WO2013063076A1 - Compositions et méthodes de modulation des complications, risques et problèmes associés aux xénogreffes - Google Patents

Compositions et méthodes de modulation des complications, risques et problèmes associés aux xénogreffes Download PDF

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WO2013063076A1
WO2013063076A1 PCT/US2012/061637 US2012061637W WO2013063076A1 WO 2013063076 A1 WO2013063076 A1 WO 2013063076A1 US 2012061637 W US2012061637 W US 2012061637W WO 2013063076 A1 WO2013063076 A1 WO 2013063076A1
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human
cell
sirpa
cells
heterologous
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Alfred Joseph TECTOR III
Leela L. PARIS
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Indiana University Research & Technology Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to the fields of molecular biology and transplantation, and more particularly relates to donor, transgenic, non-human mammals, as well as organs, tissues and cells derived therefrom that are particularly useful for xenotransplantation therapies.
  • the present invention also relates to methods of modulating xenotransplant rejection using such organs, tissues and cells derived from such transgenic, non-human mammals.
  • liver xenotransplantation may provide a solution to the shortage of organs, tissues and cells from human donors and the treatment of, for example, end stage organ failure.
  • a major barrier to clinical application of, for example, liver xenotransplantation is thrombocytopenia observed within minutes after graft reperfusion. Briefly, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) within the xenotransplant bind to and phagocytize recipient platelets, leading to thrombocytopenia.
  • LSECs liver sinusoidal endothelial cells
  • KCs Kupffer cells
  • CD47 is a ubiquitously expressed 50-kDa cell surface glycoprotein that can serve as a ligand for signal regulatory protein a (SIRPa).
  • SIRPa signal regulatory protein a
  • CD47 and SIRPa constitute a cell-cell communication system that plays important roles in a variety of cellular processes including, but not limited to, cell migration, B cell adhesion and T cell activation.
  • CD47 is expressed on the surface of several cell types (e.g., erythrocytes, leukocytes and platelets).
  • the CD47-SIRPa interaction provides a negative regulatory (i.e., inhibitory) signal to phagocytic cells such as macrophages, thereby preventing phagocytosis of normal self-cells.
  • phagocytic cells such as macrophages
  • the porcine SIRPa on the transplanted LSECs does not interact with recipient CD47 on platelets. Without the inhibitory signal from CD47-SIRPa interaction, the transplanted, porcine LSECs phagocytize the human platelets, resulting in the thrombocytopenia.
  • the present invention provides a non-human mammal comprising in its genome a nucleotide sequence encoding at least one heterologous immune- inhibitory molecule, wherein the at least one heterologous mammalian immune-inhibitory molecule is signal-regulatory protein alpha (SIRPa) or an active fragment or variant thereof.
  • SIRPa amino acid sequence comprises SEQ ID NO:l.
  • the non-human mammal is any mammal known to the art, although in one embodiment, the mammal is selected from the group consisting of a baboon, cat, chimpanzee, cow, dog, goat, horse, mouse, non-human primate, rat and pig.
  • the heterologous mammalian immune-inhibitory molecule may be from any mammal known to the art, although in one embodiment, the heterologous mammalian immune-inhibitory molecule is selected from the group consisting of a baboon, cat, chimpanzee, cow, dog, goat, horse, human, mouse, non-human primate, rat and pig.
  • the non-human mammal is a pig
  • the heterologous mammalian immune-inhibitory molecule is from a human
  • the invention also includes an organ from a non-human mammal comprising in its genome a nucleotide sequence encoding at least one heterologous immune-inhibitory molecule, wherein the at least one heterologous mammalian immune- inhibitory molecule is signal-regulatory protein alpha (SIRPa) or an active fragment or variant thereof.
  • SIRPa amino acid sequence comprises SEQ ID NO:l.
  • the organ is any organ suitable for transplantation.
  • the organ is selected from the group consisting of heart, liver, lung, kidney and pancreas.
  • the invention also includes tissue of a non-human mammal comprising in its genome a nucleotide sequence encoding at least one heterologous immune- inhibitory molecule, wherein the at least one heterologous mammalian immune-inhibitory molecule is signal-regulatory protein alpha (SIRPa) or an active fragment or variant thereof.
  • SIRPa amino acid sequence comprises SEQ ID NO:l.
  • the tissue is any tissue sutiable for transplantation, although in other embodiments, the tissue is selected from the group consisting of bone marrow, cornea, neuron and tendons.
  • the invention also includes a cell of a non-human mammal comprising in its genome a nucleotide sequence encoding at least one heterologous immune- inhibitory molecule, wherein the at least one heterologous mammalian immune-inhibitory molecule is signal-regulatory protein alpha (SIRPa) or an active fragment or variant thereof.
  • SIRPa amino acid sequence comprises SEQ ID NO:l.
  • the cell is any cell suitable for cellular graft or transplantation, including, for example, a hematopoietic cell.
  • the hematopoietic cell may be selected from the group consisting of endothelial cells, lymphocytes such as B cells and T cells, hematopoietic stem cells, Kupffer cells, machrophages, monocytes, sinusoidal endothelial cells, and platelets.
  • the invention provides a method of modulating thrombocytopenia in a transplant recipient following xenotransplantation.
  • the method comprises transplanting into a heterologous mammal an organ, tissue or cell from a non-human mammal comprising in its genome a nucleotide sequence encoding at least one heterologous immune-inhibitory molecule, wherein the at least one heterologous mammalian immune- inhibitory molecule is signal-regulatory protein alpha (SIRPa) or an active fragment or variant thereof.
  • SIRPa signal-regulatory protein alpha
  • the non-human mammal is any mammal known to the art, although in one embodiment, the mammal is selected from the group consisting of a baboon, cat, chimpanzee, cow, dog, goat, horse, mouse, non-human primate, rat and pig.
  • the heterologous mammalian immune-inhibitory molecule may be from any mammal known to the art, although in one embodiment, the heterologous mammalian immune-inhibitory molecule is selected from the group consisting of a baboon, cat, chimpanzee, cow, dog, goat, horse, human, mouse, non-human primate, rat and pig.
  • the non-human mammal is a pig
  • the heterologous mammalian immune- inhibitory molecule is from a human.
  • the invention provides a method of supplying an organ, tissue or cell suitable for transplantation.
  • the method comprises generating a transgenic, non- human mammal comprising in its genome a nucleotide sequence encoding at least one heterologous immune-inhibitory molecule, wherein the at least one heterologous mammalian immune-inhibitory molecule is signal-regulatory protein alpha (SIRPa) or an active fragment or variant thereof; and harvesting the organ, tissue or cell from the transgenic, non-human mammal.
  • SIRPa signal-regulatory protein alpha
  • the non-human mammal is any mammal known to the art, although in one embodiment, the mammal is selected from the group consisting of a baboon, cat, chimpanzee, cow, dog, goat, horse, mouse, non-human primate, rat and pig.
