WO2002092013A2 - Methods for treating liver disease and liver damage with growth hormone and foxm1b - Google Patents
Methods for treating liver disease and liver damage with growth hormone and foxm1b Download PDFInfo
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- WO2002092013A2 WO2002092013A2 PCT/US2002/015873 US0215873W WO02092013A2 WO 2002092013 A2 WO2002092013 A2 WO 2002092013A2 US 0215873 W US0215873 W US 0215873W WO 02092013 A2 WO02092013 A2 WO 02092013A2
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- liver
- foxmlb
- protein
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- nucleic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
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- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- This invention relates to methods of treating liver diseases and liver damage by inducing expression and nuclear localization of FoxMlB protein.
- the invention particularly relates to methods of inducing FoxMlB protein expression and inducing or facilitating translocation of FoxMlB protein to the nucleus of a mammalian cell, where it potentiates transcription of many essential cell cycle promotion genes.
- the invention relates to methods of preventing or ameliorating liver damage or disease comprising administering to a patient a therapeutically effective amount of growth hormone.
- the invention further relates to methods of screening compounds that induce expression of FoxMlB, induce nuclear localization of FoxMlB, or induce both expression and nuclear localization of FoxMlB protein in liver cells.
- the invention also provides such compounds that are useful for preventing or ameliorating liver damage or disease, and methods for using said compounds for preventing or ameliorating liver damage or disease.
- liver detoxify harmful compounds that enter the body.
- toxic substances may be cleared from the body by phagocytosis, secretion into the bile, or by chemical modification of the compound to facilitate elimination by the kidneys.
- Other functions of the liver include storing vitamins, producing cholesterol and bile to assist digestion, converting excess glucose into glycogen, and releasing glucose into the blood during fasting.
- the liver is also responsible for secreting all serum carrier proteins and proteins involved in blood coagulation. A healthy liver, therefore, is an important contributor to the overall health of an animal or human individual.
- liver damage increases with time and as the stress of removing these toxins increases.
- the mammalian liver is capable of completely regenerating itself in response to such liver damage (Fausto et al, 1995, FASEB J. 9: 1527-1536; Michalopoulos et al, 1997, Science 276: 60-66; Taub, 1996, FASEB J. 10: 413-427).
- excessive exposure to toxins such as alcohol or certain drugs can cause severe liver damage leading to disease.
- the ability of the liver to regenerate decreases and liver damage and disease becomes more severe and more difficult to treat.
- the ability to stimulate hepatocyte proliferation and restore the regenerative potential of these liver cells would prove invaluable in treating liver diseases.
- FoxMlB is a proliferation-specific transcription factor that shares 39% amino acid homology with the HNF-3 winged helix DNA binding domain.
- the molecule also contains a potent C-terminal transcriptional activation domain that possesses several phosphorylation sites for M-phase specific kinases as well as PEST sequences that mediate rapid protein degradation (Korver et al, 1997, Nucleic Acids Res. 25:1715-1719; Korver et al, 1997, Genomics 46:435-442; Yao et al, 1997, J. Biol. Chem. 272:19827- 19836; Ye et al, 1997, Mol. Cell Biol. 17:1626-1641).
- FoxMlB is expressed in several tumor-derived epithelial cell lines and is induced by serum prior to the GJS transition (Korver et al, 1997, Nucleic Acids Res. 25: 1715- 1719; Korver et al, 1997, Genomics 46:435-442; Yao et al, 1997, J. Biol. Chem. Til. 19827-19836; Ye et al, 1997, Mol. Cell Biol. 17: 1626-1641). In situ hybridization studies show that FoxMlB is expressed in embryonic liver, intestine, lung, and renal pelvis (Ye et al, 1997, Mol. Cell Biol. 17: 1626-1641).
- FoxMlB is not expressed in postmitotic, differentiated cells of the liver and lung, although it is expressed in proliferating cells of the thymus, testis, small intestine, and colon (Id). FoxMlB expression is reactivated in the liver prior to hepatocyte DNA replication following regeneration induced by partial hepatectomy (Id).
- RNA from wild type and transgenic regenerating livers by differential hybridization of cDNA array blots and RNase protection assays showed that FoxMlB stimulated the expression of several cell cycle regulatory genes (Id).
