WO1994015459A1 - Procede d'isolement et de transplantation d'hepatocytes specialises - Google Patents

Procede d'isolement et de transplantation d'hepatocytes specialises Download PDF

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WO1994015459A1
WO1994015459A1 PCT/US1993/012462 US9312462W WO9415459A1 WO 1994015459 A1 WO1994015459 A1 WO 1994015459A1 US 9312462 W US9312462 W US 9312462W WO 9415459 A1 WO9415459 A1 WO 9415459A1
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hepatocytes
specialized
protein
sample
cells
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PCT/US1993/012462
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WO1994015459A9 (fr
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James Michaelson
Thomas H. Fraser
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The General Hospital Corporation
Diacrin, Inc.
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Priority to AU60493/94A priority Critical patent/AU6049394A/en
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Publication of WO1994015459A9 publication Critical patent/WO1994015459A9/fr

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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
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the field of hepatocyte transplantation and human gene therapy.
  • liver tissue One of the most important characteristics of liver tissue is its ability to regenerate (see, e.g., Leffert et al., Gastroenterology 76: 1470, 1979; Harvey et al, eds, The Principles and Practice of Medicine , Twenty- first edition, Appleton-Century-Crofts, Norwalk, Co, 1984).
  • liver cells are damaged by toxins, by interference with blood supply, or by obstruction to biliary flow, the remaining cells rapidly regenerate.
  • IGF insulin-like growth factor
  • glucagon glucagon
  • epidermal growth factor are believed to be trophic substances for liver regeneration.
  • HGF hepatocyte growth factor
  • a potent mitogen for mature parenchymal hepatocytes may be a hepatotrophic factor that acts as a trigger for liver regeneration after partial
  • the present invention provides a useful method for isolating and utilizing specialized components
  • hepatocytes not only for hepatic transplantation but also for cellular transplantation and human gene therapy.
  • the invention features a method of hepatic transplantation, involving providing a sample of hepatocytes enriched for specialized hepatocytes, and transplanting the sample of hepatocytes into a mammal in need of hepatic transplant.
  • the sample of hepatocytes may be derived from self or non-self.
  • the specialized hepatocytes express a plasma protein, e.g., albumin, transferrin, complement component C3, ⁇ 2 -macroglobulin, fibrinogen.
  • a plasma protein e.g., albumin, transferrin, complement component C3, ⁇ 2 -macroglobulin, fibrinogen.
  • Factor XIII:C Factor IX, ⁇ 1 -antitrypsin or ⁇ -fetoprotein; a metabolic enzyme
  • arginosuccinate synthetase glutamine synthetase, glycogen synthetase, glucose-6-phosphatase, succinate dehydrogenase, glucokinase, pyruvate kinase, acetyl CoA carboxylase, fatty acid synthetase, alanine
  • aminotransferase glutamate dehydrogenase, ferritin or alcohol dehydrogenase
  • a membrane protein e.g., GLUT-1, or LDL receptor
  • a structural protein e.g.,
  • the specialized hepatocytes produce a carbohydrate, e.g., glycogen, glucose, or glucose-6-phosphate; a lipid, or a metabolite, e.g., urea, or glutamine.
  • a carbohydrate e.g., glycogen, glucose, or glucose-6-phosphate
  • a lipid e.g., a metabolite, e.g., urea, or glutamine.
  • a metabolite e.g., urea, or glutamine.
  • hepatocytes may be either homogeneous or heterogeneous.
  • the specialized hepatocytes express a molecule, e.g., a protein, a carbohydrate, a lipid, a metabolite, capable of
  • the invention features a method of providing a protein to a mammal, involving: (a) providing a sample of specialized hepatocytes; (b) transfecting said hepatocytes with DNA encoding the protein; and (c) transplanting the transfected
  • the method is used for treating an inherited, an acquired or metabolic
  • the hepatocyte may be any suitable hepatocyte.
  • the hepatocyte may be any suitable hepatocyte.
  • specialized hepatocyte is meant a hepatocyte that produces a greater quantity of a
  • molecule e.g., a protein, a carbohydrate, a lipid, or a metabolite, than the majority of hepatocytes.
  • a specialized hepatocyte may express a
  • a specialized hepatocyte produces at least 10%, and preferably more than 50%, and even more preferably 90% of a particular protein, e.g., albumin, relative to a non-specialized hepatocyte.
