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WO1994000560A1 - Matrice de cellules endotheliales microvasculaires et donneuses universelles - Google Patents

Matrice de cellules endotheliales microvasculaires et donneuses universelles Download PDF

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Publication number
WO1994000560A1
WO1994000560A1 PCT/US1993/006216 US9306216W WO9400560A1 WO 1994000560 A1 WO1994000560 A1 WO 1994000560A1 US 9306216 W US9306216 W US 9306216W WO 9400560 A1 WO9400560 A1 WO 9400560A1
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Prior art keywords
cells
cell
matrix
protein
molecule
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PCT/US1993/006216
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English (en)
Inventor
Joseph Madri
Peter J. Sims
Alfred L. M. Bothwell
Eileen A. Elliot
Richard A. Flavell
Scott Rollins
Leonard Bell
Scott Kennedy
Stephen Squinto
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Alexion Pharmaceuticals, Inc.
Yale University
Oklahoma Medical Research Foundation
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Application filed by Alexion Pharmaceuticals, Inc., Yale University, Oklahoma Medical Research Foundation filed Critical Alexion Pharmaceuticals, Inc.
Priority to AU46579/93A priority Critical patent/AU4657993A/en
Publication of WO1994000560A1 publication Critical patent/WO1994000560A1/fr

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Definitions

  • This invention relates to genetically engineered endothelial cells and, in particular, to endothelial cells which have been modified to resist lysis and activation by complement and evade the host's immune mechanisms for removing foreign cells, when inserted into a non-autologous host.
  • Endothelial cells are specialized cells which form the lining of the heart and the blood vessels. Because of their direct contact with the circulating blood, a number of proposals have been made to genetically engineer these cells and use them as "in vivo" drug delivery systems, for example, by Culliton, B. J. 1989.
  • Natural endothelial cells play important roles in normal physiology.
  • these cells constitute the interface between the blood and the vessel wall and the organs of the body.
  • endothelial cells secrete various natural products directly into the blood stream, maintain an antithrombotic surface on the inside of the vessel, restrict leukocytes from penetrating the vessel wall, regulate various of the biological properties of smooth muscle cells, and participate in the control of vessel wall tone. Therefore, loss of endothelial cells results in the loss of these normal physiological processes and ultimately leads to pathological conditions such as coronary artery disease, organ transplant rejection and vasculiti ⁇ .
  • microvascular capillary endothelial cells for systemic delivery of a therapeutic protein may allow for the use of either autologous or immunoprotected allogeneic or xenogeneic capillary endothelial cells.
  • Several investigators have proposed the use of implanted genetically modified fibroblasts or keratinocytes as delivery systems and, while plausible, the expressed protein must diffuse through interstitial tissues, and into the microcirculation in order to gain access to the vascular system. This diffusional barrier to the systemic circulation is a considerable impediment to achieving adequate plasma levels of the desired therapeutic protein.
  • Endothelial cells offer several advantages over fibroblasts in that they secrete their protein products directly into the bloodstream. Further, fibroblasts via their excessive production of fibrotic scar tissue, can prove highly detrimental to the host.
  • transplant rejection When a foreign cell is transplanted into a host, the immune system of the host rapidly mobilizes to destroy the cell and thereby protect the host.
  • the immune system attack on the foreign cell is referred to as transplant rejection.
  • the organism's first line of defense is through either lytic destruction or the activation of procoagulant and prothrombotic properties of the donor endothelial cell that may result from activation of the host's complement system and is generally known as the "hyperacute rejection response" or simply the "hyperacute response.”
  • the antibodies are directed against the vascular endothelial cells (Brasile et al., Trans. 40, 672-675 (1985)).
  • the complement system is a complex interaction of plasma proteins and membrane cofactors which act in a multistep, multi-protein cascade sequence in conjunction with other immunological systems of the host organism.
  • the classic complement pathway involves an initial antibody recognition of, and binding to, an antigenic site on a target cell- This surface bound antibody subsequently reacts with the first component of complement, Clq, forming a Cl-antibody complex with Ca2+, Clr, and Cls which is proteolytically active.
  • Cls cleaves C2 and C4 into active components, C2a and C4a.
  • the C4b,2a complex is an active protease called C3 convertase, and acts to cleave C3 into C3a and C3b.
  • C3b forms a complex with C4b,2a to produce C4b,2a,3b, which cleaves C5 into C5a and C5b.
  • C5b forms a complex with C6 and this complex interacts with C7 in the fluid phase thereby exposing hydrophobic domains within C5b and C6 that stabilize the C5b,6,7 ternary complex in the cell membrane.
  • Upon binding of C9 to C8 in the C5b-8 membrane complex lysis of foreign cells is rapidly accelerated.
  • C3b One of the central molecules in the complement cascade is C3b which aggregates in increasing amounts on foreign substances or organisms thereby targeting them for removal.
  • the complement precursor proteins are activated to form C3b in either of two ways: (i) by interacting with antibody bound to a foreign target (classical pathway) or (ii) non-specifically by progressive and rapidly increasing accumulation on foreign substances on the surface of foreign cells (the alternative pathway) .
  • C3b is continuously activated at a slow rate in the fluid phase by various agents including endotoxin, lipopolysaccharide, and serum proteases that convert C3 to C3b.
  • C5b can also be formed from C5 by plasmin, elastase and other serum proteases to initiate formation of the MAC.
  • MCP Membrane cofactor protein
  • Decay accelerating factor (DAF or CD55) which exists on all cells including red blood cells and prevents C3b from reacting with other complement components preventing destruction of the cell.
  • CD55 unlike CD46, does not destroy C3b.
  • Complement receptor 1 (CR1 or CD35) which exists on a select group of lymphocytes as well as erythrocytes, neutrophils, and eosinophils and causes degradation of C3b molecules adhering to neighboring cells.
  • chromosome 1, band lq32 chromosome 1, band lq32 identified as the RCA, i.e., the regulators of complement activation. They are each uniquely characterized structurally by a short consensus repeating unit (SCR) of approximately 60 amino acids composed mostly of cysteine, proline, glycine, tryptophan, and several hydrophobic residues. Reid, et al., Immunol. Today 7, 230 (1986); Coyne, et al., J. Immunol. 149, 2906-2913 (1992). For CD46 and CD55, these SCRs are known to encode the functional domains of the proteins necessary for full complement regulatory activity. Adams, et al., J. Immunol.
  • human blood cells and the vascular endothelium express a cell surface glycoprotein, CD59, that serves to prevent assembly of the C5b-9 lytic MAC and, therefore, protects these cells from complement-mediated cell activation and lysis.
  • CD59 a cell surface glycoprotein, CD59, that serves to prevent assembly of the C5b-9 lytic MAC and, therefore, protects these cells from complement-mediated cell activation and lysis.
  • U.S. Patent No. 5,135,916 issued August 4, 1992, assigned to the Oklahoma Medical Research Foundation, and U.S. serial number 07/729,926 filed July 15, 1991, assigned to the Oklahoma Medical Research Foundation and Yale University disclose that the human complement regulatory protein CD59 can be used to protect non-human endothelial cells, for example, porcine endothelial cells, from attack by human complement, either when provided in solution with the cells or expressed in genetically engineered cells.
  • CD59 does successfully address the problem of hyperacute rejection as a result of complement attack, it does not protect the cell against the overall immune attack of the host organism against foreign endothelial cells.
  • antigens In stimulating immune responses, antigens elicit many molecular and cellular changes. Lymphocytes recognize antigens as foreign and are responsible for initiating both cellular and humoral responses against the presenting antigen. B lymphocyte cells respond to antigen by the production of antibodies against the presenting antigen; T lymphocytes respond by initiating a cellular response to the presenting antigen.
  • T H cells involved in processing of antigen for presentation to B cells, characterized by the presence of a cell-surface glycoprotein called CD4, and cytolytic T lymphocytes (CTLs) , involved in recognition of antigen on cell surfaces and lysis of cells recognized as foreign, characterized by the presence of a cell-surface glycoprotein called CD8.
  • T cells recognize peptide fragments in conjunction with one of the two main classes of cell-surface glycoproteins of the major histocompatibility complex (MHC) : either class I (MHC-I) or class II (MHC-II) proteins.
  • MHC major histocompatibility complex
  • CD8+ T cells recognize antigens in conjunction with MHC-I
  • CD4+ T cells recognize them in conjunction with MHC-II.
  • the MHC contain three major classes of genes.
  • Class I genes encode the principal subunits of MHC-I glycoproteins, called -human leukocyte antigens in humans, the principle ones being HLA-A, B, and C. These are present on virtually all cells and play a major role in rejection of allografts. They also form complexes with peptide fragments of viral antigens on virus-infected cells: recognition of the complexes by CD8+ CTLs results in destruction of virus infected cells. Recognition of the complexes is by a single receptor on the T cells which recognizes antigen in combination with MHC.
  • Class II genes encode cell- surface glycoproteins that are expressed by antigen- presenting cells, principally B cells, macrophages and dendritic cells. Together with peptide fragments of antigens, the class II proteins form the epitopes that are recognized by T helper cells (CD4+) .
  • Class III genes encode at least three proteins of the complement cascade and two cytotoxic proteins, tissue necrosis factor and lymphotoxin, which are involved in diverse immune reactions that destroy cells.
  • T-cell mediated immune reactions can be organized into three sequential activation steps.
  • CD4+ and CD8+ Tlymphocytes recognize the presence of n ⁇ -autologous MHC class II and class I proteins, respectively, on the surface of the foreign cell.
  • the T-cells are activated by interaction of a ligand with the T cell receptors and other accessory stimulatory molecules, so that activation depends upon a variety of variables including humoral signals such as cytokines received by protein receptors on the surface of the cells. Most important is the interaction between the antigen specific T cell receptor and ligand, a complex of MHC and antigenic peptide on the antigen presenting cell (APC) . Other receptors present on the T cell must also be contacted by their ligands on APC to insure activation. Once activated, the T-cells synthesize and secrete interleukin-2 (IL-2) and other cytokines.