- Id cell cycle regulatory genes
- c-myc transcription factor and tumor growth factor ⁇ can also stimulate hepatocyte replication during liver regeneration.
- constitutive expression of c-myc or TGF- ⁇ increases the incidence of liver tumors (Factor et al, 1997, Hepatology 26: 1434-1443).
- Co- expression of c-myc and TGF- ⁇ in hepatocytes also stimulates oxidative stress and DNA damage leading to senescence after partial hepatectomy and the development of liver tumors between 4 to 8 months of age (Id; Factor et al, 1998, J. Biol. Chem. j . 15846- 15853).
- FoxMlB can potentiate transcription of cell cycle promotion genes and thus stimulate hepatocyte replication that can offset toxin- and age-associated liver damage, it does so only when translocated into the nucleus (Ye et al, 1999, Mol. Cell Biol. 19: 8570-8580).
- Expression of FoxMlB in adult liver followed by its induction to enter the nucleus at the appropriate time may alleviate age-related proliferation defects and avoid unwanted hepatocyte proliferation, making it a far safer candidate for therapeutic intervention than compounds that induce expression of c-myc or TGF- ⁇ .
- This invention provides methods of restoring hepatocyte DNA replication and mitosis in diseased and damaged livers.
- the invention also provides methods of inducing expression and nuclear localization of FoxMlB protein in mammalian liver cells, particularly aged or toxin-damaged liver cells.
- the invention provides methods for inducing expression, nuclear localization or expression and nuclear localization of FoxMlB protein by contacting liver cells with growth hormone.
- the invention provides screening methods to identify compounds having the ability to induce expression of FoxMlB protein, compounds that induce nuclear localization of FoxMlB protein, and compounds that induce both expression and nuclear localization of FoxMlB protein in mammalian cells.
- the invention provides pharmaceutical compositions comprising compounds identified by the screening methods of the invention.
- the invention provides methods of preventing or ameliorating liver damage in patients in need of such treatment.
- the invention provides methods for inducing nuclear localization of FoxMlB protein in a mammalian liver cell comprising the step of contacting the liver cell with growth hormone.
- the mammalian liver cell expresses FoxMlB endogenously, such as in liver cells from a young mammal.
- the liver cells have a reduced ability to express FoxMlB protein, such as liver cells in aged mammals.
- the invention provides a recombinant nucleic acid construct that can be introduced into a cell, preferably a liver cell and most preferably a hepatocyte cell to restore FoxMlB expression and regenerative potential in the cell.
- the invention provides recombinant nucleic acid constructs that comprise nucleic acid having a nucleotide sequence encoding FoxMlB protein.
- the nucleic acid encodes human FoxMlB and has the nucleotide sequence that encodes a protein as set forth in SEQ ID NO: 2.
- the recombinant nucleic acid construct also comprises an expression control sequence that is operatively linked to the nucleic acid encoding Fox M1B.
- the expression control sequence is a liver-specific promoter that is specifically active in liver cells.
- the nucleic acid comprising recombinant nucleic acid construct of the invention is transcriptionally active and expressed only in liver cells when the construct is delivered in vivo.
- Promoters useful in this aspect of the invention include, but are not limited to, human or mouse ⁇ l-antitrypsin promoter, albumin promoter, serum amyloid A promoter, transthyretin promoter, and hepatocyte nuclear factor 6 (HNF-6) promoter.
- the promoter is HNF-6, which is induced by growth hormone.
- a recombinant nucleic acid construct of the invention comprises a vector.
- the vector is a viral vector, such as an adenovirus, an adeno-associated virus, a retrovirus, herpes simplex virus, or vaccinia virus vector.
- the invention further provides methods for introducing the recombinant nucleic acid constructs of the invention into cells, most preferably mammalian cells.
- recombinant expression constructs of the invention are formulated into liposomes and introduced into mammalian liver cells.
- proliferative cell types that may benefit from FoxMlB intervention are, for example, intestinal and colonic epithelial cells, thymocytes in the thymus and lymphocytes in the spleen, and basal cells of the skin.
- Recombinant expression constructs of the invention can also be introduced into cells using, for example, the ExGen 500 reagent (MBI Fermentas).
- the invention also provides cells, preferably mammalian cells, into which have been introduced a recombinant nucleic acid construct of the invention.