  • a specialized hepatocyte may also significantly express two or more proteins, e.g., albumin and
  • enriched is meant that a population of hepatocytes, either homogeneous or heterogeneous, is composed of at least 75% specialized hepatocytes, preferably greater than 90%, more preferably 95% and even more preferably 99% specialized hepatocytes, e.g., hepatocytes expressing a plasma protein.
  • hepatocytes used for transplantation i.e., the donor cells
  • autologous source i.e, the host.
  • hepatocytes used for transplantation are derived from a non-autologous source.
  • hepatocyte sample is composed solely of a single hepatocyte sample
  • hepatocyte e.g., a population of albumin- expressing hepatocytes.
  • heterogeneous is meant that the hepatocyte sample is composed of two or more specialized hepatocytes, e.g., a population that consists of albumin- and transferrin-expressing hepatocytes.
  • the invention provides methods for the treatment of liver disorders that do not require the administration of drugs or blood-derived therapeutics.
  • the invention affords unique and critical advantages over any previous method.
  • the invention provides for treating any number of liver diseases, including, e.g., familial hypercholesterolemia, hemophilia, and cirrhosis, by transplantation of specialized hepatocytes that are capable of providing a molecule, e.g., a protein, a carbohydrate, a lipid, or a metabolite, necessary to prevent or treat a disease.
  • Transplantation of a molecule e.g., a protein, a carbohydrate, a lipid, or a metabolite
  • the specialized hepatocytes may be useful as an in vivo recombinant protein delivery system.
  • the specialized hepatocytes, especially plasma-protein expressing hepatocytes, may have both extraordinary secretory capabilities and an exceptional capacity for cell division (and therefore have a greater long-term survival in comparison to other hepatocytes).
  • the invention is intended to obviate problems commonly encountered in hepatic transplantation; for example, the limited supply of intact donor organs, the added immunological consequence of rejection against non-hepatocyte cellular elements, and the avoidance of the danger of surgical mortality associated with liver transplantation.
  • Fig. 1 is a panel of micrographs showing the immunofluorescence of mouse liver.
  • Fig. 1a is an immunofluorescent micrograph showing an albumin-containing hepatocyte (green) identified by immunofluorescence with rabbit anti-mouse albumin.
  • Fig. 1b is an immunofluorescent micrograph showing a complement component C3-containing hepatocyte in same field (red) identified by goat anti-mouse complement component C3.
  • Fig. 1c is an immunofluorescent micrograph showing a double exposure of same fields as Figs. 1 a and 1b, showing simultaneously albumin (green) and C3 (red) containing cells.
  • Fig. 1d is an immunofluorescent micrograph showing a cluster of albumin-containing hepatocytes.
  • Fig. 1e is an immunofluorescent micrograph from an A1b a /A1b c heterozygous mouse showing the reactivity of rabbit anti-mouse albumin (red).
  • Fig. If is an immunofluorescent micrograph from an A1b a /A1b c heterozygous mouse showing the reactivity of anti-A1b c (green).
  • Fig. 2 is an illustration of the percentage of hepatocytes containing various plasma proteins from the livers of mice at various ages. Identification of Specialized Hepatocytes
  • mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Rats were obtained from the Charles River Laboratories (Wilmington, MA) .
  • Antisera Rabbit anti-mouse albumin (Nordic Immunological Laboratories, Capistrano Beach, CA, Cat. Code RAM/Alb, lot 14-787); sheep anti-mouse albumin
  • antibodies useful for identifying any protein expressed by a specialized hepatocyte may be prepared according to standard techniques (see, e.g., Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). For example,
  • monoclonal antibodies may be prepared using any liver protein, e.g., estrogen receptor, glutamine synthetase, carbamoylphosphate synthetase, produced by a specialized hepatocyte and standard hybridoma technology (see, e.g., Kohler et al., Nature 256: 495, 1975; Kohler et al. Eur. J . Immunol . 6: 511, 1976; Kohler et al., Eur. J . Immunol . 6: 292, 1976; Hammerling et al. In: Monoclonal Antibodies and T Cell Hybribomas, Elsevier, NY, 1981; Ausubel et al. supra).