  • IL-2 interleukin-2
  • the cytokines secreted by the activated T-cells lead to the third, or effector, phase of the immune response which involves recruitment and activation of lymphocytes, monocytes, and other leukocytes which together lead to cell lysis, as reviewed, for example, by Pober et al., 1990 "The potential roles of vascular endothelium in immune reactions" Human Immunol. 28:258-262.
  • attempts to interrupt the T-cell immune response have generally met with limited success. For example, several strategies have tried to use reagents of various types, including antibodies and blocking proteins, to interfere with adhesion between T-cells and foreign cells.
  • Genetically engineered cells which include a DNA sequence which is expressed by the cell and which codes for a protein having complement inhibitory activity that is not normally expressed in the cell. These cells may also be engineered so that they do not express on their cell surfaces functional proteins encoded by the class II major histocompatibility complex (MHC) genes, the HLA DP, DQ, and DR genes in human cells, or their equivalent in cells of a different species. Alternatively, the genetically engineered cells do not express on their cell surfaces the proteins encoded by the class I MHC genes, the HLA A, B and C genes in human cells, or their equivalent in cells of a different species, or they do not express either the class I and class II MHC genes. In some embodiments, the cells include a genetic (DNA) sequence which is expressed by the cell and which codes for a protein which in the presence of a selected agent results in death of the cell.
  • MHC major histocompatibility complex
  • the genetic sequence which codes for a protein which has complement regulatory activity protects the cell from hyperacute rejection through attack and lysis resulting from activation of the complement system.
  • the removal of the cell surface proteins encoded by the class I (for example, HLA A, B and C) and class II (for example, HLA DP, DQ, and DR) MHC genes makes the cells substantially unrecognizable by the host's CD8+ and CD4+ T-lymphocytes, respectively.
  • the genetic sequence which codes for a protein which can produce cell death provides a mechanism for eliminating the genetically engineered cells from the host when their presence is no longer desired.
  • the cells are modified in culture using standard in vitro transfection techniques, or can be derived from transgenic animals modified as embryos. These modified cells can serve as universal donor cells for administering therapeutic agents to the host or as replacements for natural cells which have been damaged or lost. In the most preferred embodiment, the cells are dissociated endothelial cells.
  • Figure 1 shows the structure of the retroviral vector used in Example 1.
  • This vector was constructed from a defective Moloney murine leukemia virus.
  • the SV40 promoter was excised and replaced with the SRalpha promoter.
  • a 500 bp cDNA fragment containing the CD59 coding sequence was cloned into an X ol site and verified by restriction analysis.
  • the resulting plasmid was designated pRNSR ⁇ CD59.
  • Ecotropic retrovirus was produced by transfecting Psi-2 cells with polybrene and selecting in the toxic amino- glycoside G418.
  • Amphotropic virus stocks were prepared by infecting the amphotropic packaging cell line Psi-AM with the ecotropic virus, were added directly to endothelial cell cultures in the presence of polybrene, and transfectants were selected with 400 ⁇ g/ml G418.
  • Figure 2 is a graph of cell surface expression of human CD59 on porcine aortic endothelial cells (PAEC) as detected by anti-CD59 antibody and analyzed by flow cyto etric analysis.
  • the solid line represents the fluorescence intensity of PAEC infected with retrovirus shown in Figure 1 carrying only the control neomycin resistance gene.
  • the dashed line, small dotted line, and larger dotted line represent the fluorescence intensity of CD59-expre ⁇ sing PAEC cell clones 2, 9, and 1, respectively.
  • Figure 3 shows a scanning electron micrograph of CD59expressing PAEC attached to a synthetic GortexTM graft.
  • Figure 3a is the control GortexTM
  • Figures 3b, c, and d are GortexTM with CD59-expressing cells implanted thereon.
  • Figure 4 is a bar graph showing the protection of human CD59-expressing PAEC from lysis by human complement.
  • the solid bar represents the percentage of cell lysis of PAEC expressing human CD59.
  • the cross-hatched bar represents the percentage of cell lysis of PAEC expressing only the control neomycin resistance gene while the stippled bar represents the percentage of cell lysis of control (noninfected) PAEC.
  • Figure 5 is a graph demonstrating th at hCD59 expressed on porcine endothelial cells blocks assembly of a prothrombinase complex, mU thrombin/min/well versus ng C8/ml, for Lxsn, Maloney leukemia virus- based retroviral vector without CD59 insert (open circles), LxsnCD59, vector with CD59 insert (open squares) (deposited with the American Type Culture Collection, Rockville, Maryland, ATCC Accession number 69336), and LxsnCD59Flg, vector with 5'Flg epitope- flagged CD59, ATCC Accession Number 69337, insert (dark triangles) .
  • Figure 6 is a schematic of the mammalian expression vector pcDNAI/Amp used to engineer transgenic mice expressing human CD59.
  • Figure 7A shows a restriction digest map of the gene targeting vector for the mouse invariant chain gene cloned into pBS (Bluescript) .
  • the targeting vector contains the neomycin gene (neo) .
  • Figure 7B shows a partial restriction digest map of the endogenous mouse invariant chain gene and
  • Figure 7C shows a restriction digest map of the disrupted invariant chai ⁇ n gene achieved by homologous recombination. Arrows indicate the direction of transcription in all three panels.
  • the recognition region for the radiolabeled invariant gene probe used for the Southern blot shown in Figure 8 is indicated by a solid bar below Figure 7C.
  • Figure 8 is a Southern blot showing the restriction digestion pattern for two independent neomycin resistant mouse embryonic stem cell clones where the endogenous invariant chain gene has been disrupted by homologous recombination and replaced with a mutated form of the gene.
  • the two clones are designated 11.10.93 and 11.10.128. Size markers are indicated on the left side.
  • the Drain restriction pattern of the parental cells is indicated in the far right lane and is clearly different from the restriction pattern of the two clones carrying the modified invariant chain gene.
  • Figures 9a and 9b are graphs demonstrating splenocytes from mutant -/- mice are functional in presenting peptide antigens to T-cell hybrids but show diminished ability to present intact protein antigen, CPM versus peptide concentration ( ⁇ g/ml) for concentrations of 3 x 10 5 of E ⁇ 56-73 peptide (9a) and
  • Figure 10 is a schematic of the retroviral vector used in Example 5. This vector was constructed from a defective Moloney murine leukemia virus. A cDNA fragment encoding the full-length and functional human apolipoprotein E (Apo E) was subcloned into the retroviral vector. The resulting plasmid was designated Lxsn-ApoE, ATCC Accession Number 69335. Ecotropic retrovirus was produced by transfecting Psi-
  • Cells which have been genetically engineered can be transplanted into a host to allow them to both resist and evade the immune system of a host.
  • the host will normally be a human or a domesticated farm animal.
  • endothelial cells especially dissociated endothelial cells for implantation or injection into a host
  • the methods and compositions described herein are not limited to endothelial cells.
  • Other cell types can be similarly modified for transplantation. Examples of other cell types include fibroblasts, epithelial cells, skeletal, cardiac and smooth muscle cells, hepatocytes, pancreatic islet cells, bone marrow cells, astrocytes, Schwann cells, and other cell types, dissociated or used as tissue (i.e., organs).
  • endothelial cells will be construed to encompass modification of these other cell types unless otherwise specified or described specifically in the examples.
  • the cells can come from a variety of sources.
  • the cells are of non-human origin because of the ready availability of such cells in large quantities and at low cost.
  • the cells can be of porcine or bovine origin. Cells from primates, including humans, can be used if desired. Even if human cells are used, protection from hyperacute rejection will in general still be required since complement-mediated cell attack can also occur even following allotypic transplantation.
  • the genetically engineered cells are normally derived from a single clone or, for some applications, a group of individually selected clones. In this way, the characteristics of the final pharmaceutical preparation can be accurately controlled both in terms of the overall properties of the cells and their genetic make-up. Such control is of importance in evaluating the effectiveness of particular treatment protocols and in obtaining regulatory approval for such protocols.
  • the cells are genetically engineered so that they express a complement inhibitory protein or proteins on their cell surface.
  • the cells can also be genetically engineered so that they express deficient and/or dysfunctional proteins encoded by the class II, class I, or preferably the class I and class II, MHC genes on their cell surface. Even when human cells are used, it is beneficial to engineer the cells as described herein since the cell population will generally include non-autologous cells when the cells are obtained from an individual other than the one being treated.
  • the endothelial cells are obtained from the lining of a portion of the vascular system, e.g., a blood vessel or capillary, and are grown and maintained in a tissue culture or other suitable biological medium.
  • porcine large vessel endothelial cells are isolated from the thoracic aortae of male pigs.
  • the thoracic aortae is removed from the sacrificed animal using sterile techniques, cross- clamping the aortic arch and the aorta just above the renal arterial ostia using sterile clamps.
  • the organs/tissues are placed in sterile PBS buffer, containing 10X penicillin, streptomycin and fungizone. These are transported on ice.
  • the endothelium is scraped off with a sterile scalpel blade and the harvested endothelium is transferred into a sterile 15 cc conical centrifuge tube by displacing the cells with a stream of sterile PBS buffer.
  • the tubes are centrifuged at 1200 RPM and the supernatant aspirated.
  • sterile media 10% heat- inactivated fetal calf sera, penicillin (100 U/ml) , streptomycin (100 U/ml) , 5 mM Hepes, 5 mM pyruvate and 5 mM glutamine, are added to each tube and the cell pellets resuspended in the media by gently pipetting the solution up and down in a sterile five ml pipette.
  • the cells are then passaged at confluency at a 1 to 3 split ratio using 0.02% trypsin (Worthington Biochemical Corp.) in a Ca ++ and Mg ++ -free PBS containing 0.01% EDTA to dislodge the cells from the plate and dissociate the cells.
  • trypsin Worthington Biochemical Corp.
  • the cells After being genetically engineered in the manner described below, the cells are normally stored in liquid nitrogen tanks until needed for the treatment of a particular patient.
  • the ability to prepare the donor cells in advance and store them until needed is an important advantage.
  • Cells are then seeded onto a matrix for implantation.
  • dissociated endothelial cells are prepared for seeding onto the interior of GortexTM as follows.
  • the GortexTM tubing is then closed at one end and endothelial cells are introduced into the lumen of the GortexTM tubing and the other end is closed.