- the cells are hepatocytes, intestinal or colonic epithelial cells, thymocytes in the thymus and lymphocytes in the spleen, or basal cells of the skin
- the invention provides methods of stimulating liver regeneration in cells that express FoxMlB protein by inducing FoxMlB protein to translocate into the nucleus of the cells.
- the invention provides a method for inducing nuclear localization contacting the cells with growth hormone.
- the invention further provides a method of screening for compounds that induce expression of FoxMlB protein in mammalian cells, wherein the FoxMlB protein can be translocated into the nucleus.
- the inventive methods comprise the steps of contacting a plurality of cells that do not express FoxMlB under conventional culture conditions, with a candidate compound in the presence and absence of growth hormone; assaying FoxMlB expression and localization in the cells cultured in the presence and absence of growth hormone and comparing FoxMlB expression and nuclear localization in the cells, wherein a candidate compound is identified when FoxMlB is expressed in said cells and localized in the nuclei of cells in the presence but not in the absence of growth hormone.
- the invention also provides a method of screening for compounds that induce nuclear localization of FoxMlB protein.
- inventive methods comprise the steps of contacting cells that express FoxMlB protein with a candidate compound and examining the intracelluiar localization of FoxMlB protein in the cell; wherein the candidate compound is identified when FoxMlB protein is localized in the nucleus of the cell in the presence of the compound but not in the absence of the compound.
- the invention also provides methods of screening for compounds that induce both expression and nuclear localization of FoxMlB protein.
- the methods of the invention comprise the steps of (a) contacting cells that do not express FoxMlB under conventional culture conditions, with a candidate compound; and (b) assaying FoxMlB expression and localization in the cells, wherein a candidate compound is identified when FoxMlB is expressed and localized in the nuclei of cells contacted with the compound but not in cells not contacted with the compound.
- the cells are contacted with growth hormone upon induction of FoxMlB expression in the cells in the presence of the compound.
- the invention also provides methods of inducing liver cell proliferation comprising the step of contacting a liver cell with growth hormone or a compound identified in a screening method of the invention, wherein the liver cell expresses FoxMlB protein.
- the cell expresses FoxMlB protein endogenously, i.e., encoded by the cellular DNA.
- the cell expresses FoxMlB encoded by a recombinant nucleic acid construct of the invention.
- the invention further provides methods of stimulating liver regeneration in a mammal, comprising the step of contacting mammalian liver cells with growth hormone or a compound identified in a screening method of the invention.
- the cell expresses FoxMlB protein endogenously, i.e., encoded by the cellular DNA.
- the cell expresses FoxMlB encoded by a recombinant nucleic acid construct of the invention.
- the invention also provides methods of preventing or ameliorating liver damage in a mammal comprising the step of contacting mammalian liver cells with growth hormone or a compound identified in a screening method of the invention.
- the cell expresses FoxMlB protein endogenously, i.e., encoded by the cellular DNA.
- the cell expresses FoxMlB encoded by a recombinant nucleic acid construct of the invention.
- the method is a preventative measure, most preferably applied to individuals with a high susceptibility or a genetic disposition for acquiring liver damage or liver disease.
- the method is a therapeutic measure, applied to an individual who suffers from liver damage or liver disease.
- the methods of the invention prevent further damage or disease progression or reverses damage or disease progression.
- the methods are applied to an individual awaiting a liver transplant.
- the methods of the invention are applied to a liver removed from a donor to be transplanted into a recipient.
- the donor is treated with growth hormone or another compound identified in a screening method of the invention prior to surgical removal of the liver to induce expression, nuclear localization or expression and nuclear localization of FoxMlB protein.
- the liver is contacted with growth hormone or another compound identified in a screening method of the invention that induces expression, nuclear localization or expression and nuclear localization of FoxMlB protein after removal from the donor.
- the methods of the invention can also be applied to the recipient, by treating the recipient with growth hormone or another compound identified in a screening method of the invention that induces expression, nuclear localization or expression and nuclear localization of FoxMlB protein after the liver has been transplanted.
- the invention further provides methods of preventing or ameliorating liver damage in a mammal comprising the steps of introducing into the mammal liver cells that express FoxMlB protein and thereafter contacting the liver cells with growth hormone or another compound identified in a screening method of the invention.