  • the heterologous mammalian immune-inhibitory molecule may be from any mammal known to the art, although in one embodiment, the heterologous mammalian immune-inhibitory molecule is selected from the group consisting of a baboon, cat, chimpanzee, cow, dog, goat, horse, human, mouse, non-human primate, rat and pig.
  • the non-human mammal is a pig
  • the heterologous mammalian immune- inhibitory molecule is from a human.
  • the SIRPa amino acid sequence comprises SEQ ID NO:l
  • the organ is selected from the group consisting of heart, liver, lung, kidney and pancreas
  • the tissue is selected from the group consisting of bone marrow, cornea, neuron and tendon
  • the cell is a hematopoietic cell including, without limitation, endothelial cells, lymphocytes such as B cells and T cells, hematopoietic stem cells, upffer cells, machrophages, monocytes, sinusoidal endothelial cells, and platelets.
  • FIG.l shows that human platelets display variable levels of surface CD47.
  • FIG. 2 shows that human and porcine platelets display CD47.
  • FIG. 3 shows that porcine liver expresses SIRPa.
  • FIG. 4 shows that LSECs express SIRPa mRNA transcripts.
  • FIG. 5 shows that primary LSECs express SIRPa in culture.
  • FIG. 6 shows that the expression of human SIRPa by porcine LSECs reduces xenogeneic platelet phagocytosis (porcine LSECs were isolated, cultured and transfected with human SIPRa or vector alone; fluorescent human or pig platelets were incubated with transfected LSECs; and non-bound platelets removed by washing and captured fluorescence measured).
  • FIG. 7 shows SIRPa tyrosine phosphorylation in porcine LSECs incubated with human or allogeneic platelets.
  • FIG. 8. shows that domestic porcine liver expresses SIRPa.
  • FIG. 9 shows a comparison of SIRPa expression between porcine and human platelet.
  • FIG. 10 shows that primary LSECs express SIRPct in culture.
  • compositions and methods are provided for expressing heterologous nucleotide sequences in a non-human mammal and its organs, tissues and cells.
  • the compositions include nucleic acid molecules for expressing a nucleotide sequence of interest such as at least one immune-inhibitory molecule such as a SIRPa nucleotide sequence heterologous to a non- human mammal and its organs, tissues and cells.
  • compositions include expression cassettes or nucleotide constructs having a synthetic promoter as described herein operably linked to a nucleotide sequence encoding at least one an inhibitory-inhibitory molecule such as a SIRPa nucleotide sequence heterologous to a non-human mammal and its organs, tissues and cells.
  • compositions include a transformed, non-human mammal and its organs, tissues and cells having incorporated in its genome at least one of the nucleotide sequences described herein such that the transformed, non-human mammal and its organs, tissues and cells express detectable levels of heterologous SIRPa.
  • the methods involve introducing into a non-human mammal or its organs, tissues or cells an expression cassette having a synthetic promoter as described herein operably linked to a nucleotide sequence encoding a SIRPa heterologous to the mammal or its organs, tissues or cells, wherein the non-human mammal or its organs, tissues or cells are transgenic for a SIRPa heterologous to the non-human mammal or its organs, tissues or cells.
  • the methods also involve modulating complications, issues and risks associated with xenotransplant rejection in non-human and human mammals by modulating, for example, phagocytosis of recipient hematopoietic cells.
  • compositions and methods described herein therefore find use in improving the outcome of a non-human or human mammalian recipient following xenotransplantation.
  • Porcine grafts for example, are widely considered for therapeutic transplantation to humans due to their morphological compatibility with human anatomy, and due to their essentially unlimited supply.
  • the invention therefore is based, in part, on the discovery that thrombocytopenia subsequent to porcine liver xenotransplantation can be inhibited by manipulating the expression of ligands for inhibitory signaling molecules.
  • certain inhibitory receptors on donor cells do not efficiently interact with receptors on host liver effector cells (e.g., KCs and LSECs).
  • Preventing phagocytosis of platelets by the donor cells can be promoted by expressing compatible (e.g., autologous) ligands for inhibitory molecules in the xenogeneic cells.
  • compatible (e.g., autologous) ligands for inhibitory molecules in the xenogeneic cells For example, as demonstrated herein, SIRPa molecules of certain species (e.g., swine SIRPa) fail to interact with CD47 of other species (e.g., human SIRPa). Expression of human SIRPa in donor organs/tissues/cells provides an inhibitory sequence that binds to CD47 on platelets and prevents its phagocytosis.
  • xenotransplantation means the transplantation of living organs tissues or cells from one species to another. Such cross-species transplanted cells, tissues or organs are called xenografts or xenotransplants.
  • allotransplantation means a same-species transplant. Such same-species transplanted organs, tissues or cells are called allografts or allotransplants.
  • Xenotransplants have re-emerged because of a lack of available same-species organs, tissues, as well as continued allotransplant rejection. Xenotransplantation therefore offers a potential treatment for end-stage organ failure, which is a significant health problem in parts of the industrialized world.
  • Xenotransplantation using animals could alleviate the shortage of donor organs, tissues and cells and make it feasible for individuals with an end-stage organ disease to receive an organ transplant.
  • discordant liver xenotransplantation in untreated recipients is characterized by a lethal coagulopathy that results from the loss of platelets from the circulation. Platelet counts drop to near zero within minutes of reperfusion of dog-to-pig liver xenografts.
  • pig-to-non-human primate liver transplantation there is almost a complete disappearance of platelets from the circulation within one hour of reperfusion. See, Esker et al. (2010) Am. J. Transplant. 10:273-285 and Tector et al. (2002) Liver Transpl. 8:153- 159.
  • the disappearance of platelets in the immediate reperfusion period represents a barrier to xenotransplantation that must be overcome before liver xenotransplantation can be applied clinically.
  • the liver functions to clear endotoxins, cellular debris and erythrocytes from the circulation. See, Burlak et al. (2005) Transplantation 80:344-352. Under normal conditions, the liver binds and phagocytizes apoptotic neutrophils and lymphocytes from the circulation. See, Dini (1998) Biochem. Soc. Trans. 26:635-639. The liver also clears non-activated platelets, cold- stored platelets and platelets modified during sepsis. See, Grewal et al. (2008) Nat. Med. 14:648-655. These mechanisms of clearance are not mediated by platelet activation and/or coagulation.
  • CD47 also known as integrin-associated protein or SWC3 means a ligand for the extracellular region of SIRPa.
  • CD47 originally was identified in association with the integrin ⁇ 3 (hence its alternative name integrin-associated protein) and also is a member of the Ig superfamily, possessing a V-type Ig-like extracellular domain, five putative membrane- spanning segments and a short cytoplasmic tail.
  • the extracellular region of CD47 is responsible for its association with the integrin ⁇ 3 subunit.