  • the GortexTM segments are transplanted into the vasculature of the host.
  • MAC membrane attack complex
  • protection against the pore-forming activity of the C5b-9 complex can be conferred on non-primate cells by transfection of such cells with a cDNA encoding the human complement regulatory protein CD59.
  • This protein operates by limiting the incorporation of C9 into the membrane complex C5b-9, as reported by Zhao, et al., 1991 "Amplified gene expression in CD59-transfected Chinese Hamster Ovary cells confers protection against the membrane attack complex of human complement" J. Biol. Chem. 266:13418-13422; Rollins and Sims, 1990 "The complement inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9" J. Immunol.
  • CD46 also known as membrane cofactor protein (MCP), as described by Purcell, et al., 1990 "The human cell surface glycoproteins HuLy-m5, membrane cofactor protein (MCP) of the complement system, and trophoblast leucocyte common (TLX) antigen, are CD46" J. Immunol. 70:155-161; and Seya and Atkinson, 1989 "Functional properties of membrane cofactor protein of complement” Biochem. J. 264:581-588.
  • This inhibitor functions by binding to complement component C3b thereby activating molecules that cleave C3b into inactive fragments preventing accumulation of C3b and, therefore, its contribution to the formation of the MAC. See also White et al.
  • CD55 also known as decay accelerating factor (DAF), described by Nicholson-Weller et al., 1982 "Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system” J. Immunol. 129:184; Lublin and Atkinson, 1989 "Decay accelerating factor: Biochemistry, molecular biology, and function” Annu. Rev. Immunol. 7:35; Lublin et al., 1987 "The gene encoding decay-accelerating factor (DAF) is - located in the complement-regulatory locus on the long arm of chromosome 1" J ⁇ Ex . Med.
  • DAF decay accelerating factor
  • CD46, CD55, and CD59 The relative contributions of CD46, CD55, and CD59 in providing protection from complement-mediated lysis has been assessed in human amniotic epithelial cells (HAEC) by the use of specific blocking antibodies, as reported by Rooney et al., 1990 "Protection of human amniotic epithelial cells (HAEC) from complement-mediated lysis: expression on the cells of three complement inhibitory membrane proteins.” Immunology 71:308-311. The results demonstrated that CD59 provide the most protection against complement attack, as compared with CD46 and CD55.
  • Cells suitable for transplantation into a foreign host are protected from complement-mediated lysis by introducing into the cell DNA encoding a protein, or combination of proteins, inhibiting complement-mediated lysis, for example, CD59, CD55, CD46 and/or other inhibitors of C8 or C9.
  • CD59 is the preferred inhibitor, introduced into the cells by transfection or infection with a vector encoding the CD59 protein, and expressed on the surface of the transfected/infected cells.
  • the inhibitor is preferably of the same species of origin as the host into which the cells are to be transplanted.
  • the gene encoding the complement inhibitor can be introduced into a cell of a different species of origin, for example, a human CD59 gene can be introduced into a porcine cell so that the cell resists attack when transplanted into a human, or the gene can be introduced into a cell of the same species of origin so that increased amounts of the protein are expressed on the surface of the cell.
  • the gene can be placed under the control of a promoter enhancing expression of the gene which is then inserted by homologous recombination into the host cell chromosome at the site where the gene is normally located, but under the control of the promoter which enhances expression, or can be inserted into the chromosome at another locus on the chromosome.
  • DNA sequence information for CD46, CD55 and CD59 has been reported in the literature.
  • PLAIN TEXT HUMDAF cDNA
  • the amino acid sequence for the protein is: L Q C Y N C P N P T A D C K T A V N C S S D F D A C L I T K A G L Q V Y N K C W K F E H C N F N D V T T R L R E N E L T Y Y C C K K D L C N F N E Q L E N G G T S L S E K T V L L L V T P F L A A A W S L H P.
  • a cDNA sequence encoding the CD59 protein is (Sequence I.D. No. 4):
  • Matching oligonucleotide primers can be readily designed and then used to obtain full length cDNA sequences for these proteins by performing a polymerase chain reaction amplification on human CDNA.
  • the oligonucleotide primers are preferably designed with specific restriction enzyme sites so that the full length CDNA sequences can be readily subcloned into vectors for use in transfecting/infecting the target donor cells.
  • a preferred transcription cassette for introduction and stable expression in endothelial cells of the sequences encoding the complement regulatory proteins is described in U.S. Serial No. 08/021,602 entitled "Transcriptional Casette for the Expression of Complement Regulatory Proteins in Transgenic Animals.”
  • the transcriptional cassette for producing a transgenic non-human mammal having endothelial cells which express a heterologous complement regulatory protein includes: the human cytomegalovirus immediately-early gene 1 promoter and enhancer (CMV IE P/E) , a cDNA coding sequence for said heterologous complement regulatory protein, said cDNA coding sequence being operatively linked to the CMV IE P/E, the Simian Virus 40 intron donor and acceptor splice sequence, and the Simian Virus 40 polyadenylation sequence.
  • CMV IE P/E human cytomegalovirus immediately-early gene 1 promoter and enhancer
  • Plasmid pUC19-hCD59 obtained from Yale University, New Haven, CT, was used to construct this vector.
  • Human CD59 cDNA was excised from the plasmid using the restriction enzymes Ba Hl and EcoRI.
  • the human CD59 cDNA thus obtained was subcloned into the mammalian expression vector pcDNAI/Amp (Invitrogen, San Diego, CA) to yield pC8-hCD59-103.
  • the pcDNAI/Amp mammalian expression vector contains the human cytomegalovirus (CMV) immediate-early gene (IE) promoter and enhancer, the SV40 consensus intron donor and acceptor splice sequences, and a consensus polyadenylation site.
  • CMV human cytomegalovirus
  • IE immediate-early gene
  • Plasmid pC8-hCD59-103 has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852, United States of America, in E. coli straIN dh5 ⁇ , and has been assigned accession number ATCC 69231. This deposit was made under the Budapest Treaty on the International Recognition of the Deposit of Micro ⁇ organisms for the Purpose of Patent Procedure (1977) .
  • the cDNA coding sequence of the transcriptional cassette codes for CD59, CD55, and/or CD46, although the cassette can be used with other complement regulatory proteins now known or subsequently identified or developed.
  • complement regulatory protein means any protein which inhibits activation and/or lysis of cells by the complement system.
  • This "cassette” can be used to make transgenic non-human mammals all of whose germ cells and somatic cells contain the transcriptional cassette, as well as vascularized organs and endothelial cells derived from such transgenic mammals.
  • the vascularized organ may be any organ which expresses the complement regulatory protein(s) on its endothelial cells, but is preferably the heart, spleen, kidney, lung, pancreas, or liver, i.e., the organs for which substantial transplantation demand exists.
  • DNA encoding the complement inhibitors can be introduced into the cells in culture using transfection or into embryos for production of transgenic animals expressing the complement inhibitors on the surface of their cells.
  • transfection can be accomplished by electroporation, calcium phosphate precipitation, a lipofectin-based procedure, or microinjection or through use of a "gene gun".
  • CDNA for the inhibitory protein, such as CD59 is subcloned into a plasmid-based vector which encodes elements for efficient expression in the genetically engineered cell.
  • the plasmid-based vector preferably contains a marker such as the neomycin gene for selection of stable transfectants with the cytotoxic a inoglycoside G418 in eukaryotic cells and an ampicillin gene for plasmid selection in bacteria.
  • Infection which for endothelial cells is preferred, is accomplished by incorporating the genetic sequence for the inhibitory protein into a retroviral vector.
  • Various procedures are known in the art for such incorporation.
  • One such procedure which has been widely used in the art employs a defective murine retrovirus, Psi-2 cells for packaging the retrovirus, and the amphotropic packaging cell line Psi-AM to prepare infectious amphotropic virus for use in infecting the target donor cells, as described by Kohn et al., 1987 "Retroviral-mediated gene transfer into mammalian cells” Blood Cells 13:285-298.
  • a retrovirus of the self-inactivating and double-copy type can be used, such as that described by Hantzopoulos et al., 1989 "Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector" Proc. Natl. Acad. Sci. USA 86:3519-3523.
  • transgenic animals expressing a complement inhibitory protein on the surface of the cells for use as a source of modified cells for transplantation.
  • particularly useful animals include rabbits and pigs, although transgenic mice, rats, rabbits, pigs, sheep, and cattle have been made using standard techniques.
  • the most well known method for making a transgenic animal is by superovulation of a donor female, surgical removal of the egg and injection of the genetic material in the pronuclei of the embryo, as taught by U.S. Patent No. 4,873,191 to Wagner, the teachings of which are incorporated herein.
  • Another commonly used technique involves the genetic manipulation of embryonic stem cells (ES cells) , as specifically described below in Example 3.
  • ES cells embryonic stem cells
  • ES cells are grown as described, for example, in Robertson, E.J. "Embryo-derived stem cell lines" in: Teratocarcinomas and embryonic stem cells: A practical approach. E.J. Robertson, ed. 71-112 (Oxford- Washington, D.C.: IRL Press, 1987). ES cells are maintained in a plueripotent state in culture media containing recombinant Leukemia Inhibitory Factor (LIF) as described in U.S. Patent No. 5,166,065 to Williams et al. Genetic material is introduced into the embryonic stem cells, for example, by electroporation according to the method of McMahon, A.P., and Bradley, A. Cell 62, 1073-1085 (1991). Colonies are picked from day 6 to day 9 of selection into 96 or 24 well dishes (Costar) and expanded and used to isolate DNA for Southern blot analysis.
  • LIF Leukemia Inhibitory Factor
  • Chimeric mice are generated as described in Bradley, "Production and analysis of chimearic mice” in Teratocarcinomas and embryonic stem cells: A practical approach E.J. Robertson, ed. pp. 113-151 (Oxford, Washington, D.C. IRL Press 1987), the teachings of which are incorporated herein. Genetic material is injected into blastocysts. From those implanted females that become pregnant, chimaeras are selected from the offspring and bred to produce germline chimaeras for use as donor animals. III. Protection From T-Cells
  • the donor endothelial cells are genetically engineered to not express on their surface class II MHC molecules. More preferably, the cells are engineered to not express substantially all cell surface class I and class II MHC molecules.