- liver cells are removed from an individual and reintroduced into a recipient individual, most preferably the same individual to minimize immunological complications.
- the liver cells express FoxMlB endogenously.
- the liver cells are contacted ex vivo with a recombinant nucleic acid construct of the invention whereby the cells express FoxMlB protein.
- both allografts and autografts as disclosed herein are contemplated by the invention to protect or ameliorate liver damage or liver disease in a patient.
- the invention provides these methods wherein the liver cells removed from an individual are contacted with growtli hormone or a compound identified in a screening method of the invention that induces expression, nuclear localization or expression and nuclear localization of FoxMlB protein prior to or after introducing the cells into a recipient. Diminished expression of FoxMlB and its target genes mediating cell cycle regulation is associated with reduced proliferation in regenerating hepatocytes of 12- month old (old-aged) mice and in proliferating fibroblast of old-aged humans.
- FoxMlB deficiency inhibits hepatocyte proliferation even in young mice in response to liver injury, demonstrating that FoxMlB expression is essential for hepatocytes to undergo DNA replication and cell division (mitosis). FoxMlB is also essential for hepatocyte replication required to regenerate the liver in response to injury. Because FoxMlB is expressed in every proliferating cell that has been examined, FoxMlB is critical for proliferation of all cell types in the body. Taken together, the instant disclosure demonstrates that expression of FoxMlB is necessary for hepatocyte replication in response to liver injury and that increased FoxMlB levels is sufficient to restore hepatocyte proliferation in the elderly and in patients with liver diseases. Thus, the methods disclosed herein provide advantages for treating and preventing liver disease and injury.
- hepatocyte expression of the c-myc in transgenic mice which stimulates hepatocyte replication during liver regeneration.
- Constitutive c-myc expression is undesirable because it causes aberrant hepatocyte proliferation in the absence of liver injury. This is due to c-myc localizing to the nucleus in the absence of proliferative signals, and results in development of liver cancer such as hepatocellular carcinoma.
- FoxMlB nuclear localization requires proliferation-specific signals. Therefore, ectopic FoxMlB expression is insufficient to induce quiescent cells to enter the cell cycle, and thus will not induce unwanted cellular proliferation.
- This feature permits FoxMlB to be used for therapeutic intervention to ameliorate defective proliferation observed in the elderly population or patients with liver diseases exhibiting defective liver regeneration, without implicating the risk of the patients developing liver cancers such as hepatocellular carcinoma. Because increased FoxMlB expression in quiescent cells does not induce unwanted cellular proliferation leading to the development of cancer, it is much safer for administration to patients to stimulate liver regeneration.
- Figure 1A-B depicts human FoxMlB cDNA comprising a deletion of the terminal 972 nucleotides at the 3' end (SEQ ID NO: 1).
- Figure IC depicts human FoxMlB protein sequence (SEQ ID NO: 2) encoded by the nucleotide sequence as set forth in SEQ ID NO: 1.
- Figure 2 shows a graph representing 5-bromo-2'-deoxy-uridine (BrdU) incorporation (as a measure of DNA replication) at the indicated hours after partial hepatectomy (PHx) in twelve month old wild type CD-I mice (WT, solid circles), twelve month old transgenic CD-I mice (TG, solid diamonds), or two month old wild type CD-I mice (solid squares).
- PrdU 5-bromo-2'-deoxy-uridine
- Figure 3 shows a graph representing increased hepatocyte mitosis in regenerating livers of old-aged TG mice at 48 hours post PHx.
- Figure 4 shows RNase protection assays performed using total RNA isolated at the indicated hours post PHx from regenerating liver of two-month-old WT mice (A), twelve-month-old WT mice (B), and twelve month old TG mice (C).
- Figure 5 shows a western blot analysis with anti-FoxMlB antibodies performed with total liver protein extracts isolated from regenerating livers of twelve month old WT and TG mice at the indicated time points. FoxMlB protein migrates more slowly than a non-specific (NS) band.
- NS non-specific
- Figure 6 shows an RNase protection assay demonstrating increased expression of cell cycle promotion genes in regenerating liver of old TG mice compared with WT mice at the indicated hours following PHx.
