusion with cobra venom factor (CVF).
FIG. 9: Northern analysis of CD39: HUVEC following TNFα stimulation shows a decrease in the level of mRNA for CD39 [Example 7]. hEC = HUVEC = human umbilical vein endothelial cells; TNF = recombinant tumor necrosis factor.
FIG. 10: Transient transfection of COS-7 cells with pCDNA3 / CD39: FACS analysis of non-transfected COS-7 cells and COS-7 cells transfected with CD39 cDNA. Analysis by moAB (= monoclonal antibody) against CD39. Use isotype control simultaneously. Cells were stained with moAB (exact) against human CD39.
FIG. 11: EctoADPase activity of CD39-transfected COS-7 cells: Whole cell lysates of COS-7 cells transfected with CD39 cDNA express specific Ca ⁺⁺ ecto-ADPase activity (substrate = 200 μM) ADP). First bar: control; second bar: empty vector; third bar: CD39 vector.
FIG. 12: Ecto-ADPase activity of purified membranes of COS-7 cells transfected with CD39: the activity was mainly localized at the cell membrane. First bar: control COS cells; second bar: COS cells transfected with empty vector; third bar: COS cells transfected with CD39 vector.
FIG. 13: Platelet aggregation assay: Inhibition of platelet aggregation by CD39; PRP aggregation with 5 μM ADP and COS-7 cell membrane extract (27.4 μg protein). COS-7 cell membrane extract from CD39-transfected cells effectively inhibits platelet aggregation induced by 5 μM ADP and establishes the functional potential of CD39 / ectoADPase protein.
FIGS. 14-1, 14-2 : human CD39 nucleotide and amino acid sequence (from J. Immunol. 153 (8) [1994] 3577) (= SEQ ID NO: 1).

Definitions “Transplant”, “Transplant”, or “Inner Transplant” are used interchangeably and refer to biological material obtained from a donor and such biological material for transplantation to a recipient. Represents the act of placing in the entry.

  “Host” or “recipient” refers to the body of a patient into which a donor biological material is transplanted.

  “Homogeneous” refers to donors and recipients of the same species. As a subset of this, “syngeneic” refers to a condition in which the donor and recipient are genetically identical. “Self” means that the donor and the recipient are the same individual. “Xenogeneic” and “xenograft” refer to the situation where the graft donor and recipient are different species.

  “ATP diphosphohydrolase”: an enzyme capable of catalyzing the continuous hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) to adenosine monophosphate (AMP) (this enzyme is ADPase; ATPDase; ATPase; ADP monophosphatase; or apyrase; sometimes expressed as EC 3.6.1.5).

  The term “polypeptide having the activity of ATP diphosphohydrolase” includes naturally occurring ecto-ATP diphosphohydrolase proteins, as well as their oxidation resistant peptide analogs and soluble truncated forms.

  An example of ecto-ATP diphosphohydrolase is the CD39 protein. “CD39” refers to a native mammalian gene (including its cDNA) or protein, and variations in the DNA or amino acid sequence that do not damage the ATP-hydrolysis activity of the protein (eg, minor variations up to 5 amino acids or And their derivatives with deletions). The CD39 gene or protein employed in the present invention may be, for example, porcine, bovine or human, and may also be a non-human primate. Depends on target recipient species. The term “human CD39” as used herein refers to CD39 lymphocyte activity cloned from a human B cell lymphoblast cell line by CRMaliszewski et al. In J. Immunol. 153 (8) (1994) 3574-3584. The amino acid sequence of the activating marker (Genebank / NCBI accession number 765256: March 23, 1995) and at least 70%, preferably at least 80%, more preferably at least 90% (eg 95% or more, eg 99% or 100%) represents a protein that is homologous [SEQ ID NO: 1].

DETAILED DESCRIPTION OF THE INVENTION ATP diphosphohydrolases consist of a family of proteins that catalyze the continuous phosphorolysis (ie, removal of phosphate groups) of ATP to ADP or AMP. In general, this class of proteins does not show specificity for nucleoside di or triphosphate; it is activated by Ca ²⁺ or Mg ²⁺ . By converting ADP to AMP and also ATP to AMP via ADP, these enzymes inhibit or reverse platelet aggregation. The final product, AMP, is a substrate for 5 'nucleotidase, makes adenosine, and is an important platelet anti-activator and vasodilator.

  This protein is found primarily in the cellular elements of the blood and blood vessel walls. In order for such a cellular enzyme to be effective, it should be functional on the cell surface, ie as an ectoenzyme. Since ATP diphosphohydrolases are membrane-bound insoluble proteins that are expressed on the cell surface, they are conventionally referred to as ecto-ATP diphosphohydrolases. Soluble analogs of such proteins can also be prepared and injected by known methods. For example, a soluble analog can be obtained by treating the entire protein length with a standard detergent. Alternatively, a DNA construct containing DNA encoding a functional protein causes the expressed protein to pass through the endothelial cell membrane within the vasculature by causing deletion of the membrane-spanning sequence of the gene. It can also be prepared by making it soluble and / or secretable into the adjacent environment.