  • CD47-mediated cellular responses likely involve the activation of integrins, in particular that of ⁇ 3 or ⁇ 3, the molecular mechanism of such activation is not fully understood. CD47 is expressed in most cell types.
  • signal regulatory protein a or "SIRPa” (also known as CD172a, SHPS-1 SIRPA, p84 or BIT) means a transmembrane protein that contains three immunoglobulin (Ig)-like domains in its extracellular region and putative tyrosine phosphorylation sites in its cytoplasmic region.
  • Ig immunoglobulin
  • Various growth factors and events such as integrin-mediated cell adhesion to extracellular matrix (ECM) proteins induce the tyrosine phosphorylation of SIRPa.
  • ECM extracellular matrix
  • SIRPa functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli, and these phosphatases mediate the specific biological functions of SIRPa.
  • SIRPa is especially abundant in neurons and in macrophages, dendritic cells and neutrophils, although its weak expression was detected in other cell types such as fibroblasts and endothelial cells.
  • SIRPa is barely detectable in red blood cells (RBCs), T cells or B cells
  • CD47 is expressed in a variety of hematopoietic cells.
  • CD47 or SIRPa might thus mediate unidirectional signaling in the hematopoietic or immune systems.
  • RBCs red blood cells
  • SIRPa of macrophages regulates phagocytosis by macrophages in a unidirectional manner.
  • CD47 is thought to form a homodimer, and SIRPa also forms a homodimer.
  • Various monoclonal antibodies against CD47 have been shown to inhibit neutrophil migration across cell monolayers in vitro.
  • monoclonal antibodies against SIRPa or soluble fusion proteins containing the extracellular domain of CD47 inhibited the migration of neutrophils, melanoma cells or monocytes in vitro.
  • Surfactant-A (SP-A) and surfactant-D (SP-D) also are implicated as other ligands for SIRPa.
  • SP-A surfactant-A
  • SP-D surfactant-D
  • the binding of SP-A to SIRPa on alveolar macrophages prevents the activation of p38 activation, thereby preventing inflammation.
  • CD47-SIRPa signaling complex in vivo is preventing phagocytosis of RBCs or platelets by macrophages.
  • macrophage or “macrophages” mean phagocytes that have an important role in preservation of tissue integrity and function by engulfing old cells or apoptotic bodies.
  • the rate of clearance of CD47- deficient RBCs from the bloodstream was found to be markedly increased compared with that found for wild-type cells.
  • compositions of the invention include nucleic acid molecules for expressing a nucleotide sequence of interest such as a nucleotide sequence encoding at least one heterologous immune-inhibitory molecule such as SIRPct in a non-human mammal and its organs, tissues and cells.
  • a nucleotide sequence of interest such as a nucleotide sequence encoding at least one heterologous immune-inhibitory molecule such as SIRPct in a non-human mammal and its organs, tissues and cells.
  • nucleic acid molecule means a deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymer (i.e., polynucleotide) in either single- or double-stranded form that, unless otherwise limited, encompasses naturally occurring bases (i.e., adenine, guanine, cytosine, thymine and uracil) or known base analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acid molecules in a manner similar to naturally occurring nucleotides.
  • bases i.e., adenine, guanine, cytosine, thymine and uracil
  • base analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acid molecules in a manner similar to naturally occurring nucleotides.
  • Examples of known base analogues of DNA and RNA include, but are not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5- bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueos
  • isolated when used in connection with a nucleic acid molecule or amino acid molecule, means a molecule removed from its natural environment or prepared by synthetic methods such as those known to one of skill in the art. Complete purification is not required in either case.
  • the nucleic acid molecules described herein can be isolated and purified from normally associated material in conventional ways, such that in the purified preparation, the polynucleotide or even a polypeptide encoded by a nucleic acid molecule is the predominant species in the preparation. At the very least, the degree of purification is such that extraneous material in the preparation does not interfere with use of the nucleic acid molecule in the manner disclosed herein.
  • the nucleic acid molecule can be at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% pure.
  • the nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of contaminant nucleotide sequence that naturally flank it in genomic DNA of the cell from which it is derived or that are present when chemically synthesized.
  • an isolated nucleic acid molecule has a structure that is. not identical to that of any naturally occurring nucleic acid molecule or to that of any fragment of a naturally occurring genomic nucleic acid molecule spanning more than one gene.
  • An isolated nucleic acid molecule also includes, without limitation, (a) a nucleic acid molecule having a sequence of a naturally occurring genomic or extrachromosomal nucleic acid molecule, but which is not flanked by the coding sequences that flank the sequence in its natural position; (b) a nucleic acid molecule incorporated into a construct, expression cassette or vector, or into a prokaryote or eukaryote host cell's genome such that the resulting polynucleotide is not identical to any naturally occurring vector or genomic DNA; (c) a separate nucleic acid molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR) or a restriction fragment; and (d) a recombinant
  • nucleic acid molecules present in mixtures of clones for example, as these occur in a DNA library such as a cDNA or genomic DNA library.
  • An isolated nucleic acid molecule can be modified (chemically or enzymatically) or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded.
  • "about” means within a statistically meaningful range of a value or range such as a stated concentration, length, molecular weight, pH, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.
  • Methods for synthesizing nucleic acid molecules are well known in the art, such as cloning and digestion of the appropriate sequences, as well as direct chemical synthesis (e.g., ink-jet deposition and electrochemical synthesis). Methods of cloning nucleic acid molecules are described, for example, in Copeland et al. (2001) Nat. Rev. Genet. 2:769-779; Current Protocols in Molecular Biology (Ausubel et al. eds., John Wiley & Sons 1995); Molecular Cloning: A Laboratory Manual, 3 rd ed. (Sambrook & Russell eds., Cold Spring Harbor Press 2001); and PCR Cloning Protocols, 2 nd ed.
  • Methods of direct chemical synthesis of nucleic acid molecules include, but are not limited to, the phosphotriester methods of Reese (1978) Tetrahedron 34:3143-3179 and Narang et al. (1979) Methods Enzymol. 68:90-98; the phosphodiester method of Brown et al. (1979) Methods Enzymol. 68:109-151; the diethylphosphoramidate method of Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862; and the solid support methods of Fodor et al. (1991) Science 251:767-773; Pease et al. (1994) Proc.
  • compositions of the invention also include expression cassettes or nucleotide constructs having a synthetic promoter as described herein operably linked to a nucleotide sequence encoding an inhibitory signaling molecule such as SIRPct heterologous to a mammal and its organs, tissues and cells.
  • an inhibitory signaling molecule such as SIRPct heterologous to a mammal and its organs, tissues and cells.
  • expression cassette means a nucleic acid molecule having at least a control sequence operably linked to a coding sequence.
  • the nucleotide sequences for the promoters described herein are provided in expression cassettes along with the polynucleotide of interest, typically a nucleotide sequence for SIRPa heterologous to the mammal of interest.