  • the term "not express" may mean either that an insufficient amount is expressed on the surface of the cell to elicit a response or that the protein that is expressed is deficient and therefore does not elicit a response.
  • the MHC molecules are referred to as HLA molecules, specifically of classes HLA A, B and C, and class II HLA DP, DQ, and DR, and their subclasses.
  • HLA molecules specifically of classes HLA A, B and C, and class II HLA DP, DQ, and DR, and their subclasses.
  • This terminology is generally construed as specific to the human MHC, but is intended herein to include the equivalent MHC genes from the donor cell species, for example, if the cells are of porcine origin, the term HLA would refer to the equivalent porcine MHC molecules, whether MHC I or II.
  • CD4+ T cells do not recognize the genetically engineered endothelial cells; when both the class I and class II MHC molecules are removed neither CD4+ nor CD8+ cells recognize the modified cells.
  • HIV AIDS virus
  • mice inheriting this genotype remain healthy and are capable of resisting infection by foreign organisms such as viruses, as reported by Zijlstra et al., 1989 "Germ-line transmission of a disrupted ⁇ 2-microglobulin gene produced by homologous recombination in embryonic stem cells” Nature 342:435438; and Koller et al., 1990 "Normal development of mice deficient in B2M, MHC class I proteins, and CD8+ T cells” Science 248:1227-1230.
  • the preferred genetic modification performed on the endothelial cells includes 1) disrupting the endogenous invariant chain gene which functions in the assembly and transport of class II MHC molecules to the cell surface and loading of antigenic peptide, and 2) disrupting the endogenous ⁇ 2 -microglobulin gene ( ⁇ 2 M gene) which codes for a protein required for the cell surface expression of all class I MHC molecules.
  • ⁇ 2 M gene endogenous ⁇ 2 -microglobulin gene
  • the disruption of these genes is accomplished by means of a homologous recombination gene targeting technique, as described by Zijlstra et al., 1989; Koller et al., 1990; and Example 3 below showing disruption of the invariant chain gene.
  • the technique is applied to suppress expression of the class I MHC proteins on the cell surface as follows.
  • the complete ⁇ 2 M gene for the target donor endothelial cell is cloned, e.g., for porcine endothelial cells the porcine ⁇ 2M gene is cloned.
  • This is done by first obtaining cDNA for a homologous ⁇ 2 M gene, such as the mouse B2M gene.
  • DNA sequence information for the mouse ⁇ 2 M cDNA has been reported by Parnes et al., 1983 Nature 302:449-452.
  • Matching oligonucleotide primers are readily designed to hybridize by complementary base pairing to the extreme 5' and 3' ends of the mouse ⁇ 2 M cDNA.
  • oligonucleotide primers are then used to obtain full-length cDNA sequences for the mouse ⁇ 2 M protein by performing a poly erase chain reaction amplification on mouse cDNA.
  • the oligonucleotide primers are preferentially designed to encode specific restriction sites at their ends so that full-length cDNA sequences can be readily subcloned into plasmids.
  • the full-length mouse ⁇ M cDNA can then be used as a radiolabeled hybridization probe to screen cDNA libraries prepared from the source of the target donor endothelial cells, e.g., for porcine endothelial cells the mouse ⁇ 2 M cDNA is used as a hybridization probe to screen a porcine cDNA library which has been cloned into a lambda phage vector. Positive hybridizing clones are selected, purified, subcloned into plasmid vectors and then sequenced using methods known in the art.
  • the complete porcine ⁇ 2 M gene can then be cloned by screening a porcine genomic DNA library cloned into a lambda phage vector with radiolabeled porcine ⁇ 2 M cDNA as a hybridization probe. Positive clones are selected, purified, subcloned into plasmid vectors and sequenced using methods known in the art.
  • the ⁇ 2 M qene is subcloned into a plasmid based or preferentially a retroviral-based vector (the "gene targeting vector") such that the reading frame of the ⁇ 2 M gene is disrupted by insertion of a short DNA sequence which allows for positive selection of recombination in the endothelial cells, for example, a neomycin resistance gene (hereinafter referred to as the "positive selection gene”) .
  • the gene targeting vector also carries an additional selection gene (the "negative selection gene") , outside of the disrupted ⁇ 2 m gene region which allows for selection against non-homologous recombination, i.e., for selection against incorporation of the entire plasmid into the genetic information of the cell rather than just the portion of the plasmid carrying the disrupted ⁇ 2 M gene.
  • the negative selection gene can be, for example, a herpes simplex thymidine kinase gene.
  • the gene targeting vector is then transfected/infected into the cells as described above and homologous recombination events are selected by screening for clones which express the positive selection gene but not the negative selection gene.
  • the cells and their progeny in theory can exist essentially indefinitely within the host organism. Since occasions may arise when it is desirable to remove these cells from the host, further genetic engineering is preferably performed wherein the cells are provided with an internal "self-destruct" or "suicide” mechanism.
  • a self-destruct or "suicide” mechanism.
  • such a mechanism involves including in the cell a gene which expresses a protein, usually an enzyme, which confers lethal sensitivity of the cell to a specific reagent not normally present in the cell's environment.
  • the bacterial enzyme cytosine deaminase converts the non-toxic drug 5-fluorocytosine to 5-fluorouracil which in turn is converted within the cell to 5-fluorouridine 5'-triphosphate and 5- fluoro-2'-deoxyuridine 5'-monophosphate which inhibit both RNA and DNA synthesis and thereby result in cell death, as reported by Mullin, et al., 1992 "Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5— fluorocytosine: a negative selection system" Proc. Natl. Acad. Sci. USA 89:33-37.
  • cell death can be accomplished at any desired time by simply administering 5-fluorocytosine to the host organism.
  • the sequence of the bacterial CyD gene is known and thus incorporation of the gene into the donor endothelial cells can be preformed in a manner similar to that used to insert the CD59 gene.
  • the engineered cells can be used for cell replacement and for drug administration.
  • coronary artery disease is caused by a blockage inside blood vessels, reducing the delivery of oxygen and nutrients to the heart.
  • the current treatment for coronary artery blockade is either to mechanically dilate the blocked vessel from the inside with an angioplasty balloon or to use a replacement vessel, e.g., a synthetic graft or a section of the saphenous vein, to bypass or form a new channel around the blockage.
  • Coronary angioplasty involves the insertion of a catheter from the leg vessel to the coronary artery and inflation of a balloon at the tip of the catheter to dilate the atherosclerotic plaque. This balloon inflation unfortunately has the undesired side effect of removing endothelial cells from the inner lining of the blood vessel.
  • restenosis In terms of clinical practice, reocclusion of the treated vessel following coronary angioplasty, i.e., restenosis, is a significant medical problem since it occurs within six months following 30-50% of the procedures performed and is associated with substantial patient morbidity and health care expenditures.
  • the principal reasons for the restenosis are acute thrombus formation due to loss of the antithrombotic surface provided by the endothelial cells and neointima formation due to unchecked smooth muscle cell stimulation by blood-borne cells, again due to the loss of the protective endothelial cell layer.
  • Coronary bypass graft surgery does not involve removing the blockage to blood flow in the coronary artery, using instead a bypass to detour blood flow around the blocked vessel to supply the remainder of the heart muscle.
  • a bypass to detour blood flow around the blocked vessel to supply the remainder of the heart muscle.
  • the loss of the endothelial lining results in the loss of several critical endothelial properties including loss of the anticoagulant surface, loss of important smooth muscle cell regulatory force, and the loss of the protective vessel wall covering which shields smooth muscle cells from platelets, monocytes, and lymphocytes.
  • the subsequent response of the blood vessel to this pathologic injury is two-fold: 1) the physiological and beneficial migration of endothelial cells from the edge of the wound to restore luminal integrity and 2) the pathophysiological migration of smooth muscle cells from the interior of the blood vessel wall toward the lumen resulting in the neointima formation and postintervention occlusion.
  • Occlusion of peripheral arterial and coronary artery bypass grafts is a frequent and important clinical finding. Two-thirds of the saphenous vein coronary bypass grafts are either severely diseased or entirely occluded by six to eleven years following bypass surgery. Peripheral arterial bypass grafts generally suffer occlusion within two to five years.
  • Synthetic grafts also exhibit high rates of occlusion. Initially, grafts of this type are not endothelialized. This results in a substantial incidence of early occlusion due to thrombosis. With time, the grafts become partially re-endothelialized by migration of arterial endothelial cells from the proximal and distal anastomotic sites or from ingrowth of capillary endothelial cells through the porous synthetic graft onto the luminal surface. However, the process of endothelial cell migration is normally slow and does not permit total coverage of the graft by arterial endothelial cells.
  • capillary endothelial cells are less capable of inhibiting clot formation than arterial endothelial cells.
  • Attempts to reseed peripheral grafts with autologous endothelial cells have demonstrated that incomplete coverage of the graft at the time of seeding results in graft closure and lack of clinical benefit of the seeding procedure.
  • the genetically engineered cells described herein provide an important mechanism for addressing these critical problems in revascularization. These cells can be used to re-endothelialize denuded vessels or grafts without significant rejection by the patient's immune system. Moreover, since the cells can be grown in large numbers before the surgical procedure, adequate supplies are available for coverage of large areas of denuded vessel or naked graft. In this connection, further genetic engineering of the endothelial cells can be performed in accordance with copending application Serial No.
  • capillary endothelial cells can also be isolated from human sources, and can even be autologous.
  • cells can be isolated from subcutaneous fat via liposuction and cultured with known human endothelial cell growth factors.
  • a typical procedure for implanting universal donor endothelial cells in a patient's coronary artery is as follows:
  • step (1) determines that therapeutic angioplasty is appropriate, performing a standard balloon angioplasty procedure.
  • vascular graft or stent can be coated with genetically engineered endothelial cells and then implanted in a patient by:
  • a synthetic graft or stent such as a graft or stent made of DACRON or stainless steel
  • the genetically engineered endothelial cells provide an excellent mechanism for the administration of therapeutic agents either locally at the site of cell implantation or systemically. These cells might also secrete PDGF or FGF antagonists, thrombolytics, or thrombin antagonists, so as to inhibit restenosis in a vessel or graft wall.