  The activity of ecto-ATP-diphosphohydrolase is shown in endothelial cells, as well as leukocytes and platelets, and these proteins are thought to be widely distributed in mammalian vascular endothelium. Partial internal amino acid sequence information after chymotrypsin cleavage of ATP diphosphohydrolase isolated from a specific fraction of human full-term placenta is available [S. Christofiridis et al., Eur. J. Biochem. 134 (1) (1995 (Monday 15) 66-74]. Bovine aorta and iliac endothelium ecto-ATPase purification was introduced and outlined by J. Sevigny et al. (University of Sherbrooke, Canada) 24-25 October 1994 at the IBC Anticoagulant and Antithrombotic Meeting in Boston. Has been. Furthermore, SHLin and G. Guidotti, J. Biol. Chem. 264 (1989) 14408-14414 possess rat liver CAM-105 cDNA and polyclonal antibody, and at the same time consensus sequence (GPAYSGRET, amino acids 92-100 within the protein). ) And oligonucleotide primers corresponding to nucleotides −40 to −24 (5 ′) and 473 to 496 (3 ′) were prepared; further CJSippel et al., J. Biol. Chem. See also .264 (1994) 2800-2826; Cheung et al., J. Biol. Chem. 268 (1993) 24303-24310. Characterization of ATP diphosphohydrolase active in rat blood platelets, SSFrasetto et al., Molec. Cell. Biochem. 129 (1993) 47-55; ATP-diphosphohydrolase activity in the intima and media of the bovine aorta Characterization, YPCote et al., Biochimica et Biophysica Acta 1139 (1992) 133-142; Purification of ATP diphosphohydrolase from bovine aortic microsomes, K. Yagi et al., Eur. J. Biochem. 180 (1989) 509-513 And further studies related to the characterization and purification of calcium-sensitive ATP diphosphohydrolase from porcine pancreas, LeBel et al., J. Biol. Chem. 255 (1980) 1227-1233, have also been reported.

  Further useful to those skilled in the art are bovine and human liver endothelium cDNA libraries (eg, obtained and developed from Clontech, Palo Alto, California, USA).

  Isolation of porcine or human ecto-ATP diphosphohydrolase is performed utilizing FSBA labeling and immunodetection as described, for example, Y.P.Cote et al., Supra, or J.Sevigny et al., Supra. 5'-fluorosulfonylbenzoyladenosine (FSBA) is a specific antagonist of ectoADPase. The specific activity of the enzyme is measured as described above by LeBel et al.

Following protein purification, for example, bovine protein sequences can be measured using standard commercially available methods, such as an Applied Biosystems Sequenator. At the same time, polyclonal antibodies against bovine ATP diphosphohydrolase protein are produced. Monoclonal and / or polyclonal antibodies against the protein are produced, for example, by the techniques disclosed in Lin and Guidotti, supra and Cheung et al. Supra. When monoclonal and previously described polyclonal antibodies are obtained with at least some protein sequence information, there are two approaches to obtaining genes in bovine, porcine or human cells:
(I) using an expression library to detect colonies containing cDNA encoding ATP diphosphohydrolase using available antibodies; and (ii) to obtain an accurate cDNA element A predetermined oligomer corresponding to the amino acid sequence is used. See, eg, Lin and Guidotti, supra and Cheung et al. Supra.

  The porcine cDNA sequence can be obtained by techniques similar to those described above that probe with an appropriate antibody or oligomer. Similarly, human ecto-ATP diphosphohydrolase protein can be determined according to the methods described above or by probing human cDNA from endothelial cells or genomic libraries.

  Thereafter, the entire cDNA can be sequenced by known methods (N. Rosenthal, NEJ Med. 332 [March 2, 1995] 589-591). The resulting natural cDNA can also be expressed recombinantly in E. coli.

  The above method is described in detail in Sambrook, Fritsch and Maniatis, Molecular Cloning. A, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA (1989).

  Distribution of CD39 protein, activated NK cells, and certain T cell and endothelial cell lineages on B lymphocytes (Plesner, Inter. Rev. Cytology 158 (1995) 141-214; Maliszewski et al., Supra; Kansas et al., J. .Immunol. 146 (1991) 2235-44) is consistent with the known ectoADPase distribution. The cell surface glycoprotein CD39 has two potential transmembrane regions and induces signal transduction by the binding of certain antibodies. The reported molecular weight of the native CD39 protein is 70-100 kD with 6 potential N-glycosylation sites, and the molecular weight observed after enzymatic removal of the N-linked sugar is 54 kD (Maliszewski et al. ,the above). In addition, available logic deduced sequence data indicate that this protein is rich in cysteine (n = 11), methionine (n = 12) and tyrosine (n = 27), making it a target for oxidative damage. There are several parts to get.

  CD39 is defined as a B cell activation marker with the same traits as other markers (Engel et al., Leukemia & Lymphoma 1 [1994], 61-4). CD39 has been shown to have partial identity with yeast guanosine diphosphatase, but a role in mediating homotypic B cell adhesion and in antigen-specific responses has been reported (Maliszewski et al., Supra; Kansas et al., Supra. However, there is still no specific function listed. This antigen has been found to be expressed in endothelial cells that are said to be associated with activation-related changes in combination with over 120 other potential markers (Favaloro, Immun. Cell Biol. 71 (1993) 571). -581), it has been noted that it is expressed in the vascular endothelium, especially the skin blood vessels (Kansas et al., Supra).

  Once the natural protein of interest is sequenced, it is derivatized to increase resistance to oxidative insult (ie, mutated or truncated by known methods) Or other changes).

  Examples of physiological oxidants where oxidation resistance is maintained as desired include superoxide and hydroxyl radicals, hydrogen peroxide and hypohalous acid and related species. Oxygen free radical intermediates such as superoxide and hydroxyl radicals are generated by both normal and abnormal metabolic processes.

  Among the amino acids that make up proteins, histidine, methionine, cysteine, tryptophan, and arginine appear to be most easily oxidized. For example, oxidation of the native protein methionine can cause inactivation of the protein. Tyrosine is sensitive to nitric oxide and peroxonitrate and can thereby inactivate enzyme function. Therefore, in such a case, different amino acids can be used instead of natural methionine, as described in C.B.Glaser et al., USP 5,256,770.