  • nucleotide construct means an oligonucleotide or polynucleotide composed of deoxyribonucleotides, ribonucleotides or combinations thereof having incorporated therein the promoters described herein. The nucleotide construct also can be used in the methods described herein to transform the mammal of interest.
  • control sequences i.e., promoters
  • operably linked means that the elements of the expression cassette are configured so as to perform their usual function.
  • control sequences i.e., promoters
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • control sequences means promoters, polyadenylation signals, transcription and translation termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for replication, transcription and translation of a coding sequence in a recipient host cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • a “promoter” means a nucleotide region comprising a nucleic acid (i.e., DNA) regulatory sequence, wherein the regulatory sequence is derived from a gene or synthetically created that is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence.
  • Promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc. ) and “constitutive promoters” (where expression of a polynucleotide sequence operably linked to the promoter is unregulated and therefore continuous).
  • the promoters can be organ-, tissue- or cell-specific promoters.
  • the expression cassette can include other control sequences 5' to the coding sequence.
  • the expression cassette can include a 5' leader sequence, which can act to enhance translation.
  • the expression cassette also can include a coding sequence for a SIRPct heterologous to a non-human mammal or its organs, tissues or cells of interest.
  • a "coding sequence” means a nucleotide sequence that encodes a particular polypeptide, and is a nucleotide sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at a 5' (amino) terminus and a translation stop codon at a 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, viral nucleotide sequences, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • heterologous to the non-human mammal or its organs, tissues or cells of interest means a nucleotide sequence for SIRPct other than the one naturally or natively found in the non-human mammal of interest. Likewise, the SIRPct is heterologous to the synthetic promoter.
  • Nucleic and/or amino acid sequences for SIRPct from human and non-human mammals are well known in the art and can be used in the expression cassette. Examples of such sequences include, but are not limited to, those disclosed in: Genbank ® Accession Nos.
  • XP_851803.1 (canine); NM_001037831.2, (chicken); NP_786982.1 (cow); NM_175788.1 (cow); EF576852.1 (horse); AAH26692.1 (human); CAM28335.1 (human); NM_001122962.1 (human); NM_001039508.1 (human); NM_080816.2 (human), NM_018556.3 (human), NM_080792.2 (human), NM_001040023.1 (human), BC075849.1 (human), BC038510.2 (human), BC033092.1 (human), BC026692.1 (human); NM_007547.2 (mouse); AGIZ01000017.1 (mouse); NM_001177647.1 (mouse); NM_001177646.1 (mouse); Nlvl_007547.3 (mouse); BC025886.1 (mouse); NM_013016.2 (rat); AF055065.1
  • the expression cassette also can include a transcriptional and/or translational termination region that is functional in non-human mammals.
  • the termination region can be native with the transcriptional initiation region ⁇ i.e., promoter), can be native with the operably linked coding sequence, can be native with the mammal of interest, or can be derived from another source ⁇ i.e., foreign or heterologous to the promoter, the coding sequence, the mammalian host cell, or any combination thereof). Termination regions are typically located downstream (3'-direction) from the coding sequence.
  • the expression cassette also can include one or more linkers.
  • linker means a nucleotide sequence that functions to link one element of the expression cassette with another without otherwise contributing to the transcription or translation of a nucleotide sequence of interest when present in the expression cassette.
  • the linker can comprise plasmid sequences, restriction sequences and/or sequences of a 5'-untranslated region (5'-UTR).
  • the length and sequence of the linker can vary and can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 nucleotides or greater in length.
  • the expression cassette also can include a signal and/or targeting peptide sequence.
  • An expression cassette therefore can have at least, in the direction of transcription, a synthetic promoter that is functional in a non-human mammal or one of its organs, tissues or cells operably linked to a nucleotide sequence encoding a SIRPct heterologous to the non- human mammal, organ tissue or cell and a nucleotide sequence encoding a terminator.
  • the expression cassettes described herein can be used to generate transgenic, non-human mammals, organs, tissues or cells.
  • the expression of the immune-inhibitory molecule can be under the control of a heterologous promoter.
  • the promoter can be a tissue-specific promoter.
  • the expression cassette can be incorporated into a vector.
  • vector means a replicon, such as a plasmid, phage or cosmid, to which the nucleotide sequence of the expression cassette can be attached so as to bring about replication of the attached sequence.
  • Vectors typically contain one or a small number of restriction endonuclease recognition sites where a nucleotide sequence of interest can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a selectable marker that can be used for identifying and selecting cells transformed with the vector.
  • Vector construct all refer to an assembly that is capable of directing the expression of a sequence or gene of interest.
  • expression vector all refer to an assembly that is capable of directing the expression of a sequence or gene of interest.
  • the terms include cloning and expression vehicles.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • a vector can be linear.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • vectors for use in creating transgenic, non- human mammals can be found, for example, in US Patent Application Publication Nos. 2009/0239259 and 20110065779; US Patent Nos. 6,025,192 and 6,989,268; as well as Boyd & Samid (1993) J. Anim. Sci. 71:1-9 and Hofmann et al. (2003) EMBO reports 11:1054-1058.
  • a vector therefore can be capable of transferring nucleotide sequences to target cells (e.g., bacterial plasmid vectors, particulate carriers and liposomes).
  • target cells e.g., bacterial plasmid vectors, particulate carriers and liposomes.
  • Selectable markers also can be used to identify and select transformed mammals, organs, tissues or cells.
  • compositions of the invention also include a transformed/transgenic, non- human mammal and its organs, tissues and cells having incorporated in its genome at least one of the nucleotide sequences described herein such that the transformed, non-human mammal and its organs, tissues and cells express detectable levels of heterologous SIRPa.
  • the transgenic, non-human mammal's genome therefore can include a nucleotide sequence encoding a heterologous immune-inhibitory molecule (e.g., a SIRPa polypeptide of a different species, such as a human SIRPa polypeptide).
  • transgenic refers to a non-human mammal, organ, tissue or cell thereof containing a transgene.
  • a transgenic animal is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell or by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • transgenic pigs that include one or more transgenes encoding one or more molecules are within the scope of this invention.
  • a double or triple transgenic animal which includes two or three transgenes, can be produced.
  • the mammalian source of the heterologous SIRPa is not so limited and can include human and non-human mammals alike.
  • examples of mammals as a source for the heterologous SIRPa include, but are not limited to, baboons, cats, chimpanzees, cows, dogs, goats, horses, humans, mice, non-human primates rats and pigs.
  • the non-human mammal can be a pig ⁇ i.e., a swine species or a miniature swine species.
  • Porcine grafts are widely considered for therapeutic transplantation to humans due to their morphological compatibility with human anatomy and their essentially unlimited supply. Consequently, the mammalian source for the heterologous SIRPa can be a human.