  • Systemic drug delivery via universal donor endothelial cells might be most effectively accomplished by the use of genetically engineered microvascular (capillary) endothelial cells which offer several advantages including a relatively large surface area to volume ratio, especially when the cells are seeded into a capillary network as described below, and direct secretion of therapeutic protein products without any barrier to diffusion.
  • agents which can be administered in this way include blood clotting factors, clot dissolving factors, hormones, growth factors, cytokines, enzymes, and cholesterol binding or removing proteins.
  • an appropriate gene or combination of genes is inserted into the genome of the donor endothelial cells prior to transplantation.
  • a solution to the limitations of the relatively low density of endothelial cells which are available when seeded on a two dimensional surface is to take advantage of the biological properties of microvascular capillary endothelial cells. For example, several groups have demonstrated in vitro formation of stable capillary networks when microvascular capillary endothelial cells are maintained in a three dimensional culture system consisting of extracellular matrix components such as collagen in the presence of angiogenic factors.
  • microvascular endothelial cells cultured in three dimensions as compared to cells cultured in two dimensions.
  • this culture system provides for the maintenance of large numbers of cells in a relatively small volume.
  • this endothelial cell culture system to be useful for systemic protein delivery in vivo, several important additional properties must be demonstrated.
  • these cells must be amenable to genetic engineering and should continue to express their recombinant protein even when maintained in a three dimensional matrix of extracellular matrix components.
  • these three dimensional cultures must be transplantable into recipients and, preferably, demonstrate vascular anastomosis to the recipient circulation in vivo .
  • capillary endothelial cells can also be isolated from human sources, including autologous sources, as described by U.S. Patent No. 4,820,626 to Williams and Jarrell Sources of human capillary endothelial cells include omental fat, subcutaneous fat, or perinephric fat.
  • cells can be isolated from subcutaneous fat via liposuction and cultured with known endothelial cell growth factors.
  • a typical procedure for isolating cells of this type, for example, from a porcine source, is as follows:
  • Porcine microvascular endothelial cells are isolated by first removing the epididymal fat pads and/or kidneys from male pigs using sterile techniques. To do this, the organs or tissues are placed in sterile HEPES buffer (pH 7.4) which contains 140 mM NaCl, 10 mM HEPES, 10 mm KC1, 0.1 mm CaCl 2 , 0.2 mm MgCl 2 , 11 g/liter NaHC0 3 , 5.0 g/liter glucose, 100 U/ml penicillin, and 100 U/ml streptomycin. For kidneys, the peri-renal fat is dissected away and the kidneys are placed in sterile HEPES buffer as above.
  • the large visible vessels are dissected away from the epididymal fat and the fat is then placed into sterile HEPES buffer.
  • the fatty tissue is placed into 50 ml sterile Falcon "Blue Max” tubes containing a small amount of sterile HEPES buffer and the fat is minced for 3 to 5 minutes with a scissors.
  • the minced tissue is then placed into 50 ml Erlenmeyer flasks containing equal volumes of sterile HEPES buffer containing 5 mg/ml of collagenase and 5 mg/ml of bovine serum albumin (BSA) .
  • the flasks are incubated at 37°C with agitation for 20 minutes.
  • a small aliquot (0.1 ml) is removed from each flask every 20 minutes and then examined for the appearance of tube-like fragments of the capillary bed. The incubation is continued until the majority of the minced tissue contains tube-like fragments and single cells.
  • the cell suspension is centrifuged at 200 x g for 7 minutes in 15 ml sterile conical tubes.
  • the top white fatty layer is then aspirated off and the pellets are resuspended in 10 ml of HEPES buffer containing 10% BSA and then recentrifuged and resuspended an additional two times.
  • the resultant pellets are resuspended in 45% Percoll and centrifuged at 15,000 x g for 20 minutes at 4°C in a SS34 fixed angle rotor.
  • the tufts of the PMECs are in a milky off-white layer beneath the top-most adipocyte-containing layer and above a translucent layer containing larger vessel fragments.
  • the microvascular tufts and free endothelial cells are collected with a sterile pipette and then pelleted by centrifugation in HEPES-BSA at 200 x g for 3 minutes.
  • the tufts are resuspended in media (Medium 199E containing 20% heat-inactivated FBS, Penicillin, streptomycin, 5 mM HEPES, 5 M Pyruvate, and 5 mm glutamine mixed 1:1 with the same medium containing 10% FBS which has been conditioned for 48 hours by incubating over confluent endothelial cell cultures) .
  • media Medium 199E containing 20% heat-inactivated FBS, Penicillin, streptomycin, 5 mM HEPES, 5 M Pyruvate, and 5 mm glutamine mixed 1:1 with the same medium containing 10% FBS which has been conditioned for 48 hours by incubating over confluent endothelial cell cultures
  • the cells are then seeded into tissue culture flasks that have been coated with 1.5% gelatin in PBS overnight. 7.
  • the PMEC cultures are then incubated in a 5% C0 2 , 95% humidified atmosphere at 37°C.
  • the PMEC are routinely passaged at confluency using 0.02% trypsin in a Ca 2+ and Mg 2+ -free PBS containing EDTA to dislodge the cells from the plate and to dissociate cell aggregates.
  • the genetically engineered endothelial cells can be frozen under liquid nitrogen and stored until needed.
  • the engineered cells are first dispersed in a 5 mg/ml solution of neutralized acid soluble type I collagen (isolated from calf dermis) at a concentration of 3.0 x 10 6 cells per ml of collagen solution at 4°C. This mixture is plated into 24-well cluster dishes in 0.75 ml aliquots and placed in a
  • the cells are engineered to result in the expression or production of molecules encoding therapeutic proteins or nucleotide molecules, respectively.
  • therapeutic proteins include hormones, enzymes, receptors, immunomodulators, and neurotrans itters.
  • therapeutic nucleotide molecule examples include antisense, ribozymes, and molecules binding to viral and bacterial nucleic acids to inhibit translation thereof.
  • These capillary endothelial cell networks can then be implanted subcutaneously into a recipient patient, where the cells will secrete the therapeutic proteins systemically.
  • the engineered donor endothelial cells avoid graft rejection normally associated with the transplantation of non-autologous cells and thus can be used to administer their encoded therapeutic agent for substantial periods of time until, for example, removed from the host by a self-destruction mechanism of the type described above.
  • the genetically engineered capillary endothelial cells are seeded into a biological or synthetic matrix for implantation into the subcutaneous tissue of the recipient.
  • the matrix may preferably be of commercially available biocompatible materials such as collagen types I through XII, thrombospondin, entactin, proteoglycans, glycosa inoglycans, vitronectin, laminin, fibronectin, fibrinogen, MatrigelTM, or a combination of these and other natural extracellular matrix (ECM) components that allow capillary endothelial cells to form capillary networks in three dimensions since capillary endothelial cells, as opposed to large vessel endothelial cells or non-endothelial cells, form differentiated capillary networks in three dimensional collagen gels in vitro via the process of angiogenesis (Madri and Williams, "Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the cell matrix
  • the matrix is preferably in the form of a gel, prepared by modulation of matrix concentration, pH, temperature, salt content, or other physico-chemical properties known to those skilled in the art in order to induce gel formation.
  • the gel matrix should be in a form suitable for seeding with cells and implantation into the body. There should also be sufficient porosity for diffusion of gases and nutrients prior to vascularization and, ideally, to allow anastomosis of recipient blood vessels to the donor capillary networks. It may be advantageous to add angiogenic factors to the matrix prior to, or at the time of, implantation.
  • Such angiogenic factors will increase capillary network formation of the transplanted capillary endothelial cells and may also increase ingrowth of host capillaries.
  • Other materials may serve as support structure for the three dimensional ECM gel such as ethylene vinyl acetate, polylactide- glycolide, polyanhydride, fibrous suture material, or other biocompatible synthetic polymers.
  • the gel and, if present, the support structure may be enclosed within a porous polymeric framework prior to implantation into the recipient.
  • attachment molecules are any molecules for which there is a receptor on the cell surface. These include natural and synthetic molecules having one or more binding sites.
  • Extracellular matrix molecules include compounds such as laminin, fibronectin, thrombospondin, entactin, proteoglycans, glycosaminoglycans and collagen types I through XII.
  • Other natural attachment molecules include simple carbohydrates, complex carbohydrates, asialoglycoproteins, lectin ⁇ , growth factors, low density lipoproteins, heparin, poly-lysine, thrombin, vitronectin, and fibrinogen.
  • Synthetic molecules include peptides made using conventional methods to incorporate one or more binding sites such as R G D from fibronectin, L I G R K K T from fibronectin and Y I G S R from laminin. Attachment molecules are bound to a surface by ionic or covalent binding, or by association (from solution or by drying) . In some embodiments, the polymer may be modified to include one or more binding sites. Alternatively, the polymer may itself be formed in whole or in part by crosslinked attachment molecule or synthetic peptide.
  • Example l Expression of Human CD59 in Porcine Endothelial Cells Protects them from Hyperacute Rejection by Human Complement.
  • PAEC PAEC
  • DMEM fetal bovine serum
  • P/S penicillin and streptomycin
  • the cells Prior to retroviral infection, the cells were grown to 50% confluence.
  • Subconfluent PAEC were infected by using the amphotropic helper-free retroviral vector pRNSRalphaCD59+. The structure of this retroviral construct is shown in Figure 1.
  • PAEC were also infected with a control retroviral vector containing the drug selection marker gene neomycin or were uninfected.
  • amphotropic retroviral stocks were added to subconfluent cells growing in a T-25 tissue culture flask in a total volume of 3 ml. Polybrene was added to the flasks and the cultures were incubated at 37°C for 2 to 5 hours. The cell culture media was then removed, onolayers were rinsed two times in 5 ml of media and then 5 ml of media was added to the cells which were incubated at 37°C in 8% C02.
  • Neomycin resistant colonies were assayed for the cell surface expression of human CD59 by flow cytometric techniques (FACS analysis) .
  • FACS analysis flow cytometric techniques
  • the cells were then rinsed two times in staining buffer and then incubated for 30 minutes at 23°C with an FITC-conjugated goat anti-rabbit IgG or an anti-mouse IgG diluted 1:50 in staining buffer.