  Methods for making amino acids oxidation resistant are generally known. A preferred method is to remove the affected amino acid or replace it with one or more different amino acids that do not react with the oxidizing agent. For example, amino acids such as leucine, alanine and glutamine are preferred substituted amino acids in terms of their size and neutrality characteristics. Methods by which amino acids can be removed or substituted in protein sequences are also known to those skilled in the art. Genes encoding peptides having partially altered amino acid sequences can be made synthetically [see, eg, Higuchi, PCR Protocols, Acad. Press., San Diego USA (1990) 177-183]. A preferred method involves site-specific in vitro mutagenesis, and synthetic oligodeoxyribonucleotides containing the desired nucleotide substitutions, insertions or deletions designed to specifically alter the nucleotide sequence of the target single-stranded DNA Including the use of This primer, when hybridized to a single-stranded template by primer extension, yields a heteroduplex DNA that encodes a protein sequence with the intended mutation when replicated in transformed cells.

  Mutant ecto-ATPase analogs that retain at least about 60%, more preferably at least 70%, more desirably at least 90% of normal activity after exposure to an oxidant are considered substantially resistant to oxidation. Can do.

  The present invention also includes a pharmaceutical composition having platelet aggregation inhibitory activity comprising a unit dosage sterile formulation of a soluble, preferably oxidation-resistant ecto-ATP diphosphohydrolase analog, in a pharmaceutically acceptable carrier. I will provide a.

  Administration of such analogs can be by bolus intravenous injection, constant rate intravenous infusion, or a combination of both routes.

  The present invention also contemplates biocompatible devices such as prosthetic devices coated with an oxidation resistant ecto-ATP diphosphohydrolase analog. See, for example, R.K.Ito et al., USP 5,126,140.

  The invention broadly includes a method of treating a dysfunctional or activation response of a mammalian cell (eg, an endothelial cell) to inflammation or other platelet-mediated activation stimulus, the method comprising: DNA encoding a polypeptide having ATP diphosphohydrolase activity is modified therein by inserting it in operative association with an appropriate promoter to secrete functional ecto-ATPase from the cell at an effective level and Inhibiting platelet aggregation on the cell surface by expressing.

  The invention also includes cells so modified and tissues or organs containing such cells.

  Cells or cell populations can be treated in vivo or in vitro (ex vivo) in accordance with the present invention. For example, for in vivo treatment, the ecto-ATP diphosphohydrolase vector can be inserted by direct infection in situ of a cell, tissue or organ. In that case, at least some cells of the organ are genetically modified by temporarily clamping the blood vessels of the organ (eg, kidney) from the patient's blood circulation and inserting the vector construct into the organ. A sufficient amount of time to perfuse the blood vessel with a solution containing a transmissible vector construct containing the ecto-ATP diphosphohydrolase gene; and when the clamp is removed, blood flow can be restored to the organ, Normal function resumes.

  Adenovirus-mediated gene transfer into a tube or organ by transduction perfusion is a method for genetically modifying cells in vivo as just described.

  The present invention, in a further aspect, includes a method of inhibiting platelet aggregation or thrombus formation in a subject in need of treatment, the method comprising: ATP diphosphohydrolase in a cell of a subject in a platelet mediated activated state or inflammatory state. A DNA encoding an active polypeptide is inserted in an operatively linked state with a promoter, and the polypeptide is expressed at an effective level of platelet aggregation (thrombosuppression).

  In other embodiments, the cells can be removed from the subject or donor animal, genetically modified ex vivo by insertion of vector DNA, and then reimplanted into the subject or transplanted to another recipient. In that case, for example, the organ can be removed from the patient or donor and subjected to the perfusion process described above ex vivo, and then the organ can be reimplanted into the patient or transplanted to a different recipient of the same or different species. can do.

  Ex vivo genetically modified endothelial cells may be administered to patients under certain conditions by intravenous or intraarterial injection.

In yet another embodiment, the present invention provides donor cells, or tissues or organs containing such cells, to a mammalian recipient, where these cells are sensitive to platelet-mediated activation stimuli. There are methods for transplanting, including:
(A) modifying donor cells, or their progenitor cells, by introducing into them DNA encoding a protein having ATP diphosphohydrolase activity;
(B) transplanting such modified donor cells, tissues, or organs into a recipient to express a polypeptide having ATP diphosphohydrolase activity, thereby reducing or inhibiting platelet aggregation on the recipient's cell surface To
Consists of.

  The donor species can be the same or different from the recipient species and can be any suitable species that can provide endothelial cells, tissues or organs suitable for transplantation or (tissue) transplantation.

  In a preferred embodiment, human ecto-ATP diphosphohydrolase is expressed from cells of different mammalian species that have been placed or transplanted into a human recipient. The donor may be the same or different from the recipient. The recipient is a mammal, such as a primate, and is primarily a human. However, other mammals such as non-human primates can also be suitable recipients. For human recipients, human (ie, allogeneic) and porcine (ie, xenogeneic) donors are considered appropriate, but any other mammalian species (eg, bovine or non-human primate) can serve as a donor. May be appropriate. For example, porcine arterial endothelial cells (PAEC) or their progenitor cells can be obtained from a porcine subject and genetically modified and re-transplanted (to an appropriate time to replenish for transplant) Or transplanted into other mammalian (ie, human) subjects.

  Since donor cells or tissues have been or will be transplanted into different types of transplant recipients, they will receive DNA encoding the xenograft recipient's ecto-ATP diphosphohydrolase protein. It can be somatic recombinant or transgenic in that it contains and expresses. Such cells or tissues can continue to express the desired ecto-ATP diphosphohydrolase indefinitely during its cell life. For example, when transplanted into a human recipient, porcine arterial endothelial cells (PAEC) or their progenitor cells can be genetically modified to express porcine or human ATP diphosphohydrolase protein at an effective level.