  • liver protein e.g., estrogen receptor, glutamine synthetase, carbamoylphosphate synthetase
  • polyclonal antibodies can, if desired, also be produced (see, e.g., Garvey et al., Methods in Immunology, W. A. Benjamin, Northampton, MA, 1977). Once produced, monoclonal or polyclonal
  • antibodies are tested for specific recognition of their antigen by Western blot or immunoprecipitation analysis (see, e.g, methods described in Ausubel et al., supra) .
  • a mixture of rabbit anti-mouse albumin and goat anti-mouse transferrin was absorbed as follows: 25 microliters of the mix of the two antibodies were combined with a 2.5 microliter volume of protein, at concentrations of 1, .1, .01, .001 and .0001 mg/ml.
  • Hepatocytes are Heterogeneous. Immunofluorescence tests on mouse and rat liver were carried out with more than 20 antisera to individual plasma proteins. In every case, each antiserum was found to react with only a small number of hepatocytes (Fig. 1). This narrow reactivity was identified using antisera to albumin, transferrin, complement component C3, fibrinogen, ⁇ 2 -macroglobulin, and ⁇ -fetoprotein in the mouse, as well as with antisera to albumin, transferrin, fibrinogen and ⁇ -fetoprotein in the rat. Albumin-containing hepatocytes constitute somewhat less than 1% of the liver, while cells
  • the reaction of the rabbit antisera was visualized by the green fluorescence of FITC while the reaction of the goat antisera were revealed by the red fluorescence of TRITC.
  • Two-color immunofluorescence reactions also showed a cellular identity between the hepatocytes identified by rabbit anti-mouse fibrinogen, goat anti-mouse fibrinogen, rabbit anti-rat fibrinogen and goat anti-rat fibrinogen.
  • Other anti-mouse plasma protein antisera showed only partial cross reactivity with their rat counterparts.
  • Each Plasma Protein Seen by Immunofluorescence is Contained in a Separate Population of Hepatocytes. More than 250 two-color immunofluorescence comparisons were carried out on mouse liver, which demonstrated that each plasma protein is located in separate population of hepatocytes, (see Fig. 1, Table I). Proteins assayed include albumin, transferrin, complement component C3, ⁇ 2 -macroglobulin, fibrinogen, and ⁇ -fetoprotein in the mouse liver. For the rat liver, albumin, transferrin, fibrinogen and ⁇ -fetoprotein were examined (Table II).
  • Hepatocytes containing each protein are scattered throughout the liver without any obvious regional orientation. Other than containing different proteins, there are no obvious histological differences between each class of hepatocyte in adult liver.
  • Cells containing plasma protein are scattered throughout the liver individually and in clusters ( Figure 1d).
  • Serial sections of mouse liver revealed that generally clusters can be followed on adjacent sections; usually these clusters are not large (25 or fewer cells) and have a globular shape. This size and shape is roughly in accord with the estimate of liver clonal size found by West (J. Embryol . Exp. Morpho . 36:151, 1976) from studies of aggregation chimeras.
  • Hepatocytes Suspensions of Hepatocytes.
  • Starvation-induced Suppression of Plasma-protein Synthesis Can be Seen by Immunofluorescence. Starvation is known to induce a shutdown of plasma protein synthesis (see, e.g. Morgan et al., J. Biol . Chem . 246:3500, 1971). Over a three-day period, albumin-, transferrin-, and fibrinogen-containing cells fade and disappear from the livers of mice deprived of food. Within a few hours of refeeding these mice, the plasma protein-containing cells come back to their usual appearance, with both single cells and clusters visible in the usual quantities.
  • Hepatocyte Heterogeneity In most experiments, livers were perfused with saline to remove the blood. Some experiments revealed, however, that with respect to the visualization of all the plasma proteins except albumin, the perfusion step could be eliminated without any apparent effect on the outcome. Albumin-containing hepatocytes are readily visualized in unperfused liver, although a markedly increased background is evident.
  • intracellular albumin contributed to the liver as a whole by the albumin-containing hepatocyte would appear to exceed 0.2 mg/gram of liver, a value that is close to the amount of pro-albumin that Morgan et al. supra measured biochemically as 0.4 mg/gm.