  • the cells were rinsed two times in staining buffer, once in PBS and then resuspended in 1% paraformaldehyde in PBS and analyzed by FACS. Positive cell surface expression of human CD59 (as measured by fluorescence intensity on the x axis) is demonstrated in Figure 2 for cell clones 1, 2 and 9 but not for control PAEC infected with control containing only the neomycin resistance gene.
  • CD59-infected PAEC were not different from either uninfected PAEC or PAEC infected with control vector. For example, they maintained proliferation rates identical to uninfected cells and they did not overgrow monolayers or proliferate in suspension, and were contact inhibited. Additionally, CD59-infected porcine endothelial cells were capable of attaching to a synthetic GortexTM graft, as demonstrated in the scanning micrograph shown in Figure 3. Two centimeters square of synthetic GortexTM sheets were steam-sterilized, placed in sterile 35 mm bacteriological petri dishes and overlaid with sterile stainless steel fences having a one centimeter square well.
  • CD59-infected PAEC were then seeded into the center wells of the fences at a density of 1 x 10 5 cells in a volume of 0.5 ml of culture media as described above and incubated at 37°C in 5% C0 2 . After two days, the cultures were refed with media and after an additional two days the media was aspirated off and the cultures were washed with PBS and then fixed with buffered 2% glutaraldehyde, 4% paraformaldehyde for one hour. The fences were then removed and the GortexTM was processed for scanning electron microscopy.
  • Figure 3 demonstrates that PAEC expressing cell surface human CD59 attach as well to synthetic GortexTM grafts as normal endothelial cells.
  • CD59— infected PAEC were assayed for their sensitivity to cytolysis by complement in human serum.
  • CD59-infected PAEC, control PAEC infected with vector alone, and uninfected PAEC were plated into 48-well tissue culture plates at a density of 1.25 x 10 5 cells/well in DMEM with 10% FBS, 2 mM glutamine and P/S. The culture media was removed and the cells were washed three times with media without FBS.
  • human serum diluted in DMEM at various concentrations was added to the cultures for 2 hours at 37°C. The percentage ⁇ of viable cells remaining in the cultures was assessed by staining the cells with 0.1% trypan blue.
  • Figure 4 demonstrates that greater than 80% of uninfected or control (vector alone infected) PAEC were killed by human serum whereas less than 10% of CD59-infected PAEC were killed.
  • the cells were then assayed to determine if the hCD59 would block complement activation, as well as the subsequent complement-mediated cell lysis.
  • the following prothrombinase assay was used.
  • Porcine aortic endothelial cells were stably infected with retroviral vectors carrying either a neomycin resistance gene alone (LXSN) or carrying human CD59 cDNA (LXSNCD59) or human CD59 cDNA engineered to carry a FLAG peptide epitope at its carboxy terminus (LXSNCD59Flg) .
  • LXSN neomycin resistance gene alone
  • LXSNCD59 human CD59 cDNA
  • LXSNCD59Flg human CD59 cDNA engineered to carry a FLAG peptide epitope at its carboxy terminus
  • Figure 5 is a graph demonstrating that hCD59 expressed on porcine endothelial cells blocks assembly of a prothrombinase complex, Lxsn, Maloney leukemia virus-based retroviral vector without CD59 insert (open circles), LxsnCD59, vector with CD59 insert (open squares) , and LxsnCD59Flg, vector with 5'Fig epitope-flagged CD59 insert (dark triangles) .
  • Example 2 Stable Expression of hCD59 in mouse cells and which protects the cells from complement mediated lysis.
  • Mouse Balb/3T3 cells were transfected with the pC8-hCD59-103 plasmid described in Figure 6. The transfection was performed using the calcium phosphate precipitation method with 10 ⁇ g of plasmid pC8-hCD59- 103 applied to approximately 10 6 cells. Stable transfectants were selected by cotransfecting the Balb/3T3 cells with a neomycin resistance plasmid (pSV2-neo; Yale University, New Haven, CT; 1 ⁇ g per 10 6 cells) and then selecting stably expressing cell clones in the presence of geniticin (G418; 500 ⁇ g/ml). Balb/3T3 cells transformed with just the neomycin resistance plasmid and not the pC8-hCD59-103 plasmid were used as controls.
  • a neomycin resistance plasmid pSV2-neo; Yale University, New Haven, CT; 1 ⁇ g per 10 6 cells
  • Stable transfectants were assessed for hCD59 expression by flow cytometry using 10 ⁇ g/ml anti-CD59 monoclonal antibody MEM-43 (Accurate Chemical and Scientific Corp., Westbury, NY) or 20 ⁇ g/ml of an anti-CD59 polyclonal serum (Southeastern Wisconsin Blood Center, Milwaukee, Wl) .
  • the cells were incubated with the primary antibodies at 4°C for 30 minutes.
  • FITC-conjugated-anti-mouse IgG was then added and allowed to incubate an additional 30 minutes at 4°C.
  • the cells were analyzed using a FACSort (B ' ecton Dickenson) flow cyto eter with the FL1 fluorescence channel (520 nm) set at logarithmic gain. The results show that stable transfection was achieved.
  • hCD59-expressing Balb/3T3 clones were analyzed for the expression of functional CD59 by using a complement-dependent cell killing assay designed to monitor the cellular release of a fluorescent dye.
  • the dye release assay was performed essentially as described by the manufacturer, Molecular Probes, Inc. (Eugene, Oregon) . Briefly, the assay relies on the uptake of calcein-AM and its subsequent hydrolysis to the fluorescent calcein molecule.
  • Calcein-AM is an esterase substrate that is membrane permeable and virtually non-fluorescent. Substrate hydrolysis by intracellular esterase activity yields the intensely fluorescent product, calcein. Calcein is a poly-anionic molecule that is retained in the living cell and results in the cell generating an intense uniform green fluorescence.
  • Complement-mediated dye release was determined from cell supernatants after incubation of the cells with a reactive antibody (polyclonal antiserum raised in rabbits to 3 types of mouse cells; L-cells, Balb/3T3 cells and MOP-8 cells, in the presence of complement C8-deficient human serum for 15 minutes at 37°C. EDTA-treated human serum was added back to the reaction at increasing concentrations (0-15%) for 30 minutes at 37°C. The percent dye release at the end of the incubation period was calculated from total uptake, corrected for non-specific dye release. Dye release was measured at 490nm using a cytofluor fluorescence plate reader (Millipore) .
  • mice Balb/3T3 cells which stably express human CD59 are protected from the complement killing effects of human serum whereas neomycin-resistant control clones not expressing hCD59 are sensitive to human serum-dependent cell killing.
  • Example 3 Generation of Transgenic Mice Expressing hCD59.
  • the CMV-hCD59-SV40 transcriptional unit was removed from pC8-hCD59-103 using the restriction enzymes Spel and Seal. This restriction digest selectively extracts the transcription unit from the overall vector and permits purification of only the essential elements required for expression.
  • the 2300bp restriction fragment resulting from the digest was gel isolated, extensively purified through an ELUTIP column (Schleicher & Schuell, Keene, NH) , dialyzed against pyrogen free injection buffer (lOmM Tris, pH7.4 + O.lmM EDTA in pyrogen free water), and used for embryo injection to generate transgenic mice in accordance with the methods of Hogan et al. 1986 and Brinster et al., 1985.
  • mice were determined to contain the transcriptional cassette of the invention.
  • Transgene positive mice were then analyzed further for expression of hCD59 in various mouse cells and tissues.
  • Several cell and/or tissue types were isolated from the hCD59 transgenic mice in order to determine the extent of transgene expression in these transgenic animals.
  • the initial analysis of each positive transgenic mouse involved isolating whole blood from the animal by retro-orbital bleeding. Clotting was prevented by the addition of ACD-2 (71.4mM citric acid, 85mM sodium citrate, lllmM dextrose) at a ratio of 6 parts blood to 1 part ACD-2.
  • the different cell types i.e., erythrocytes, lymphocytes and monocytes
  • Erythrocytes were washed with HBSS + 2% fetal calf serum and then processed directly.
  • Total leukocytes were isolated from whole blood by hypotonic lysis of the erythrocytes followed by centrifugation and washing with HBSS + 2% fetal calf serum.
  • the cells were incubated in HBSS + 2% fetal calf serum with the addition of 10 ⁇ g/ml anti-CD59 monoclonal antibody (MEM-43, Accurate Chemical and Scientific Corp., Westbury, NY; or YTH53.1, Serotec, Indianapolis, IN) or 20 ⁇ g/ml of an anti-CD59 polyclonal serum (Southeastern Wisconsin Blood Center, Milwaukee, Wl) for 30 minutes at 4°C.
  • FITC-conjugated-anti-mouse IgG was added and allowed to incubate an addit l 30 minutes at 4°C.
  • the cells were then anal ⁇ _td using a FACSort (Becton Dickenson) flow cytometer with the FL1 fluorescence channel (520 nm) set at logarithmic gain.
  • FACSort Becton Dickenson
  • Y-3 specific for a common epitope shared by murine class I molecules H2Kb and H2Kk which represent the 2 possible haplotypes of the offspring, was used to assay erythrocytes for a known cell surface marker.
  • Another antibody, T200 which recognizes the common leukocyte antigen CD45, was used as a control antibody to assess lymphocyte staining.
  • Flow cytometric analysis was performed on cells from several of the transgenic mice obtained as described above.
  • hDC59 was expressed on the monocytes of all of the CD59-positive transgenic animals tested. Litter mates negative for the hCD59 cDNA by Southern blotting did not express hCD59. Neutrophils and lymphocytes were negative for hDC59. These results demonstrate that hCD59 cDNA was expressed in transgenic animals.
  • Dissected tissues i.e., abdominal aorta, heart, lung, and fat pad
  • O.C.T. tissue Tek II, Miles, Elkhart, Indiana
  • Tissue sections were cut on a cryotome and mounted on slides. Duplicate sections were then processed for immunohistochemistry. The tissue sections were stained with anti-CD59 antisera (MEM-43 or YTH 53.1) and anti-MHC Class I (Y-3, as a control), washed several times with PBS, and then incubated with an Rhodamine-conjugated goat anti-mouse IgG secondary antibody.