  A heterologous gene can be inserted into a germ cell (eg, an egg) to produce a transgenic animal with that gene, which is then passaged to offspring. For example, DNA encoding ATP diphosphohydrolase can be inserted into the animal or its mother ancestor at the single cell stage or early morula stage. The preferred stage is the single cell stage, but the process may be performed between 2 and 8 cell stages. Methods for producing transgenic pigs are described in W.L.Fodor and S.P.Squinto, Xeno 3 (1995) 23-26 and references cited therein.

  In other embodiments, a gene can be inserted into a somatic cell of a donor animal to provide a somatic cell recombinant animal, and the DNA construct from that animal is not capable of being passaged to offspring [eg, ADMiller and See GT Rosman, Biotechniques 7, No. 9 (1989) 980-990].

  Preferably, the inserted DNA sequence is integrated into the cell's genome. Alternatively, the inserted sequence can be maintained outside the chromosome within the cell, either stably or for a limited period of time.

  Cells, tissues, or organs can be removed from the donor and transplanted into the recipient by well-known surgical techniques. Although any mammalian cell can be targeted for ecto-ATP diphosphohydrolase gene insertion, endothelial cells are the preferred cells for operation. The modification of the endothelial cells can be by any of a variety of methods known in the art. For example, cells or tissues can be directly injected in vivo with DNA. Suitable methods for inserting foreign cells or foreign DNA into animal tissue include microinjection, embryonic stem (ES) cell manipulation, electroporation, cell cancer, transfection-k, transduction, retroviral infection, etc. .

  In other embodiments, the gene is inserted into a specific locus, such as a thrombomodulin locus or a locus containing von Willebrand factor. In order to produce a transgenic animal with such a gene, the construct is introduced into embryonic stem (ES) cells and the resulting offspring expresses the construct on its vascular endothelium.

  For gene delivery, retroviral vectors, particularly replication-deficient retroviral vectors lacking one or more of the gag, pol and env sequences required for retroviral replication, are well known in the art and transform endothelial cells. Can be used to PA317 or other producer cell lines that produce helper-free viral vectors are well described in the literature.

  Exemplary retroviral constructs include at least one viral long terminal repeat and promoter sequence upstream of the therapeutic substance nucleotide sequence and at least one viral long terminal repeat and polyadenyl downstream of the therapeutic sequence. Contains the activation signal.

  Vectors derived from adenoviruses, i.e. viruses that cause upper respiratory tract disease and also present in latent infections in primates, are also generally known in the art, and in certain situations, In particular, it is useful in terms of its ability to infect non-replicating somatic cells. The ability of adenovirus to bind cells at low ambient temperatures is also advantageous in transplants designed to facilitate gene transfer during cold storage.

  Prior to transplantation, the treated endothelial cells or tissues may be screened for genetically modified cells that contain and express the construct. To that end, the vector construct can be provided with a second nucleotide sequence encoding an expression product that confers resistance to a selectable marker substance. A suitable selectable marker for screening is the neo gene that confers resistance to neomycin or the neomycin analog G418.

  Alternative targeted gene delivery methods include DNA-protein conjugates, liposomes, and the like.

  The protein coding region and / or promoter region of the inserted DNA may be heterologous, i.e. not native to the cell. Alternatively, one or both of the protein coding region and the promoter region can be native to the cell, provided that the promoter is other than a promoter that normally regulates ATP diphosphohydrolase expression in the cell. .

  The protein coding sequence may include a suitable signal sequence, eg, a sequence encoding a nuclear specific signal sequence.

  A means of achieving expression of an anti-thrombotic effective (ie “stable”) level of ATP hydrolyzed protein, eg, CD39, under endothelial activation conditions is also available.

  Preferably, the protein coding region is under the control of a constitutive or inducible (ie, “regulatory” subset) promoter.

  The advantage of using an inducible promoter for transplantation purposes is that the desired high level transcription / expression of the active gene / protein can be delayed for an appropriate period prior to transplantation. For example, transcription can be obtained as needed in response to a predetermined stimulus, such as the presence of tetracycline in the cellular environment. Examples of tetracycline inducible promoters suitable for use in the present invention are disclosed in Furte et al., PNAS USA 91 (1994) 9302-9306. Alternatively, a regulatable promoter system that initiates transcription upon withdrawal of tetracycline has been described by Gossen and Bujard, PNAS USA 90 (1992) 5547-51.

  Preferably, the transcription / expression of the ATP diphosphohydrolase gene / protein is induced in response to a predetermined external stimulus, and the stimulus is applied just prior to applying an activation stimulus to the cell, so that expression Is already at an effective level for platelet aggregation-suppression purposes. For example, cells of a donor mammal (eg, pig) can be genetically modified according to the present invention by insertion of an ATP diphosphohydrolase gene (eg, porcine or human) under the control of a promoter induced by an agent such as tetracycline. Can be modified. Until the cells, or the tissues or organs containing the cells, are surgically removed for transplantation purposes, somatic recombinant animals or transgenic animals can be grown to the desired level of maturity under tetracycline-free conditions . In such cases, the tetracycline may be administered to the donor animal prior to surgical removal of the organ to initiate induction of high level transcription / expression of the ATP hydrolyzing gene / protein. The organ can then be transplanted into a recipient (eg, a human) and tetracycline is allowed to remain for a sufficient amount of time to maintain the desired level of ATP diphosphohydrolase protein in the transplanted cells to inhibit platelet aggregation in the recipient. Administration to the recipient may be continued. Alternatively, after surgical removal from the donor, the organ may be maintained ex vivo in a tetracycline-containing medium until suitable for transplantation into the recipient.