  • immunofluorescence reactivities was tested by a number of methods. First, immunoelectrophoresis with each anti- mouse plasma protein antiserum showed reactivity with only the expected band. Second, immunoprecipitation with each of these antisera yielded protein on SDS-PAGE with the expected molecular weight. Third, as indicated in Tables I and II, the specificity of each antibody was seen by two-color immunofluorescence experiments
  • pairs of antisera with the same specificity e.g., rabbit anti-albumin versus goat anti-albumin
  • the minimal concentration of transferrin required to absorb all anti- transferrin activity was found to be .01 mg/ml, with a general diminution of anti-transferrin reactivity at .001 mg/ml; the minimal concentration of albumin required to remove all anti-albumin reactivity was .1 mg/ml, with some absorption evidence at .01 and .001 mg/ml. "No" absorption indicates no diminution of reactivity even at highest protein concentrations (1 mg/ml).
  • Source of purified proteins mouse transferrin #1 (Research Plus), mouse transferrin #2 (Cappel); mouse albumin (Sigma), cytochrome C (Sigma) mouse ⁇ -fetoprotein (Calbiochem). Essentially identical results were found in 3 separate albumin/transferrin absorption experiments. The ⁇ - fetoprotein absorption experiment utilized liver from a 7 day old mouse as its target tissue.
  • anti-plasma protein antisera become completely unreactive against hepatocytes.
  • Plasma protein-containing cells were not observed in a number of mouse tissues where synthesis is either absent or low. Tissues examined included brain, gut, testes, salivary gland, bone marrow, thyroid and lymph node. The lactating mammary gland synthesizes transferrin (but not albumin) in amounts comparable to the liver (see, e.g., Jordan et al., Biochim . et Biophys . Acta 174:373, 1969), and preliminary experiments have revealed that a subpopulation of these cells react with antisera to transferrin, but not with anti-albumin antisera.
  • transferrin but not albumin
  • hematopoietic cells are present in the liver of mice and rats. Included among these hematopoietic cells are
  • megakaryocytes which are known to contain fibrinogen (see, e.g., Uzan et al., Biochem . Biophys . Res . Comm . 140:543, 1986; Handagama et al. Prog . Clin . Biol. Res . 356:119, 1990; and Louache et al., Blood 15 , 77:311, 1991).
  • megakaryocytes in the livers of neonatal rats were found to contain fibrinogen, as indicated by reactivity with antisera to fibrinogen, but not with antisera to albumin, ⁇ - fetoprotein or transferrin. The same reactivities were found with respect to megakaryocytes in rat bone marrow.
  • antisera see, e.g., Michaelson, Immunogenetics 17:219, 1983).
  • anti-actin antisera showed immunofluorescence reactivity throughout the liver.
  • actin is present in all mouse hepatocytes, where it is concentrated on the periphery of the cell.
  • carbamoylphosphate synthetase is known to be located in all hepatocytes except those located in the few rows of cells adjacent to the terminal hepatic venules (see, e.g., Moorman et al., supra) , and a similar pattern of widespread expression is found using the methods described above.
  • Additional liver-specific products in human liver can be determined by the methods outlined above.
  • Other methods for identifying proteins, lipids, carbohydrates and other metabolites are known in the art (see, e.g. Jungermann et al., supra)
  • the invention involves the use of specialized subpopulations of hepatocytes in transplantation for the treatment of human diseases, e.g., disorders of hepatic metabolism, ranging from ailments associated with
  • Hemophilia A and factor IX deficiency (Hemophilia B).
  • Familial hypercholesterolemia is an autosomal dominant disorder in human patients caused by a deficiency of the receptor that mediates the uptake of low density lipoprotein (see, e.g., Scriver et al. (eds) The Metabolic Basis of Inherited Disease , McGraw-Hill, NY, pp 1215-1250). The disease leads to elevated levels of serum cholesterol and premature development of coronary artery disease.
  • lipoprotein receptors have severe hypercholesterolemia that is refractory to medical therapy.
  • Alpha 1 -antitrypsin deficiency is a hereditary disorder characterized by reduced serum levels of ⁇ 1 - antitrypsin, a protease inhibitor that provides the major defense for the lower respiratory tract against the ravages of neutrophil elastase, a powerful destructive protease.
  • the loss of this protective screen of the fragile alveolar walls results in emphysema (see, e.g., Wilson et al., supra) .