  • the results of the immunohistochemical staining for abdominal aortic tissue demonstrate positive cellular expression of CD59 in the tissue section in the transgenic animals.
  • the immunohistochemical stain reveals bright staining of the intimal layer of abdominal aortic endothelial cells and bright staining of the aortic adventitial capillary endothelial cells. Some low level staining of medial smooth muscle cells could be seen. Similar results were obtained for the other tissues tested.
  • Bright staining of endothelial cells lining the coronary arteriolar region was observed, along with staining of yocardial cells.
  • Reduced staining of alveolar epithelial cells was seen in combination with bright staining of adjacent capillary endothelial cells.
  • For the mouse epididymal fat pad which is an example of a systemic capillary network, there was bright capillary endothelial cell staining and present, but reduced, interstitial staining.
  • Example 4 Generation of recombinant mice bearing a deletion of the invariant chain (li) having reduced cell surface MHC class II expression and diminished ability to present exogenous protein antigen.
  • Major histocompatibility complex (MHC) class I and class II molecules bind and present peptide antigen to T-cells. While class I molecules predominantly bind peptides generated from endogenously synthesized cytosolic proteins prior to their release from the endoplas ic reticulum (ER) , class II MHC molecules present exogenously derived internalized antigen.
  • MHC major histocompatibility complex
  • Class II molecules are expressed on the cell surface as heterodimers composed of a 34kD ⁇ and 28kD ⁇ chains.
  • the ⁇ and ⁇ chains also interact with a third glycoprotein, the invariant chain (li) , following their translocation into the ER.
  • the ⁇ li complex is then transported through the Golgi apparatus to an acidic compartment where the li is proteolytically removed. It is within this same compartment that class II molecules may come in contact with exogenous antigens that have been internalized by endocytosis. Although many important features of this pathway remain unknown, it appears that a coordinated series of events may simultaneously generate both peptide antigens and li-free- ⁇ /5 dimers leading to class II-peptide interaction. Finally, class II ⁇ -peptide complexes are transported to the cell surface for presentation to antigen-specific T- cells.
  • invariant chain gene can be disrupted in mouse embryonic stem cells (ES cells) , and in transgenic mice produced using the ES cells, by specifically and stably replacing it in the genome with a mutated and non-functional form of the invariant chain gene and that replacement of the native invariant chain gene with a non-functional mutant can be achieved in a given cell by gene targeting technology which takes advantage of a homologous recombination event between the mutated gene and the native invariant chain gane.
  • a partial restriction enzyme map for the mouse invariant chain gene is shown in Figure 7B.
  • a gene targeting vector was constructed, so as to replace a sequence of the invariant chain gene between nucleotides 661 and 1064 with the neomycin gene.
  • This genetic engineering leads to the elimination of most of exon 1 including the translation initiation codon ATG, and a large portion of the promoter including the TATA box and CAAT box which function as regulatory elements required for accurate and efficient transcription of the invariant chain gene, as reported by Zhu and Jones, 1989 "Complete sequence of the murine invariant chain (Ii) gene" Nucleic Acids Res. 17:447-448.
  • This gene targeting vector is shown in Figure 7A as a general example of the disruption strategy.
  • deletion of li chain causes a significant reduction of cell surface class II expression, accumulation of class II molecules in the endoplasmic reticulum and a significantly diminished ability to present exogenous protein antigen.
  • li chain "knock-out" mice show a profound reduction in their CD4 + T-cell subpopulation.
  • Embryonic stem cells were routinely passaged every other day in ES growth media containing DMEM (high glucose) with 15% FBS and 0.1 mM 2-mercaptoethanol. The ES cells were maintained on a confluent layer of primary embryonic fibroblasts. Two days prior to the transfection of the ES cells with the gene targeting vector the cells were expanded in culture.
  • DNA corresponding to the invariant chain targeting vector were introduced into 1 x 10 7 ES cells by electroporation using a BioRad electroporator set at 250 ⁇ F and 0.32 kV.
  • the ES cells were then seeded onto 10 x 100 mm NuncTM tissue culture plates and stable transfectants were selected for chromosomal integration by way of neomycin resistance in G418 (170 ⁇ g/ml) and/or gangcyclovir in some experiments where the herpes-simplex virus thymidine kinase gene was included in the targeting vector.
  • Genomic DNA was isolated from 55 individual stable transfectants and digested overnight with either the _BcoRI or Drain restriction endonucleases. Digested DNA was resolved by electrophoresis, blotted to GeneScreen+TM nylon membranes and then hybridized with a radiolabeled DNA probe specific for the mouse invariant chain gene.
  • mice deficient in expression of the MHC class II-associated li gene by introducing a deletion into the li gene of embryonic stem cells.
  • mice deficient in expression of the MHC class II- associated li gene was used to generate mice deficient in expression of the MHC class II- associated li gene by introducing a deletion into the li gene of the embryonic stem cell line ES-D3, described by Doetschman, J. Embryo1. Exp. Morph. 87,27-45 (1985) .
  • a 0.4 kb Stul fragment containing exon 1 and a portion of the li promoter region was replaced with a neomycin resistance (neo r ) cassette to create an li targeting vector, pliKO, as shown in Figure 8.
  • the targeting vector contained 5 kb of homologous flanking sequence and two copies of the herpes simplex virus (HSV-1) thymidine kinase (tk) gene inserted at the 5' end of the li fragment.
  • HSV-1 herpes simplex virus
  • tk thymidine kinase
  • a 2.1 kb Bglll-EcoRI fragment encoding the promoter and exon 1 was subcloned into pSK Bluescript (Stratagene, LaJolla, CA) .
  • the internal 0.4 kb StuI fragment was deleted and the neomycin resistance cassette (pMClneo PolyA, Stratagene) was introduced in place of the StuI fragment.
  • the cells were plated on ten 100-mm plates in media (Dulbecco's modified Eagle medium with 15% fetal calf serum, 2 mM glutamine and 0.1 mM 2-mercaptoethanol) supplemented with leukemia inhibitory factor (Gibco-BRL) .
  • Genomic DNA was purified from individual clones and analyzed by Southern blot with a 5' probe indicated by the solid box, as shown in Figure 9.
  • This probe is a 0.6 kb Bglll-Xbal fragment from the 5' end of the li gene. It hybridizes to a 3.0kb EcoRl fragment of the endogenous li gene and to a 2.5 kb fragment of the disrupted allele.
  • the Southern blot analysis is a hybridization of the li probe to EcoRI digested tail DNA from littermates derived from the mating of two heterozygous +/ ⁇ li mice.
  • Hybridization with the 5' li probe identifies animals which are homozygous wildtype (+/+) , heterozygous (+/-) , or homozygous for the disrupted li allele (-/-).
  • LPS-treated splenocytes from wildtype, heterozygous (+/-), or mutant (-/-) mice were examined by immunofluorescence confocal microscopy using the li-specific antibody, ln-1. Confocal microscopy of LPS treated splenocytes from heterozygous (+/-) and homozygous (-/-) was performed with an invariant chain specific antibody, ln-1.
  • Splenocytes were attached to Alcian Blue coated cover slips, permeabilized with 0.01% saponin and stained with the invariant chain specific mAb ln-1 (A gift from Jim Miller, Chicago University) followed by affinity purified donkey anti- rat-TRITC (Jackson Labs) .
  • the cells were then imaged on either a Zeiss confocal microscope.
  • the +/- splenocytes expressed normal levels of li as compared to C57BL/6 or 129Sv/J parental mice. As expected, no li expression was detected either on the plasma membrane or intracellularly in LPS-treated splenocytes from the -/- mice.
  • the cells were washed twice with staining buffer (1 x PBS, 1% fetal calf serum, 0.1% NaN 3 ) and incubated with the second step reagent fluorescein-conjugated mouse anti-rat antibody (M5/114) or fluorescein-conjugated F(ab')2 fragment of rabbit antibody to mouse IgG(Y-Ae) .
  • M5/114 fluorescein-conjugated mouse anti-rat antibody
  • F(ab')2 fragment of rabbit antibody to mouse IgG(Y-Ae) were washed, fixed with 1% paraformaldehyde and analyzed by flow cytometry on a Becton Dickinson FACS Star.
  • M5/114 other class II specific Ab Y3P, Y248, Y219, AF120.6, and Y237) were used to stain splenocytes. All antibodies tested gave similar results.
  • LPS-treated splenocytes were double-labeled for class I and various antigenic markers known to be localized to distinct organelles.
  • LPS stimulated splenocytes were attached to Alcian Blue coated cover slips, permeabilized with 0.01% saponin and stained with primary antibody and affinity purified secondary antibody.
  • the cells were then imaged on a Zeiss confocal microscope with either staining with a Rabbit anti-serum raised to affinity purified I-A d (Rb anti-I- A d , a gift of Dr. Ralph Kubo, Cetus, Corp., LaJolla, CA) and affinity purified FITC-goat anti rabbit; staining with Rb anti-I-A d (Green) and the FcR specific mAb 2.4G2 (Red), or staining with Rb anti-I-A d (Green) and an anti-BiP mAb (Red) .
  • Rb anti-I- A d a gift of Dr. Ralph Kubo, Cetus, Corp., LaJolla, CA
  • affinity purified FITC-goat anti rabbit staining with Rb anti-I-A d (Green) and the FcR specific mAb 2.4G2 (Red)
  • Rb anti-I-A d Green
  • splenocytes from the homozygous mutant mice exhibited a 5-10 fold reduction in the level of class II staining on the plasma membrane. Relative to the staining observed in splenocytes from parental or heterozygous
  • (+/-) mice class II molecules in cells isolated from homozygous
  • Mutant splenocytes (open circles) are far better at presenting exogenous peptide antigens then are the control +/+ (open squares) or +/- (dark diamonds) cells. In contrast the -/- cells are at least 10 times less efficient at presenting E ⁇ fusion protein. No stimulation of the T-cells was observed when a non-relevant fusion protein, Conpep (described by Nakagawa, et al., Eur. J. Immun. 21, 2851-2855 (1991) , was substituted for the E ⁇ Sl/2 protein.