  • the organs, tissues or cells comprise a nucleotide sequence (e.g., a transgene) encoding at least one heterologous immune-inhibitory molecule such as SIRPa.
  • the immune-inhibitory molecule can include a SIRPa polypeptide, or fragment or variant thereof, of a second mammal.
  • Useful fragments and variants include those which retain the ability to bind with the appropriate receptor on a liver cell (e.g., a fragment which binds to CD47 on a platelet) and mediate at least one biological activity of the molecule (e.g., inhibition of phagocytosis, CD47-SIRPct intracellular signaling).
  • a liver cell e.g., KCs and LSECs
  • a cell e.g., KCs and LSECs
  • a cell e.g., KCs and LSECs
  • a cell e.g., KCs and LSECs
  • a cell e.g., KCs and LSECs
  • pig KCs and LSECs that express human SIRPa or a functional fragment or variant thereof.
  • the heterologous immune-inhibitory nucleic or encoded amino acid molecule can be at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the heterologous SIRPa nucleic or amino sequence, or a fragment thereof (e.g., the molecule has a sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heterologous SIRPa nucleic or amino sequence or a fragment thereof).
  • the immune-inhibitory molecule can gave a sequence that differs from the sequence in at least about 1 amino acid position, but not more than about 35 nucleic or amino acid positions (e.g., the amino acid sequence can differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 residues).
  • the differences can be conservative and/or non-conservative amino acid substitutions.
  • amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic
  • a predicted nonessential amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • the organ from a transgenic, non-human mammal whose genome comprises a nucleotide sequence encoding an immune-inhibitory molecule can express the immune-inhibitory molecule in an amount sufficient to decrease immune recognition of the organ by a cell of the heterologous mammal.
  • organs for transplant include, but are not limited to, heart, liver, lung, kidney and pancreas.
  • tissues for transplant include, but are not limited to, bone marrow, cornea, neuron, skin and tendon.
  • the cells from a transgenic, non-human mammal whose genome comprises a nucleotide sequence encoding an immune-inhibitory molecule can be cells of a transgenic, non-human mammal, such as a germ cell line transgenic, non-human mammal, for example, a germ cell line transgenic pig.
  • the at least one heterologous immune-inhibitory molecule e.g., SIRPa
  • the at least one heterologous immune-inhibitory molecule can be expressed in a cell, tissue and/or organ of the non-human mammal in an amount sufficient to interact with a SIRPa ligand such as CD47 on a different cell (e.g., on a human platelet) and/or decrease recognition of the cell and/or organ by the different cell.
  • the organs, tissues and cells can be graftable, such as pig KCs or LSECs.
  • the recombinant non-human mammalian organs, tissues and cells express a heterologous SIRPa polypeptide, or a fragment thereof (e.g., a fragment that mediates inhibition of an immunological reaction, such as a LSEC-mediated phagocytosis of platelets).
  • a heterologous SIRPa polypeptide e.g., a fragment that mediates inhibition of an immunological reaction, such as a LSEC-mediated phagocytosis of platelets.
  • Methods of the invention include introducing and expressing in a non-human mammal, organ, tissue or cell a nucleotide sequence such as an expression cassette or construct as described herein.
  • introducing means presenting to the non- human mammal, organ, tissue or cell, the nucleotide sequence in such a manner that the sequence gains access to the interior of a cell within the non-human mammal, organ, tissue or cell.
  • the methods and compositions do not depend on the particular method for introducing the nucleotide sequence, only that it gains access to the interior of at least one cell of the non- human mammal, organ, tissue or cell.
  • Methods of introducing nucleotide sequences, selecting transformants and regenerating whole non-mammals, which may require routine modification in respect of a particular species, are well known in the art.
  • the methods include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods and sexual breeding.
  • stable transformation means that the nucleotide sequence of interest is introduced and integrates into the genome of the non-human mammal, organ, tissue or cell and is capable of being inherited by the progeny thereof.
  • transient transformation means that the nucleotide sequence of interest is introduced, but does not integrate into the genome of the non-human mammal, organ, tissue or cell.
  • Methods of transforming non-human mammals, organs, tissues and cells by introducing a polynucleotide sequence of interest can and will vary depending on the species of non-human mammal targeted for transformation.
  • methods of introducing nucleotide sequences into non-mammals therefore include, but are not limited to ballistic particle acceleration/cell gun, electroporation, embryonic stem (ES) cell manipulation, direct gene transfer, microinjection, transfection, transduction and viral/retrovirial-mediated transfer (e.g., retroviruses).
  • retroviruses are useful vehicles for gene transfer. See, e.g., Eglitis et al. (1988) Adv. Exp. Med. Biol. 241:19.
  • the structural genes of the virus can be replaced by a single gene (e.g., a human SIRPa gene) that is then transcribed under the control of regulatory elements contained in the viral long terminal repeat (LTR).
  • LTR viral long terminal repeat
  • a variety of single-gene-vector backbones have been used, including the Moloney murine leukemia virus (MoMuL V).
  • Retroviral vectors that permit multiple insertions of different genes such as a gene for a selectable marker and a second gene of interest, under the control of an internal promoter can be derived from this type of backbone. See, e.g., Gilboa (1988) Adv. Exp. Med. Biol. 241:29-33. The elements of the construction of vectors for the expression of a protein product are known to one of skill in the art. The most efficient expression from retroviral vectors is observed when "strong" promoters are used to control transcription, such as the SV promoter or LTR promoters. See, e.g., Chang et al. (1989) Int. J. Cell Cloning 7:264-280. These promoters are constitutive and do not generally permit tissue- specific expression. Other suitable promoters are discussed above.
  • the recombinant expression cassettes or constructs described above can be used to produce a transgenic pig by any method known in the art, including, but not limited to, microinjection, ES cell manipulation, electroporation, cell gun, transfection, transduction, retroviral infection, etc.
  • Transgenic non-human mammals can be produced by introducing transgenes into the germline of the animal.
  • Embryonal target cells at various developmental stages can be used to introduce the human transgene construct. As is generally understood in the art, different methods are used to introduce the transgene depending on the stage of development of the embryonal target cell.
  • One technique for transgenically altering a non-human mammal is to microinject a recombinant nucleic acid molecule into the male pronucleus of a fertilized egg so as to cause one or more copies of the recombinant nucleic acid molecule to be retained in the cells of the developing animal.
  • the recombinant nucleic acid molecule of interest is isolated in a linear form with most of the sequences used for replication in bacteria removed. Linearization and removal of excess vector sequences results in a greater efficiency in production of transgenic mammals. See, e.g., Brinster et al. (1985) PNAS 82:4438-4442. In general, the zygote is the best target for micro-injection.
  • the male pronucleus reaches a size which allows reproducible injection of DNA solutions by standard microinjection techniques.