  • Approximately 10 percent of children homozygous for ⁇ 1 -antitrypsin deficiency will develop significant liver disease including neonatal hepatitis and progressive
  • cirrhosis (Wilson et al. supra) .
  • ⁇ 1 -antitrypsin deficiency is asymptomatic cirrhosis, which may progress from a micronodular to a macronodular state and may be complicated by the development of hepatocellular carcinoma.
  • Hemophilia A and hemophilia B are sex-linked inherited plasma coagulation disorders due to defects in factors VIII and factor IX, respectively (see, e.g., Wilson et al., supra) .
  • Factor VIII coagulant protein is a single- chain protein (265 kDa) which regulates the activation of factor X by proteases generated in the intrinsic coagulation pathway. It is synthesized in liver parenchymal cells and circulates complexed to the von Willebrand protein. One in 10,000 males is born with deficiency or dysfunction of the factor VIII molecule.
  • Factor IX is a vitamin K-dependent serine protease precursor (55 kDA) involved in the intrinsic blood coagulation pathway.
  • Factor IXa active protease
  • factor XIa Factor XIa
  • tissue factor-Vila complex see, e.g., Wilson et al. supra
  • Factor IXa then activates factor X in conjunction with activated factor VIII.
  • Factor IX is synthesized in the liver. Factor IX deficiency or dysfunction occurs in 1 in 100,000 male births.
  • hemophilia A Treatment of patients with hemophilia A involves administration of plasma products enriched in factor VIII.
  • the current treatment of patients with hemophilia B involves administration of either frozen plasma or crude preparations of plasma, different from plasma fractions used to treat hemophilia A, enriched in the prothrombin complex proteins. Both methods are complicated by the potential risk of exposing patients to viral contaminants, such as viral hepatitis and human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Hepatocytes Isolation of Hepatocytes.
  • mammalian organ donors may be used to provide specialized hepatocytes.
  • mammalian organ donors may be used to provide specialized hepatocytes.
  • hepatocytes may be obtained, without limitation, from hepatic tissue of a chicken, a mouse, a rat, a dog, a baboon, a pig, or a human.
  • the tissue may come from liver fragments of differing sizes provided by a living (self or non-self) or a deceased donor, e.g., from a needle biopsy, a small wedge biopsy, or a partial hepatectomy. If the donor hepatocytes are from non-self, then donor-recipient
  • Class I and class II histocompatibility antigens are determined and individuals closely matched immunologically to the patient are selected as donors. Furthermore, all donors are screened for the presence of transmissible viruses (e.g., human
  • hyperlipidemia are regarded as suitable donors.
  • hepatocytes from hepatic tissue suitable for culturing and transplanting are described, e.g., in Langford et al., In Vitro Cellular & Developmental Biology 25: 174, 1989; Strom et al., Journal National Cancer Institute , 68:, 771, 1982; Kay et al., Proc . Natl . Acad . Sci . , USA 89:, 89, 1992; and Enat et al., Proc . Natl . Acad . Sci . , USA 81: 1411, 1984.
  • surgically isolated liver from a mammalian donor is
  • hepatocytes are displaced by gentle shaking in a collagenase solution.
  • the resulting hepatocytes are either plated for culturing or enriched, e.g., by cell sorting (as described below) to obtain populations of specialized hepatocytes.
  • Cell viability is examined by trypan blue exclusion.
  • hepatocytes can be isolated and enriched from the mixed hepatocyte cell suspension, described above, by the
  • fractionation method employed to enrich for a particular specialized hepatocyte e.g., a plasma-protein, a metabolic protein, a membrane protein or a structural protein,
  • FACS fluorescence-activated cell sorting
  • one suitable cell-separation technique employs the preparation of single cell suspensions of mixed hepatocytes, followed by sorting of these cells on a
  • fluorescence-activated cell sorter One sorting method involves labeling specific cells with an antibody coupled to a fluorescent dye, followed by separating the labeled cells from the unlabeled cells in an electronic fluorescence- activated cell sorter (see e.g., Horan et al., Science , 198, 148, 1977; Miller et al., J. Immunol . Methods , 47: 13, 1981; and Loken et al., J . Immunol . Methods , 50:R85, 1982).