  In other embodiments, transcription can also occur by removing tetracycline from the cellular environment. In this case, the cells of the donor animal can be genetically modified according to the invention by inserting a gene encoding an ATP diphosphohydrolase protein under the control of a promoter blocked by tetracycline. In such cases, the animal can be grown to the desired level of maturity while administering tetracycline until the time the cells, tissues or organs are collected. Prior to surgical removal, the administration of tetracycline to the donor animal is stopped to initiate the induction of ATP diphosphohydrolase protein expression, after which patients who are transplanted with cells, tissues or organs are treated with appropriate ATP diphosphohydrolase levels It can also be maintained tetracycline-free for a time sufficient to maintain the expression of.

  In addition to the use of constitutive or inducible promoters that promote high level expression, multiple copies of DNA encoding ATP diphosphohydrolase are operably linked to a promoter that further increases gene transcription and protein expression. Can also be arranged.

  In xenotransplantation, the modified cells and donor tissues and organs defined above have the complementary function of preventing transplant rejection because the main response is hyperacute rejection. Therefore, the genetic material of the cells of the donor organ also typically changes to prevent activation of the recipient's complement pathway. This can also be done by providing a transgenic animal that expresses the complement inhibitor of the recipient species. Endothelial cells of donor organs obtained from such animals can be modified by gene therapy techniques to provide endothelial cells as defined above. Alternatively, a vector containing a DNA encoding a protein having ATP diphosphohydrolase activity can be introduced into a transgenic animal at the single cell stage or the early morula stage. In this method, the resulting transgenic animal expresses a complement inhibitor and has endothelial cells as defined above. Accordingly, in a further aspect, the present invention also relates to an endothelial cell, tissue, donor organ and non-human transgenic animal or somatic recombination as defined above that expresses one or more human complement inhibitors. Provide animals.

  Although any mammalian cell such as monocytes, NK cells, lymphocytes or islet cells can be targeted for ATP diphosphohydrolase gene insertion, the preferred cell for operation is an endothelial cell.

  In another embodiment of the invention, a polypeptide having ATP diphosphohydrolase activity can be directly acted in vivo on a cell, tissue or organ in a pharmaceutically acceptable carrier.

  In that case, the present invention also includes a method of inhibiting platelet aggregation in a warm-blooded mammal, comprising a polypeptide having ATP diphosphohydrolase activity (eg, CD39) or theirs in a pharmaceutically acceptable carrier. The method comprises administering to the animal a pharmaceutically acceptable salt in an amount effective to inhibit platelet aggregation.

  The present invention further provides antiplatelet aggregation activity comprising a unit dose of a polypeptide having ATP diphosphohydrolase activity (eg, CD39) or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier. Having a pharmaceutical composition.

  The polypeptides according to the invention or their hydrohalic acid derivatives are typically administered as pharmaceutical compositions in the form of solutions or suspensions. However, as is well known, peptides can also be formulated as tablets, pills, capsules, delayed release formulations, or powders for therapeutic administration. The production of therapeutic compositions containing polypeptides as active ingredients is well understood in the art. Typically, such compositions are made in injectable form, for example, as a liquid solution or suspension.

  Pharmaceutical compositions useful in the practice of the present invention can contain a polypeptide having ATP diphosphohydrolase activity formulated into a therapeutic composition as a pharmaceutically acceptable neutral salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide), which are inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid or oxalic acid, tartaric acid, or mandel Formed with organic acids such as acids. Salts formed with free carboxyl groups can also be inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron (III) hydroxide, or isopropylamine, trimethylamine, (2-ethyl It can also be obtained from organic bases such as amino) ethanol, histidine, or procaine.

  The therapeutic peptide-containing composition is usually administered intravenously, for example, by unit dose injection. The term “unit dose” represents a physically discrete unit suitable for a single dose to a human, each unit being pre-determined to produce the desired therapeutic effect in association with the required excipients. Containing a large amount of active substance.

  The composition is administered in a manner consistent with the dosage form and in a therapeutically effective amount. The amount to be administered will depend on the subject being treated, the ability of the subject's blood clotting system to utilize the active ingredient, and the degree of platelet aggregation inhibition desired. The exact amount of active ingredient required for administration will vary according to the judgment of the worker and is specific to each individual. However, suitable dosage ranges are on the order of 1 to 100 nanomole polypeptide per kilogram body weight per minute and will vary depending on the route of administration.

  An artificial blood vessel coated with a polypeptide having ATP diphosphohydrolase activity (eg, CD39) is also within the scope of the present invention. Commercially available materials suitable for producing such prostheses include polyesters such as Dacron® (C.R. Bard) or polyfluorocarbons such as Teflon® (Gore-Tex).

The present invention relates to a wide range of disease states in mammals that exhibit an increased tendency to aggregate platelets (eg, ischemic heart disease, atherosclerosis, multiple sclerosis, intracranial tumors, thromboembolic and hyperlipidemia, thrombus Therapeutic treatment of phlebitis, venous thrombosis, cerebral thrombosis, atherosclerosis and thrombotic symptoms such as coronary and retinal thrombosis), and surgery such as postpartum or coronary artery bypass surgery, angioplasty, or prosthetic heart valve transplantation Can be applied to surgery.
The following examples are merely illustrative and do not limit the present invention.