  • Stimulation of the T-cell hybrids was measured by incubating 1 x 10 5 1H 3 , hybrid cells (Rudensky, 1991) with 1 or 3 x 10 5 spleen cells per well of 96-well plates in the presence or absence of peptide/protein. After 24 hour of incubation 50 ⁇ l aliquots of supernatants were removed and tested for lymphokine production using the CTLL-2 cell line at 5 x 10 3 cells per well. Cells were pulsed for 4 hours with 1 ⁇ Ci per well of 3 H-thymidine and collected.
  • the E ⁇ recombinant fusion protein was generated by subcloning oligonucleotides encoding the nucleotide sequence of the naturally processed E ⁇ -derived 17mer peptide (E ⁇ 56-73) into the BamEI site of pGEXTAG (Nakagawa, et al. 1991) .
  • the E ⁇ Sl/2 fusion protein has a stop codon incorporated into the oligonucleotide such that the TAG peptide is not translated.
  • Recombinant fusion proteins were expressed and purified as described by Smith and Johnson, Gene 67, 31-40 (1988).
  • a nonspecific fusion protein, conpep which encodes a conalbumin 13 amino acid peptide (amino acids 134-146) was produced and used to establish specificity of the T-cell response.
  • both the mutant and wildtype splenocytes are able to functionally bind and present exogenously added E ⁇ peptide to an E ⁇ specific T-cell hybrido a.
  • the ability of the -/- cells to present intact E ⁇ - containing fusion protein was markedly reduced as compared to +/+ cells, as shown by Figure 10B. Since the li deficient mice were generated by targeted mutation of the li gene, -/- cells should not be defective at internalizing or degrading exogenous protein into antigenic peptides.
  • mutant cells to present whole protein was likely to reflect a failure of the class II molecules to acquire processed peptide antigens either due to the inability of the ⁇ and ⁇ chains to reach to the appropriate endoso al compartment or their inability to bind processed antigen.
  • the class II molecules on the cell surface of -/- cells were transported from the ER to the cell surface without passing through a putative processing compartment, the peptide binding groove of the class II molecules might be "empty.”
  • the ability of +/- and -/- splenocytes to bind E ⁇ peptide by measuring cell surface staining with Y-Ae was compared.
  • Y-Ae is a monoclonal alloantibody that detects a determinant expressed on a subset of class II I-A b molecules when complexed with an E ⁇ -derived peptide E ⁇ 56-73.
  • Example 5 Genetic engineering of microvascular capillary endothelial cells, implantation, and expression of protein in vivo.
  • Rat microvascular capillary endothelial cells were isolated by first removing the epididymal fat pads using sterile technique.
  • the fat pads were placed in sterile HEPES (pH 7.4) which contains 140 mM NaCl, 10 mM HEPES, 10 mM KCl, 0.1 mM CaCl 2 , 0.2 mM MgCl 2 , 11 g/1 NaHC0 3 , 5.0 g/1 glucose, 100 U/ml penicillin, and 100 U/ml streptomycin.
  • the fat was then minced for 3 to 5 minutes with a scissors.
  • the minced tissue was placed into flasks containing equal volumes of sterile HEPES buffer with 5 mg/ml collagenase and 5 mg/ml bovine serum albumin. The incubation was continued until the majority of the minced tissue contained tube-like fragments and single cells. The cell suspension was then centrifuged at 200 x g for 7 minutes in 15 ml conical tubes. The top white fatty layer was then aspirated off and the pellets resuspended in 10 ml of HEPES buffer containing 10% BSA and recentrifuged and resuspended an additional two times.
  • the resultant pellets were resuspended in 45% Percoll and centrifuged at 15,000 x g for 20 minutes at 4°C in a SS34 fixed angle rotor.
  • the tufts of the RFCs are in a milky white layer beneath the top-most adipocyte-containing layer and above a translucent layer containing large vessel fragments.
  • the tufts were resuspended in media (Medium 199E containing 20% heat inactivated fetal bovine serum, 5 mM HEPES, penicillin and streptomycin, 5 mM pyruvate, and 5 mM glutamate mixed with 1:1 with the same medium containing 10% FBS which has been conditioned for 48 hours by incubating over confluent endothelial cells) .
  • media Medium 199E containing 20% heat inactivated fetal bovine serum, 5 mM HEPES, penicillin and streptomycin, 5 mM pyruvate, and 5 mM glutamate mixed with 1:1 with the same medium containing 10% FBS which has been conditioned for 48 hours by incubating over confluent endothelial cells.
  • the cells were then seeded into tissue culture flasks that have been coated with 1.5% gelatin in PBS overnight.
  • the microvascular endothelial cell cultures were incubated in 5% C02 at 37°C.
  • Retroviral vectors for the expression of human ApoE were constructed from a defective Moloney murine leukemia virus. A cDNA fragment encoding the full-length and functional human apolipoprotein E (Apo E) was subcloned into the retroviral vector. The resulting plasmid was designated Lxsn-ApoE. Ecotropic retrovirus was produced by transfecting Psi-2 cells with polybrene and selecting in G418. Amphotrophic virus stocks were prepared by infecting Psi-AM packaging cells and stable microvascular capillary endothelial cell transfectants were selected in G418.
  • the engineered cells were first dispersed in a 5 mg/ml solution of neutralized acid-soluble type I collagen (isolated from calf dermis) at a concentration of 3.0 x 10 6 cells per ml in DMEM with 10% fetal calf serum and 25% bovine aortic endothelial cell-conditioned media. The cells were maintained at 37°C with 5% C0 2 .
  • the media was harvested and analyzed by Western blot analysis using an antibody specific for human ApoE.
  • the analysis shows that rat epididymal fat pad capillary endothelial cells stably infected with the retroviral construct and cultured in a three dimensional collagen matrix express and secrete human ApoE as determined by Western blotting analysis using an antibody to human ApoE protein.
  • microvascular capillary cells have undergone a phenotypic change during their formation of capillary networks in three dimensional collagen gels, they continue to express and secrete levels of human ApoE equivalent to that of cells maintained in two dimensional cultures.
  • NAME Pabst, Patrea L.
  • CTATATACCT CCTCTTGCCA CCCATACTAT TTGTGATCGG AATCATACAT GGCTACCTGT 300
  • CTCTATTAAA ATCTTCAATA GTTGTTATTC TGTAGTTTCA CTCTCATGAG TGCAACTGTG 1380
  • TTCTCTCTAC AGTCAGTCTG GAGTAATCCC AAAGTGGTGT CTTTCGTAAA TAAGGAGAAC 60
  • GCAGAGCCCC AGCCCAGACC CCGCCCAAAG CACTCATTTA ACTGGTATTG CGGAGCCACG 720
  • TTTTTTTCCA CAAGATCTGA AATGATATTT CCACTTATAA AGGAAATAAA AAATGAAAAA 2340
  • TTACATGTAA AACAAGAAAA GTTGAAGAAG ATATGTGAAG AAAAATGTAT TTTTCCTAAA 2820

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Abstract

L'invention concerne des cellules produites par ingénierie génétique et pouvant servir de cellules donneuses universelles dans des utilisations telles que la reconstruction de parois vasculaires ou l'administration d'agents thérapeutiques. Les cellules comprennent une région codante réalisant une protection contre une lyse à base de complément, c'est-à-dire, un rejet hyperaigu. De plus, le génome naturel de la cellule est modifié, de façon que les protéines fonctionnelles codées par les gènes du complexe d'histocompatibilité majeure de classe II ou à la fois de classe I et de classe II n'apparaissent pas sur la surface de la cellule. De cette façon, on évite l'attaque de cellules T. Les cellules peuvent éventuellement comporter un mécanisme d'autodestruction, de façon à être supprimées de l'hôte quand elles ne sont plus nécessaires.
PCT/US1993/006216 1992-06-29 1993-06-29 Matrice de cellules endotheliales microvasculaires et donneuses universelles WO1994000560A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843182A (en) * 1994-03-14 1998-12-01 Cryolife, Inc. Treated tissue for implantation and methods of preparation
WO2000056898A1 (fr) * 1999-03-24 2000-09-28 The Board Of Trustees Of The Leland Stanford Junior University Cellules endotheliales microvasculaires et immortelles, et utilisation de celles-ci
US6430148B1 (en) 1997-12-22 2002-08-06 Lsi Logic Corporation Multidirectional communication systems
EP2061873A2 (fr) * 2006-09-21 2009-05-27 Tissue Genesis Matrices d'administration de cellules
US10633434B2 (en) 2016-06-14 2020-04-28 Regeneron Pharmaceuticals, Inc. Anti-C5 antibodies
US11365265B2 (en) 2017-12-13 2022-06-21 Regeneron Pharmaceuticals, Inc. Anti-C5 antibody combinations and uses thereof

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US5843182A (en) * 1994-03-14 1998-12-01 Cryolife, Inc. Treated tissue for implantation and methods of preparation
US6430148B1 (en) 1997-12-22 2002-08-06 Lsi Logic Corporation Multidirectional communication systems
WO2000056898A1 (fr) * 1999-03-24 2000-09-28 The Board Of Trustees Of The Leland Stanford Junior University Cellules endotheliales microvasculaires et immortelles, et utilisation de celles-ci
EP2061873A2 (fr) * 2006-09-21 2009-05-27 Tissue Genesis Matrices d'administration de cellules
EP2061873A4 (fr) * 2006-09-21 2009-10-21 Tissue Genesis Matrices d'administration de cellules
US10633434B2 (en) 2016-06-14 2020-04-28 Regeneron Pharmaceuticals, Inc. Anti-C5 antibodies
US11479602B2 (en) 2016-06-14 2022-10-25 Regeneren Pharmaceuticals, Inc. Methods of treating C5-associated diseases comprising administering anti-C5 antibodies
US11492392B2 (en) 2016-06-14 2022-11-08 Regeneran Pharmaceuticals, Inc. Polynucleotides encoding anti-C5 antibodies
US11365265B2 (en) 2017-12-13 2022-06-21 Regeneron Pharmaceuticals, Inc. Anti-C5 antibody combinations and uses thereof
US12084516B2 (en) 2017-12-13 2024-09-10 Regeneron Pharmaceuticals, Inc. Anti-C5 antibody combinations and uses thereof

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