  • enrichment of a specialized hepatocyte expressing LDL receptor protein would involve incubating the mixed population of hepatocytes, as described above, with an antibody, e.g., a fluorescein-conjugated monoclonal antibody against LDL receptor, followed by FACS.
  • the subpopulation of hepatocytes enriched for cells expressing LDL receptor is then processed as described below.
  • the FACS procedure might also be modified to enrich for other types of specialized hepatocytes, such as plasma-protein
  • the FACS procedure may be modified to sort specialized hepatocytes, e.g., a plasma- protein expressing hepatocytes, based on spectroscopic characteristics , i.e., the ultraviolet (UV) or fluorescence properties of a specific plasma-protein hepatocyte or more generally simply on the premise that plasma-protein
  • spectroscopic characteristics i.e., the ultraviolet (UV) or fluorescence properties of a specific plasma-protein hepatocyte or more generally simply on the premise that plasma-protein
  • expressing cells will have a higher A 280 than non-plasma- protein expressing hepatocytes that do not contain secretory granules.
  • An alternative FACS-based protocol may be applied that utilizes methods of cytoplasmic vital staining, i.e., stains that specifically react with a specialized hepatocyte that expresses e.g, a plasma-protein, a membrane protein, a metabolic protein or a structural protein. After staining, cells are then sorted by FACS to enrich for positively stained cells.
  • subpopulations of hepatocytes involves manipulating
  • hepatocyte growth conditions in tissue culture media to selectively induce specific subpopulations of hepatocytes to proliferate. For example, mice undergo a renewal of ⁇ - fetoprotein synthesis after they have been injected with high levels of estradiol (Hau et al. Acta Endocrinologica 106: 141-144, 1984). Thus, mixed hepatocytes may be
  • the mitotic expansion of fibrinogen-expressing hepatocytes might be selectively obtained by inducing hepatocyte cultures with stimuli known to effect
  • inflammation such as IL-1 or TNF or turpentine
  • turpentine e.g., 0.1 pg- lmg/ml
  • mitotic expansion of albumin-expressing hepatocytes might simply be achieved by culturing mixed populations of hepatocytes in the absence of albumin.
  • an additional separation technique based on centrifugation methodologies can be utilized to enrich for the generic class of plasma- protein expressing hepatocytes (see, e.g., Sharpe, Methods of Cell Separation , Elsevier, 1988) . Since it appears that approximately 1% of total liver hepatocytes produce any of the 100 or so plasma proteins, it might reasonably be expected that these cells have significantly higher
  • cytoplasmic protein due to higher amounts of intracellular secretory granules, relative to non-plasma- protein-expressing hepatocytes. Consequently, these cells may vary in their density compared with other non-secretory cells. This difference may be exploited to separate these cells by density sedimentation or centrifugation.
  • a variant on this invention involves the isolation by sorting of this generic class of plasma protein producing cells.
  • this group may be considered homogeneous with respect to the general quality of plasma protein synthesis. Consequently, these cells may also constitute a superior population of cells for hepatocyte transplantation.
  • Hepatocytes are generally known in the art to be difficult cells to adapt to tissue culture, but this may not apply to specialized hepatocytes, e.g., subtypes of plasma-protein expressing hepatocytes, which may harbor a potential for cell division not evident in ordinary unspecialized hepatocytes.
  • hepatocytes isolated as described above may be plated at a density of 3-4 X 10 4 cells per cm 2 onto plates with a polycationic matrix (Primaria; Falcon, Oxnard, CA) in a hormonally defined medium (see, e.g., Enat et al., supra) supplemented with 10% fetal bovine serum; 1-6 hr later the medium is replaced with fresh hormonally defined medium, which is subsequently changed every 6-24 hr during the duration of culturing. Culture media may be further
  • mitogens e.g., IL-6, IGF-1, prolactin, EGF,
  • glucocorticoids to augment growth and cell division of the hepatocytes.
  • Isolated hepatocytes may be stored and preserved by any standard method, e.g., by freezing. These methods are highly
  • mammalian cells see, e.g., Rajotte et al., Cryobiology 18: 357, 1981; Daniel, ed, Methods of Mammalian Reproduction , New York, Academic Press, 1979, p. 179; and Sandier et al., Horm . Metabol . Res . 12: 71, 1982 and should be most useful for establishing deposits of specialized hepatocytes for subsequent utilization in transplantation.