Example 1 (a) :
Xenostatic porcine arterial endothelial cells (PAEC) exert an inhibitory effect on the human platelet activation response to standard platelet agonists in the absence of plasma xenoreactive antibodies and complement.
Factors that inhibit human platelet activation in an in vitro system are bound to cells and are not found in cell culture supernatants. This cell-binding factor is expressed in monolayers, bead cultures or cell suspensions in the presence of PAEC in the presence of ADP (2-10 μM), collagen (2-10 μg / ml) and low concentrations of thrombin (<1 U / ml). Completely blocks the human platelet response to.
The importance of prostacyclin metabolism, thrombomodulin (by thrombin neutralization) and NO has been evaluated by several methods and has been shown not to be important for the inhibition of platelet activation performed by this PAEC.
In the experimental system performed, the non-inhibitory effect of ADP-β-S (which is an analog of non-hydrolyzable ADP and therefore not degraded by ecto-ADPase) on the human platelet response associated with PAEC can be demonstrated. Thus, the inhibitory endothelial cell binding factor is identified as ecto-ATP diphosphohydrolase (apyrase).

Example 1 (b) : Loss of PAEC inhibitor phenotype after PAEC activation Within 30 to 60 minutes with the development of an environment that accepts platelet activation by activation of PAEC by standard human recombination in vitro. Causes a rapid loss of the EC anti-aggregating phenotype.

Example 1 (c): Regulation of ecto-ATP diphosphohydrolase in PAEC by rTNFa Endothelial cell ecto-ATP diphosphohydrolase is markedly regulated by the EC activation response.
Ecto-ATP diphosphohydrolase kinetics: PAEC ecto-ATP diphosphohydrolase Vmax is on the order of ADP 50-55 nmol converted per 1 × 10 ⁶ cells / min as determined by the catabolism of ¹⁴ C-ADP ( Km approximately 200 μM). These numbers are measured by other methods [AJMarcus et al., J. Clin. Invest. 88 (1991) 1690-1696; ELGordon et al., J. Biol. Chem. 261 (1986) 15596-15507]. Consistent with that described for EC and pig EC described above.
Endothelial cells lose ecto-ADPase activity after 60 minutes of incubation when activated by 10 and 50 ng / ml TNFα. FIG. 1 shows enzyme activity levels at 4 hours measured by biochemical methods (D. LeBel et al., Supra) and TLC measurements of ¹⁴ C-ADP or AMP cell degradation (AJMarcus et al., Supra). Once EC is activated, platelet activation can occur because this ability to inhibit is lost. This inhibitory activity is mainly related to ecto-ATP diphosphohydrolase expressed in PAEC.

Example 1 (d) :
PAEC ecto-ATP diphosphohydrolase kinetics after activation of intact cells was also measured by TLC: V max 15 nmol ADP / 1 × 10 ⁶ cells / min (Km 70 μM). The reciprocal plot shows a non-antagonistic inhibition process. This new observation is consistent with either an inhibitor that binds to the enzyme-substrate complex (but not the free enzyme itself) or an inhibitory process that disrupts the enzyme catalytic function independently of the substrate binding (Fig. 2).

Example 2 (a) : Oxidative invasion is incubated with HOOH, which inhibits porcine endothelial cell ecto-ATP diphosphohydrolase, at a concentration of 5 μM and 10 μM that may be produced by activated endothelial cells without catalase activity. It then has a marked effect on the activity of non-additive ecto-ATP diphosphohydrolase that is comparable to that observed in subsequent cellular activation by cytokines. FIG. 3 represents the loss of enzyme activity after 4 hours incubation and 5 μM HOOH treatment.
HOOH production by PAEC following activation with cytokines such as TNF in vitro was determined to be on the order of about 0.015 nmoles / min / 10 ⁶ cells.
Thus, ecto-ATP diphosphohydrolase may be sensitive to oxidative processes facilitated by cytokine activation of PAEC. Endogenous xanthine oxidase and others such as NADPH oxidase, an enzyme system in PAEC, assimilate significant levels of reactive oxygen intermediates after cell activation, which have a profound effect on membrane-bound ectoenzymes have.

Example 2 (b) :
In the reverse format for drugs that induce oxidative invasion, β-mercaptoethanol, a potent reducing agent at micromolar concentrations, protects enzyme activity. This also supports the situation where endothelial cells are activated by cytokines (FIG. 4).

Example 2 (c) :
Loss of ecto-ATP diphosphohydrolase activity in PAEC is typically phosphate free with TNFα activation in vitro followed by HOOH (hydrogen peroxide, 5 μM) and xanthine oxidase / xanthine (XO / X, xanthine 200 μM). Expressed as a result of incubation and perturbation of endothelial cells with xanthine oxidase (100 mU / ml combination). XO / X causes oxidative damage to cells and their membrane proteins and lipids by both peroxide and superoxide radicals. In the presence of iron, toxic hydroxyl radicals are formed. Note the late decline in enzyme activity after exposure to oxygen radicals (FIG. 5).

Example 3 :
An antioxidant strategy with the addition of SOD / catalase to a similar test system has been shown to protect the maintenance of endothelial cell ecto-ATP diphosphohydrolase activity after the activation process. Superoxide dismutase (Cu-Zn form from bovine erythrocytes) removes oxygen radicals and was used at a concentration of 330 U / ml. Catalase degrades HOOH and a preparation from bovine liver was used at a final concentration of 1000 U / ml.
Zinc has a variety of effects on cell membranes, but at concentrations already shown to maintain porcine endothelial integrity after cytokine perturbation in vitro, as demonstrated by its potential here. It can also work as a powerful antioxidant. Addition to these systems appears to protect the maintenance of endothelial cell ecto-ATP diphosphohydrolase activity as well (FIG. 6).

Example 4 :
Direct oxidation of endothelial cell ecto-ATP diphosphohydrolase is responsible for regulating endothelial cell-platelet interactions in a cell activation setting.
Experiments similar to those described above are performed on purified proteins to further assess direct loss of post-oxidation activity with or without proteolytic modification [Rivett, Curr. Top. Cell Requl. 28 (1986) 291].