  • the use of zygotes as a target for gene transfer has a major advantage in that, in most cases, the injected DNA will be incorporated into the host genome before the first cleavage.
  • the animals developing from the injected eggs contain at least one copy of the recombinant nucleic acid molecule in their tissues.
  • These transgenic animals will generally transmit the gene through the germ line to the next generation.
  • the progeny of the transgenically manipulated embryos may be tested for the presence of the construct by Southern blot analysis of a segment of tissue. Typically, a small part of the tail is used for this purpose.
  • the stable integration of the recombinant nucleic acid molecule into the genome of transgenic embryos allows permanent transgenic mammal lines carrying the recombinant nucleic acid molecule to be established.
  • Alternative methods for producing a non-human mammal containing a recombinant nucleic acid molecule as described herein include infection of fertilized eggs, embryo-derived stem cells, to potent embryonal carcinoma (EC) cells, or early cleavage embryos with viral expression vectors containing the recombinant nucleic acid molecule. See, e.g., Palmiter ef al. (1986) Ann. Rev. Genet. 20:465-499; and Capecchi (1989) Science 244:1288-1292.
  • a second technique for transgenically altering a non-human mammal is retroviral infection.
  • the developing embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection.
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida.
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene. See, Jahner et al.
  • Virus or virus-producing cells can be injected into the blastocoele. See, Jahner et al. (1982) Nature 298:623-628. Most of the founders will be mosaic for the transgene since incorporation typically occurs only in a subset of the cells which formed the transgenic pig. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the mid-gestation embryo. See, Jahner et al., supra.
  • a third technique for transgenically altering a non-human mammal can be to target transgene introduction into an embryonic stem cell (ES).
  • ES cells are obtained from pre- implantation embryos cultured in vitro and fused with embryos. See, Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gessler et al. (1986) PNAS 83:9065- 9069; and Robertson et al. (1986) Nature 322:445-448.
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.
  • Such transformed ES cells could thereafter be combined with blastocysts, for example, from a peg.
  • the ES cells could be used thereafter to colonize the embryo and contribute to the germ line of the resulting chimeric animal. See, Jaenisch (1988) Science 240:1468-1474.
  • Introducing the recombinant gene at the fertilized oocyte stage ensures that the gene sequence will be present in all of the germ cells and somatic cells of the transgenic "founder” animal.
  • "founder” or “F” means the animal into which the recombinant gene was introduced at the one cell embryo stage.
  • the presence of the recombinant gene sequence in the germ cells of the transgenic F animal in turn means that approximately half of the F animal's descendants will carry the activated recombinant gene sequence in all of their germ cells and somatic cells.
  • pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 weeks. Nuclear donor cells remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs.
  • Fetal liver-derived cells can be used to perform gene targeting and create genetic knockout or transgenic pigs. See, Waghmare ef. al. (2011) J. Surgical Res. Epub 2011., which describes a fetal liver-derived cells (FLDC) that have maintained normal karyotype through 84 population doublings.
  • FLDC fetal liver-derived cells
  • Transgenic pigs can be produced by: (1) microinjecting a recombinant nucleic acid molecule into a fertilized pig egg to produce a genetically altered swine egg; (2) implanting the genetically altered pig egg into a host female; (3) maintaining the host female for a time period equal to a substantial portion of the gestation period of the fetus; and (4) harvesting a transgenic pig having at least one cell that has developed from the genetically altered egg, which expresses the recombinant nucleic acid molecule.
  • transgenic animal production is typically divided into four main phases: (1) preparation of the animals; (2) recovery and maintenance in vitro of one or two-celled embryos; (3) microinjection of the embryos; and (4) reimplantation of embryos into recipient females.
  • the methods used for producing transgenic, non-human mammals, particularly pigs do not differ in principle from those used to produce transgenic mice. Cf., e.g., Gordon ef al. (1983) Methods Enzymol. 101:411 and Gordon et al. (1980) PNAS 77:7380, concerning generally transgenic mice with Hammer et al. (1985) Nature 315:680, Hammer et al. (1986) J. Anim. Sci.
  • One step of the preparatory phase includes synchronizing the estrus cycle of at least the donor females and inducing superovulation in the donor females prior to mating.
  • Superovulation typically involves administering drugs at an appropriate stage of the estrus cycle to stimulate follicular development, followed by treatment with drugs to synchronize estrus and initiate ovulation.
  • pregnant mare's serum is typically used to mimic the follicle-stimulating hormone (FSH) in combination with human chorionic gonadotropin (hCG) to mimic luteinizing hormone (LH).
  • FSH follicle-stimulating hormone
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone
  • the efficient induction of superovulation in pigs depend, as is well known, on several variables including the age and weight of the females, and the dose and timing of the gonadotropin administration.
  • one or two-cell fertilized eggs from the superovulated females are harvested for microinjection.
  • a variety of techniques useful in collecting eggs from pigs are known. For example, in one approach, oviducts of fertilized superovulated females can be surgically removed and isolated in a buffer solution/culture medium, and fertilized eggs expressed from the isolated oviductal tissues. See, Gordon et al.
  • the oviducts can be cannulated and the fertilized eggs can be surgically collected from anesthetized animals by flushing with buffer solution/culture medium, thereby eliminating the need to sacrifice the animal. See, Hammer et al. (1985) Nature 315:600.
  • the timing of the embryo harvest after mating of the superovulated females can depend on the length of the fertilization process and the time required for adequate enlargement of the pronuclei. This temporal waiting period can range from, for example, up to 48 hours for larger breeds of swine.
  • Fertilized eggs appropriate for microinjection such as one-cell ova containing pronuclei, or two-cell embryos, can be readily identified under a dissecting microscope.
  • the equipment and reagents required for microinjection of isolated pig embryos are similar to those used for the mouse. See, for example, Gordon et al. (1983), supra and Gordon et al. (1980), supra, which describe equipment and reagents for microinjecting embryos. Briefly, fertilized eggs are positioned with an egg holder (fabricated from 1 mm glass tubing), which is attached to a micro-manipulator, which is in turn coordinated with a dissecting microscope optionally fitted with differential interference contrast optics. Where visualization of pronuclei is difficult because of optically dense cytoplasmic material, such as is generally the case with pig embryos, centrifugation of the embryos can be carried out without compromising embryo viability.
  • a recombinant nucleic acid molecule of the present invention is provided, typically in linearized form, by linearizing the recombinant nucleic acid molecule with at least 1 restriction endonuclease, with an end goal being removal of any prokaryotic sequences as well as any unnecessary flanking sequences.
  • the recombinant nucleic acid molecule containing the tissue specific promoter and the sequence encoding the immune inhibitory molecule may be isolated from the vector sequences using 1 or more restriction endonucleases.
  • the linearized, recombinant nucleic acid molecule may be microinjected into the pig egg to produce a genetically altered mammalian egg using well known techniques.