  • transplantation are not provided by the transplant
  • concomitant immunosuppression therapy is necessary, e.g., administration of the immunosuppressive agent cyclosporine (see, e.g., Physician 's Desk Reference , Medical Economics Data, Montvale, N.J., 1992) or, e.g., by masking antigens of non-self donor hepatocytes according to methods described by Faustman and Coe (Science 252: 1700, 1991) to prevent rejection of transplanted cells.
  • hepatocytes Several standard methods are available for the transplantation of specialized hepatocytes either delivered directly to the liver, e.g., via the portal vein, or for deposition within other locations throughout the body; for example, some investigators have implanted hepatocytes within the spleen (see, e.g, Vroemen et al.,
  • pancreas see, e.g., Jaffe et al., Transplantation , 45: 497, 1988
  • subrenal capsule see, e.g., Ricordi et al., Transplantation 45, 1148, 1988
  • microcarrier beads in the peritoneum see, e.g., Demetriou et al., Proc. Natl . Acad . Sci . 83: 7475, 1986).
  • a patient diagnosed with Hemophilia A may be treated by providing specialized hepatocytes that express Factor VIII:C by transplantation.
  • Donors are chosen as described above.
  • the preferable donor has no family history of hemophilia or any other liver disease, and is closely matched to the recipient immunologically.
  • Liver tissue is obtained, as described above, and Factor VIII:C expressing hepatocytes are isolated and cultured.
  • the specialized Factor VIII:C expressing cells then may be introduced to the recipient by standard surgical procedures. Between 10 2 to 10 9 cells may be transplanted in a session. Additional transplants can be performed as required (based on assaying blood coagulation). If necessary, immunosuppressive therapy is also administered as described above.
  • the specialized populations of hepatocytes may provide an excellent source of hepatocytes that can stably produce and deliver recombinant proteins into the circulatory system.
  • the specialized hepatocytes would be isolated from a recipient, cultured and expanded, transfected or transformed with a recombinant gene in vitro, and subsequently transplanted into the donor organism. Once situated, the hepatocytes would manufacture and secrete the desired recombinant protein for in vivo delivery.
  • a gene encoding for example a human low density lipoprotein receptor (see, e.g., Wilson et al., Proc . Natl . Acad . Sci . , USA 87: 8437, 1990), human ⁇ 1 -antitrypsin gene (see, e.g., Kay et al., Proc . Natl . Acad . Sci . USA 89: 89, 1992), human factor VIII (see e.g., Wood et al., Nature 312:330, 1984) or human factor IX (see, e.g., Armentano et al., Proc . Natl .
  • hepatocytes (as discussed above), either obtained from a patient suffering from a cellular defect or genetic disease such as familial hypercholesterolemia, ⁇ 1 -antitrypsin deficiency, factor VIII or factor IX deficiency, respectively, or from an acceptable donor human (as described above) or animal.
  • any of a variety of conventional gene transfer methods may be used for introducing genes into cells.
  • the precise method used to introduce a replacement gene e.g., a human low density lipoprotein receptor, a human ⁇ 1 -antitrypsin gene, a human factor VIII gene or a human factor IX gene to the specialized subpopulation of hepatocytes is not critical to the invention.
  • physical methods for the introduction of DNA into cells include microinjection (see, e.g., Capecchi et al., Cell 22:479, 1980), electroporation (see, e.g., Reiss et al., Biochem . Biophys. Res . Commun . 137: 244, 1986) and
  • nucleic acids into mammalian cells can be executed through the use of viral vectors, in particular those derived from murine and avian retroviruses (see, e.g., Gluzman et al., Viral Vectors, Cold Spring Harbor
  • hepatocytes This example is provided for the purpose of illustrating, not limiting, the invention.
  • standard recombinant DNA methods see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989
  • a full-length human factor IX cDNA or complete genomic sequence with associated
  • regulatory sequences or sequences e.g., promoters
  • enhancers, terminators, provided by a heterologous source is cloned into a retroviral vector.