Example 5 :
Figure 7 represents the loss of activity in vivo after 60 minutes warm ischaemic time and after 5, 15, 30 and 60 minutes warm reperfusion. Note the loss of activity after 30 minutes of reperfusion in vivo. An initial increase in ATP diphosphohydrolase activity may represent leukocyte adhesion bound to damaged endothelium in vivo.

Example 6 :
FIG. 8 shows that pretreatment of rats with Cobra Toxin Factor (CVF) to deplete complement from animals also results in systemic complement activation damage with induction of oxidative insult, causing glomeruli to collapse. It represents an increase in the loss of ATP diphosphohydrolase activity as a result when bleeded and then reperfused for 30 minutes.

Example 7: Northern analysis of CD39 in HUVEC after cytokine activation Human umbilical vein endothelial cells (HUVEC) were incubated with TNFα (final concentration 10 ng / ml) for 2, 6 and 24 hours. Cells were washed twice with phosphate buffer and RNA was purified and analyzed by Northern blot. A total of 10 ug of RNA per well was applied to a TAE-agarose gel (TAE = Tris / acetic acid / EDTA buffer). Electrophoresis was performed at 40 mA for 2 hours. RNA was transferred to a charge modified nylon membrane and UV-crosslinked. The CD39 cDNA fragment (pCDNA3-CD39) cleaved from the plasmid DNA was labeled with [α ³² P] -dCTP to a specific activity of 2 × 10 ⁹ cpm / μg DNA by a random hexamer labeling method. Pre-hybridization, hybridization, washing, and membrane stripping were performed using Stratagene's rapid hybridization protocol. Final wash at 60 ° C. in 0.1-x sodium citrate saline (SSC) /0.1% sodium dodecyl sulfate (SDS). Blots were exposed to Kodak XAR-2 film at -80 ° C for 1 day with the help of the screen. The results shown in FIG. 9 indicate that CD39 / ecto-ADPase mRNA levels were significantly reduced after EC was stimulated with TNFα for more than 6 to 24 hours.

Example 8 : COS-7 cells transfected with CD39 were analyzed for COS-7 cells transfected with CD39 cDNA having ecto-ADPase biochemical and functional activity as determined by FACS analysis. Expressly identified CD39 (FIG. 10).
Whole cell lysates (FIG. 11) and membrane preparations (FIG. 12) of COS-7 cells are significant only when COS-7 cells are transfected with CD39 vector compared to empty vector or control COS-7 cells. Shows activity. Evaluation of ecto-ADPase activity was measured by hydrolysis of 200 μM ADP under Ca ⁺⁺ -dependent conditions.
Membrane preparations of COS-7 cells transfected with CD39 cDNA successfully eliminated platelet aggregates for ADP in vitro (FIG. 13).

Sequence Listing (1) General information:
(I) Patent applicant:
(A) Name: Sand Limited (B) Street: Lichtstrasse 35
(C) City: Basel (E) Country: Switzerland (F) Zip code (ZIP): CH-4002
(G) Telephone: 61-324 5269
(H) Fax: 61-322 7532

(A) Name: New England Deacones Hospital Corporation (B) Street: Pilgrim Road 185
(C) City: Boston (D) State: Massachusetts (E) Country: United States (F) Zip Code (ZIP): 02215

(A) Name: Bach, Fritz H (B) Street: Blossom Lane 8
(C) City: Boston, Manchester-by-the-Sea (D) State: Massachusetts (E) Country: United States (F) Zip Code (ZIP): 01966

(A) Name: Robson, Simon (B) Street: Longwood Avenue 45, Apartment 705
(C) City: Brooklyn (D) State: Massachusetts (E) Country: United States (F) Zip Code (ZIP): 02146

(Ii) Title of invention: transplantation and gene therapy for inflammatory or thrombotic conditions (iii) Number of sequences: 1
(Iv) Computer decoding format:
(A) Media type: floppy disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS
(D) Software:
(V) Application data:
Application number: WO PCT / EP96 / .....
(Vi) Priority application data:
(A) Application number: US 08/410371
(B) Application date: March 24, 1995 (A) Application number: US .....
(B) Application date: February 12, 1996

(2) Information of SEQ ID NO: 1
(I) Sequence characteristics (A) Length: 1818 base pairs (B) Type: Nucleic acid (C) Number of strands: Single strand (D) Topology: Linear (ii) Sequence type: mRNA
(Iii) Hypothetical: NO
(Iii) Antisense: NO
(Vi) Origin: human MP-1 B lymphoblast cell line (A) Organism name: Homo sapiens (x) Public information:
CRMaliszewski et al., J. Immunol. 153 (8) (1994) 3574-3583 (Figure 2 on page 3577)
(Xi) Sequence: SEQ ID NO: 1:

Regulation of ecto-ADPase: Bar graph showing the inhibitory effect of human γTNFα on ecto-ATP diphosphohydrolase activity: ■ = TLC nmol ADP / million cells / min; □ = LeBel / Fiske μmol phosphate / hour / mg cell protein [ Example 1 (c)]. γTNFα = recombinant tumor necrosis factor α. LWB (Rheinweaver Burke) ectoADPase (double reciprocal plot of enzyme kinetics): This shows the kinetics of stationary phase and cytokine-mediated PAEC. ■ = Control; □ = TNF [Example 1 (d)]. Inhibition of ecto-ADPase activity by oxidative invasion and cell activation (HOOH 5 μM / ecto-ADPase): bar graph showing peroxide and cytokine-mediated loss of ecto-ATP diphosphohydrolase activity against PAEC [Example 2 (a) ]. BME = β-mercaptoethanol. Protective effect of β-mercaptoethanol on ectoADPase activity: Bar graph showing that β-mercaptoethanol (BME) protects the cytokine-mediated loss of ectoATP-diphosphohydrolase activity against PAEC. [Example 2 (b)]. BME = β-mercaptoethanol. Ecto-ADPase regulation kinetics: Bar graph showing ecto-ATP diphosphohydrolase regulation kinetics by TNFα and oxidizing agents: (in order on the graph) ■ = control; □ = XO / X; ■ = HOOH; ■ = TNF [ Example 2 (c)]. XO / X = xanthine oxidase / xanthine; HOOH = hydrogen peroxide. Modulation of ecto-ADPase activity by antioxidants: Plot of ecto-ATP diphosphohydrolase activity of activated PAECs treated with antioxidants [Example 3]. SOD = superoxide dismutase; Cat = catalase. Reperfusion injury: Bar graph showing ecto-ATP diphosphohydrolase activity in purified rat glomeruli as a function of in vivo reperfusion time [Example 5]. Isch = ischemic time (minutes); Reperf = reperfusion time (minutes). Effect of CVF: Bar graph showing the effect of pretreatment of rat glomeruli with ischemia followed by reperfusion with cobra venom factor (CVF). Northern analysis of CD39: HUVEC following TNFα stimulation shows a decrease in the level of mRNA for CD39 [Example 7]. hEC = HUVEC = human umbilical vein endothelial cells; TNF = recombinant tumor necrosis factor. Transient transfection of COS-7 cells with pCDNA3 / CD39: FACS analysis of non-transfected COS-7 cells and COS-7 cells transfected with CD39 cDNA. Analysis by moAB (= monoclonal antibody) against CD39. Use isotype control simultaneously. Cells were stained with moAB (exact) against human CD39. EctoADPase activity of CD39-transfected COS-7 cells: Whole cell lysates of COS-7 cells transfected with CD39 cDNA express specific Ca ⁺⁺ ecto-ADPase activity (substrate = 200 μM ADP). First bar: control; second bar: empty vector; third bar: CD39 vector. EctoADPase activity of purified membranes of COS-7 cells transfected with CD39: the activity was mainly localized at the cell membrane. First bar: control COS cells; second bar: COS cells transfected with empty vector; third bar: COS cells transfected with CD39 vector. FIG. 13: Platelet aggregation assay: Inhibition of platelet aggregation by CD39; PRP aggregation with 5 μM ADP and COS-7 cell membrane extract (27.4 μg protein). COS-7 cell membrane extract from CD39-transfected cells effectively inhibits platelet aggregation induced by 5 μM ADP and establishes the functional potential of CD39 / ectoADPase protein. Human CD39 nucleotide and amino acid sequence (from J. Immunol. 153 (8) [1994] 3577) (= SEQ ID NO: 1). Human CD39 nucleotide and amino acid sequence (from J. Immunol. 153 (8) [1994] 3577) (= SEQ ID NO: 1). (Continued from FIG. 14-1)

Claims (15)

Characterized in that it comprises a DNA encoding the human CD39 protein having ATP diphosphohydrolase hydrolase activity, the cells genetically modified by inserting into mammalian cells or their precursor cells, cell activation or oxidation manner under invasive condition to confer the ability to stably express the human CD39 protein is used to reduce the susceptibility to inflammatory or immunological stimulus and platelet adhesion, gene modification reagent for. Characterized in that it comprises a DNA encoding the human CD39 protein having ATP diphosphohydrolase hydrolase activity, the cells genetically modified by inserting into mammalian cells or their precursor cells, cell activation or oxidation specifically the human CD39 protein was expressed in inhibiting platelet aggregation effective levels in invasive conditions, it used to inhibit platelet aggregation, reagents for genetic modification. DNA encoding human CD39 protein is operably linked to a inducible promoter, agent according to claim 1 or 2. Characterized in that it comprises a DNA encoding the human CD39 protein having ATP diphosphohydrolase hydrolase activity, the cells genetically modified by inserting into mammalian cells or their precursor cells, cell activation or oxidation specifically the human CD39 protein was expressed in inhibiting platelet aggregation effective levels in invasive conditions to control platelet aggregation, used to prevent or reduce thrombotic conditions, genetic modification reagent for. The reagent according to claim 4 , wherein the cell is an endothelial cell. The reagent according to claim 4 or 5 , wherein the subject is a human. Characterized in that it comprises a DNA encoding the human CD39 protein having ATP diphosphohydrolase hydrolase activity, the cell by inserting operably linked to a promoter in mammalian cells or their precursor cells in the donor the genetically modified, resulting modified donor cells, tissues, or organs transplanted into a recipient, the resulting modified cells or tissues or organs, in order to express the human CD39 protein in platelet aggregation inhibition effective levels Reagents used for gene modification. The reagent according to claim 7 , wherein the cell is an endothelial cell. The reagent according to claim 7 or 8 , wherein the recipient is a human. The reagent according to any one of claims 7 to 9 , wherein the donor is heterogeneous with respect to the recipient. The reagent according to any one of claims 7 to 10 , wherein the donor cell, tissue or organ is a pig. In the manufacture of a medicament for reducing platelet aggregation, the use of recombinant human CD39 protein having ATP diphosphohydrolase hydrolase activity. Characterized in that it comprises a DNA encoding the human CD39 protein having ATP diphosphohydrolase hydrolase activity in cellular activation or oxidative insults conditions, the endothelial cells of mammals and genetically modified, inflammation of the cells Alternatively, a genetic modification reagent used to reduce sensitivity to immunological stimulation and platelet adhesion. The reagent according to claim 13 , wherein the mammal is a pig. The reagent according to claim 13 , wherein the mammal is a human.

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