  • the linearized, nucleic acid molecule is microinjected directly into the pronuclei of the fertilized eggs as has been described by Gordon et al. (1980), supra. This leads to the stable chromosomal integration of the recombinant nucleic acid molecule in a significant population of the surviving embryos. See, e.g., Brinster et al. (1985) PNAS 82:4438-4442 and Hammer et al. (1985) Nature 315:600-603.
  • the microneedles used for injection can also be pulled from glass tubing.
  • the tip of a microneedle is allowed to fill with plasmid suspension by capillary action.
  • the microneedle is then inserted into the pronucleus of a cell held by the egg holder, and plasmid suspension injected into the pronucleus. If injection is successful, the pronucleus will generally swell noticeably.
  • the microneedle is then withdrawn, and cells that survive the microinjection (e.g., those which do not lyse) are subsequently used for implantation in a host female.
  • the genetically altered non-human mammalian embryo is then transferred to the oviduct or uterine horns of the recipient host female.
  • Microinjected embryos are collected in the implantation pipette, the pipette inserted into the surgically exposed oviduct of a recipient female, and the microinjected eggs expelled into the oviduct.
  • any surgical incision can be closed, and the embryos allowed to continue gestation in the foster mother. See, e.g., Gordon et al. (1983) Method Enzymol. 101:411; Gordon et al. (1980) PNAS 77:7390; Hammer et al. (1985) Nature 315:600; and Wall et al. (1985) Bioi. Reprod. 32:645.
  • the host female mammal containing the implanted genetically altered eggs are maintained for a sufficient time period to give birth to a transgenic non-human mammal having at least 1 cell, for example, a bone marrow cell, for example, a hematopoietic cell, which expresses the recombinant nucleic acid molecule of the present invention that has developed from the genetically altered mammalian egg.
  • a transgenic non-human mammal having at least 1 cell, for example, a bone marrow cell, for example, a hematopoietic cell, which expresses the recombinant nucleic acid molecule of the present invention that has developed from the genetically altered mammalian egg.
  • tail sections are taken from the piglets and digested with Proteinase K. DNA from the samples is phenol-chloroform extracted, then digested with various restriction enzymes.
  • the DNA digests are electrophoresed on a Tris- borate gel, blotted on nitrocellulose, and hybridized with a probe consisting of the at least a portion of the coding region of the recombinant eDNA of interest which had been labeled by extension of random hexamers. Under conditions of high stringency, this probe should not hybridize with the endogenous pig gene, and will allow the identification of transgenic pigs.
  • transgenic pigs For additional guidance and methods for producing transgenic pigs, see generally Martin et al., "Production of transgenic swine, transgenic animal technology: a laboratory handbook,” 3151-388 (Carl A. Pinkert, ed., Academic Press 1994); as well as US Patent Nos. 5,523,226 and 6,498,285.
  • transgenic cells, organs, tissues, and animals described herein can include additional genetic modifications, such as modifications that render the cells and organs more suitable for xenotransplantation.
  • transgenic pigs expressing inhibitors of complement are described, for example, in US Patent No. 6,825,395.
  • compositions and methods for depleting xenoreactive antibodies are described, for example, in US Patent No. 6,943,239.
  • compositions described herein can be used as part of a transplantation therapy.
  • Therapies that promote tolerance and/or decrease immune recognition of transplanted organs, tissues or cells include use of immunosuppressive agents (e.g., cyclosporine and FK506), antibodies (e.g., anti-T cell antibodies such as polyclonal anti-thymocyte antisera (ATG)), irradiation and protocols to induce mixed chimerism.
  • immunosuppressive agents e.g., cyclosporine and FK506
  • antibodies e.g., anti-T cell antibodies such as polyclonal anti-thymocyte antisera (ATG)
  • ATG polyclonal anti-thymocyte antisera
  • Various agents and regimens for inducing tolerance are described, for example, in US Patent Nos. 6,911,220; 6,306,651; 6,412,492; 6,514,513; 6,558,663 and 6,296,846; see also, Kuwaki et
  • Natural antibodies can be eliminated by organ perfusion, and/or transplantation of tolerance-inducing bone marrow. Preparation of the recipient for transplantation, and maintenance of the recipient after transplantation, can include any or all of the following steps. Certain aspects described below are particularly useful for primate (e.g., human) recipients. Recipients are treated with a preparation of horse anti-human thymocyte globulin (ATG) injected intravenously (e.g., at a dose of about 25-100 mg/kg or 50 mg/kg at days -3, -2, -1 prior to transplantation). The antibody preparation eliminates mature T cells and natural killer cells. The ATG preparation also eliminates natural killer (NK) cells. Anti-human ATG obtained from any mammalian host can also be used.
  • ATG horse anti-human thymocyte globulin
  • the recipient may be treated with a monoclonal anti-human T cell antibody, such as LoCD2b (Immerge BioTherapeutics, Inc.; Cambridge, MA).
  • a monoclonal anti-human T cell antibody such as LoCD2b (Immerge BioTherapeutics, Inc.; Cambridge, MA).
  • Thymic irradiation can be used (e.g., as an alternative to thymectomy).
  • the recipient can be administered low dose radiation in order to make room for newly injected bone marrow cells (if bone marrow is to be administered).
  • the recipient can be treated with an agent that depletes complement, such as cobra venom factor (at about 5-10 mg/day, at days -1).
  • an agent that depletes complement such as cobra venom factor (at about 5-10 mg/day, at days -1).
  • Natural antibodies can be absorbed from the recipient's blood by hemoperfusion of a liver of the donor species.
  • the cells, tissues, or organs used for transplantation may be further genetically modified such that they are not recognized by natural antibodies of the host (e.g., the cells are ot-l,3-galactosyltransferase deficient).
  • maintenance therapy includes treatment with a human anti- human CD154 mAb (e.g., ABI793, Novartis Pharma AG; Basel, Switzerland; ⁇ 25 mglkg).
  • a human anti- human CD154 mAb e.g., ABI793, Novartis Pharma AG; Basel, Switzerland; ⁇ 25 mglkg.
  • Mycophenolate mofetil MMF; 25-110 mg/kd/day
  • Methylprednisolone may also be administered, beginning on the day of transplantation, tapering thereafter over the next 3-4 weeks.
  • agents useful for supportive therapy include anti-inflammatory agents such as prostacyclin, dopamine, ganiclovir, levofloxacin, cimetidine, heparin, antithrombin, erythropoietin, and aspirin.
  • Organ xenotransplants can include whole hearts, lungs, livers, kidneys or pancreases.
  • Tissue xenotransplants can include skin grafts for burn patients, corneal transplants for the visually impaired, or bone transplants for limb reconstruction.
  • Cellular xenotransplants may provide treatment for people with diabetes, Alzheimer's or Parkinson's diseases.