  • Numerous vectors useful for this purpose are generally known and have been described (see, e.g. Miller, Human Gene Therapy, 1:5, 1990; Friedman, Science , 244:1275, 1989; Eglitis and Anderson,
  • the human coagulation factor IX gene can be isolated from a human liver cDNA library (prepared according to standard techniques, see, e.g., Ausubel et al. supra , or from a commercially available library see, e.g, Stratagene Catalog, La Jolla, CA, #937200 human liver cDNA library from a Normal male, 49 years old) or a human genomic library (prepared according to standard techniques, see, e.g., Ausubel et al., supra , or from a commercially
  • the factor IX cDNA is cloned into any suitable vector used for retroviral- mediated gene transfer as described above.
  • Factor IX cDNA expression is directed from any suitable promoter, e.g., the human cytomegolvirus, simian virus 40, or metallothionein.
  • Factor IX cDNA expression may be driven and regulated by any natural mammalian regulatory sequences and elements.
  • enhancers/promoters known to direct preferential gene expression in the liver might be used to direct Factor IX cDNA gene expression.
  • enhancers/promoters include, without limitation: ⁇ -fetoprotein (see, e.g., Hammer et al., Science , 235: 53, 1987), transthyretin (see, e.g., Yan et al., EMBO J . 9: 869, 1990), ⁇ 1 -antitrypsin (see, e.g.,
  • Kelsey et al. Genes & Development 1:161, 1987
  • albumin see, e.g., Pinkert et al. Genes & Development 1: 268, 1987
  • metallothionein see, e.g., Karin et al. Nature 308:
  • Factor IX genomic clone if a Factor IX genomic clone is utilized, its expression may be regulated by its cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, e.g., a SV40 promoter region. Regardless of how Factor IX gene expression is regulated from the transduced specialized hepatocytes the final level of protein, after transplantation (see above), should fall within physiological levels of circulating factor IX found in non-affected individuals.
  • Clotting activity of purified recombinant factor IX secreted by transduced hepatocytes may be determined by a one-stage clotting assay with factor IX-deficient plasma as described by Thompson , J . Clin . Invest . , 59: 900, 1977.

Abstract

Procédé de transplantation hépatique selon lequel on utilise un échantillon d'hépatocytes enrichis destinés à des hépatocytes spécialisés et on transplante l'échantillon d'hépatocytes dans un mammifère nécessitant une transplantation hépatique. On peut également administrer une protéine à un mammifère (a) en utilisant un échantillon d'hépatocytes enrichis pour des hépatocytes spécialisés; (b) en transfectant les hépatocytes spécialiés avec de l'ADN qui code la protéine; et (c) en transplantant les hépatocytes transfectés dans le mammifère.
PCT/US1993/012462 1993-01-05 1993-12-21 Procede d'isolement et de transplantation d'hepatocytes specialises WO1994015459A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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WO1999014352A2 (fr) * 1997-09-12 1999-03-25 University Of Dundee Dosage

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WO1989007136A2 (fr) * 1988-02-05 1989-08-10 Whitehead Institute For Biomedical Research Hepatocytes modifies et leurs utilisations

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WO1989007136A2 (fr) * 1988-02-05 1989-08-10 Whitehead Institute For Biomedical Research Hepatocytes modifies et leurs utilisations

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CELL SEPARATION: METHODS AND SELECTED APPLICATIONS, Volume 4, issued 1987, H. PERTOFT et al., "Separation and Characterization of Liver Cells", pages 1-23. *
HEPATOLOGY, Volume 14, Number 1, issued 1991, S. GUPTA et al., "Permanent Engraftment and Function of Hepatocytes Delivered to the Liver: Implications for Gene Therapy and Liver Repopulation", pages 144-149. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Volume 87, issued August 1990, D. ARMENTANO et al., "Expression of Human Factor IX in Rabbit Hepatocytes by Retrovirus-Mediated Gene Transfer: Potential for Gene Therapy of Hemophilia B", pages 6141-6145. *
SCIENCE, Volume 254, issued 20 December 1991, J.R. CHOWDHURY et al., "Long-Term Improvement of Hypercholesterolemia after ex Vivo Gene Therapy in LDLR-Deficient Rabbits", pages 1802-1805. *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014352A2 (fr) * 1997-09-12 1999-03-25 University Of Dundee Dosage
WO1999014352A3 (fr) * 1997-09-12 1999-06-24 Univ Dundee Dosage

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