  • methods of the invention include modulating, inhibiting, treating or preventing rejection or the complications, issues and/or risks of xenotransplant such as organs, tissues or cells in a host by increasing expression of at least one immune inhibitory molecule, such as SIRPa, in the graft.
  • at least one immune inhibitory molecule such as SIRPa
  • expression of the at least one immune inhibitory molecule is increased by expressing a transgene encoding the molecule.
  • xenotransplanted organs, tissues or cells include a transgene encoding a SIRPa polypeptide of the host mammal.
  • complications, issues and/or risks of xenotransplantation include, but are not limited to, immunological hyperacute rejection, chronic rejection of the xenotransplant, acute vascular rejection and cellular rejection (e.g., thrombocytopenia).
  • module means that transfusion- related complications, issues and/or risks can be decreased in a transplant recipient by a statistically significant amount including, but not limited to, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% when provided a xenotransplant organ, tissue or cell expressing at least one immune inhibitory molecule such as SIRPa when compared to an appropriate control.
  • SIRPa immune inhibitory molecule
  • inhibitor means that transfusion-related complications, issues and/or risks can be decreased in a transplant recipient by a statistically significant amount including, but not limited to, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% when provided a xenotransplant organ, tissue or cell expressing at least one immune inhibitory molecule such as SIRPa when compared to an appropriate control.
  • SIRPa immune inhibitory molecule
  • treat means a slowing, stopping or reversing of the complications, issues and/or risks observed in xenotransplantation in a recipient when provided a xenotransplant organ, tissue or cell expressing at least one immune inhibitory molecule such as SIRPa when compared to an appropriate control.
  • the term also means a reversing of the progression of such complications, issues and/or risks ate to a point of eliminating rejection of the xenotransplant.
  • prevent means an absence of the complications, issues and/or risks observed in xenotransplantation in a recipient when provided a xenotransplant organ, tissue or cell expressing at least one compared to an appropriate control subject.
  • a xenotransplant organ, tissue or cell expressing at least one compared to an appropriate control subject Although there can be overlap between “treating” and “preventing,” it is intended that latter is an even more drastic (i.e., less subtle) decrease, to the point of elimination, in the complications, issues and/or risks than would be observed during the former.
  • “treating” can occur in a recipient having the complications, issues and/or risks, whereas “preventing” can occur in a recipient merely susceptible to or not yet exhibiting overt complications, issues and/or risks of rejection.
  • the methods of invention also include supplying a graft, such as a kidney, liver, heart, thymus, hematopoietic stem cell or pancreatic islet cell, that expresses a heterologous immune-inhibitory molecule (e.g., SIPRa polypeptide); and implanting the graft in a recipient; thereby supplying a graft.
  • a heterologous immune-inhibitory molecule e.g., SIPRa polypeptide
  • the methods can reduce hematopoietic-cell-mediated rejection of the graft and/or prolongs acceptance of the graft.
  • the donor and recipient are of different species, for example, the donor is a non-human mammal (e.g., a pig) and the recipient is a human.
  • Example 1 shows the construction of a human SIRPa vector for use in generating transgenic non-human mammals.
  • Human SIRPA transgenic vector Homo sapiens SIRPa transcript variant 3 cDNA (GenBank ® Accession No. NM_080792.1; SEQ ID NO:l) operably linked with a Myc-DDK tag and poly A sequence was cloned to the downstream of pig ROSA26 promoter and CMV promoter, respectively. LoxP flanked enhanced green fluorescent protein (EGFP) cDNA was inserted between the promoter and human SIRPA cDNA to facilitate selection of transgenic cell line.
  • EGFP enhanced green fluorescent protein
  • Example 2 This example describes the generation of transgenic pigs expressing human SIRPa.
  • Example 1 The vector described in Example 1 will be delivered into porcine cells, and EGFP expressing cells can be obtained by flow sorting. A Cre-expressing plasmid then will be transfected into the EGFP expression cells to remove EGFP gene and allow human SIRPa expression. EGFP negative cells will be collected by flow sorting and used for somatic cell nuclear transfer (SCNT) to generate a human SIRPa transgenic pig.
  • SCNT somatic cell nuclear transfer
  • Example 3 This example describes a method of modulating thrombocytopenia in a human host recipient follow a liver transplant from a pig described in Example 2.
  • a human recipient is transplanted with a porcine liver having a transgene that encodes a human SIRPa polypeptide. Following the transplant, the platelet levels of the human recipient are measured at regular intervals.
  • the human recipient transplanted with a porcine liver having a transgene that encodes a human SIRPa polypeptide has significantly elevated platelet levels and is considered to not have thrombocytopenia.

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Abstract

La présente invention concerne des compositions comprenant des mammifères non humains transgéniques ainsi que des organes, des tissus et des cellules dérivés de ceux-ci exprimant au moins une molécule hétérologue inhibitrice de la réponse immune telle que la protéine régulatrice des signaux alpha. L'invention concerne également une méthode d'utilisation desdits mammifères non humains transgéniques ainsi que des organes, tissus et cellules dérivés de ceux-ci pour moduler les complications, problèmes et risques associés aux xénogreffes. AA Comptages BB Isotope
PCT/US2012/061637 2011-10-25 2012-10-24 Compositions et méthodes de modulation des complications, risques et problèmes associés aux xénogreffes WO2013063076A1 (fr)

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CN106535914A (zh) * 2014-08-08 2017-03-22 阿列索治疗公司 SIRP‑α变体构建体及其用途
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
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US11613564B2 (en) 2019-05-31 2023-03-28 ALX Oncology Inc. Methods of treating cancer

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106535914A (zh) * 2014-08-08 2017-03-22 阿列索治疗公司 SIRP‑α变体构建体及其用途
CN113621075A (zh) * 2014-08-08 2021-11-09 Alx肿瘤生物技术公司 SIRP-α变体构建体及其用途
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10278372B2 (en) 2014-12-10 2019-05-07 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10993419B2 (en) 2014-12-10 2021-05-04 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US11234418B2 (en) 2014-12-10 2022-02-01 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
EP3128005A1 (fr) * 2015-08-07 2017-02-08 Alexo Therapeutics Inc. Constructions de variants sirp-alpha et leurs utilisations
US10259859B2 (en) 2015-08-07 2019-04-16 ALX Oncology Inc. Constructs having a SIRP-α domain or variant thereof
US10696730B2 (en) 2015-08-07 2020-06-30 ALX Oncology Inc. Constructs having a SIRP-alpha domain or variant thereof
US11208459B2 (en) 2015-08-07 2021-12-28 ALX Oncology Inc. Constructs having a SIRP-alpha domain or variant thereof
US11639376B2 (en) 2015-08-07 2023-05-02 ALX Oncology Inc. Constructs having a SIRP-α domain or variant thereof
US11613564B2 (en) 2019-05-31 2023-03-28 ALX Oncology Inc. Methods of treating cancer

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