- Figure 7 shows an RNase protection assay of total RNA isolated from regenerating livers of twelve-month-old WT or TG mice using an antisense RNA probe for p21.
- Figure 8 shows a graph representing the number of p21 positive nuclei per 2500 hepatocytes per regenerating mouse liver, + the standard deviation (SD).
- Figure 9A depicts a Western blot with anti-p53 antibodies showing p53 protein expression in regenerating livers of old-aged TTR-FoxMlB TG mice and old-aged WT mice.
- Figures 9B-C show graphs depicting relative p53 and p21 protein levels in old aged TTR-FoxMlB transgenic mice compared to levels in old-aged WT mice at various times after PHx.
- Figure 10 shows immunohistochemical staining of FoxMlB protein with FoxMlB antibody and nuclear expression of FoxMlB protein in CCl 4 -treated regenerating liver from WT (A-C) or TG (D-F) mice.
- Figure 11 shows a graph representing BrdU incorporation in hepatocytes at various time points after CCl 4 -induced liver damage in WT and TG mice. BrdU positive cells were counted in three viewing fields, each field containing about 250 nuclei.
- Figure 12A shows a statistical analysis of p21 -staining hepatocytes in WT and TG liver regeneration.
- Figure 12B shows a graph representing levels of p21 mRNA expression in regenerating livers from WT and TG mice, normalized to glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) and large ribosomal L32 protein levels.
- Figure 13 shows a graph representing Cyclin Dl (A), Cyclin E (B), Cyclin Bl
- FIG. 14A shows FoxMlB mRNA levels in regenerating livers of old Balb/c mice infected with either AdCon (adenovirus control) or AdFoxMlB (adeno viral vector with FoxMlB) two days prior to PHx operation or left uninfected. Expression of FoxMlB mRNA was normalized to cyclophilin levels. Shown below the panel is the fold induction compared to expression levels at the beginning of the experiment (the 0- hour time point).
- Figure 14B shows a graph representing hepatocyte BrdU incorporation during mouse liver regeneration induced by PHx in twelve month-old Balb/c mice infected with either AdFoxMlB or AdCon or left uninfected. The mean of the number of BrdU positive nuclei per 1000 hepatocytes and the standard deviation (SD) was calculated for each time point.
- Figure 14C shows a graph representing increased hepatocyte mitosis in regenerating livers of old mice infected with AdFoxMlB between 36 to 44 hours post
- hepatocyte mitosis is expressed as the mean of the number of mitotic figures found per 1000 hepatocytes + SD.
- Figure 15 shows immunohistochemical staining with FoxMlB antibody showing hepatocyte nuclear expression of FoxMlB protein in regenerating liver of old mice infected with AdFoxMlB but not with AdCon.
- Figure 16 shows a graph representing stimulated expression of cyclin genes in regenerating liver of old mice infected with AdFoxMlB. Expression levels of cyclin expression levels were normalized to the GAPDH and ribosome large subunit L32 protein mRNA levels. Graphic presentation of normalized mean mRNA levels of Cyclin A2 (A), Cyclin Bl (B), Cyclin Bl (C), Cyclin Dl (D), Cyclin D3 (E), Cyclin E (F), Cyclin F (G), and Cyclin GI (H).
- Figure 17 is a schematic representation of triple-LoxP FoxMlB targeting vector used to generate the conditional FoxMlB knockout mice.
- Figure 18A depicts a graph showing BrdU incorporation in FoxMlB deficient hepatocytes after partial hepatectomy.
- Figure 18B depicts a graph showing hepatocyte mitosis at various time points after partial hepatectomy in FoxMlB -/- and FoxMlB fl/fl mice.
- Figure 19A depicts RNase protection assays performed in duplicate showing expression of cell cycle regulatory genes in regenerating liver of FoxMlB -/- and FoxMlB fl/fl mice.
- Figure 19B depicts a Western blot analysis showing p21 protein levels in regenerating FoxMlB -/- and FoxMlB fl/fl hepatocytes.
- Figure 19C depicts a Western blot analysis with cdk-1 specific phospho-Tyrosine 15 antibodies and kinase assays using HI protein as a substrate in FoxMlB -/- and FoxMlB fl/fl hepatocytes during liver regeneration.
- Figure 20 shows hepatocyte nuclear expression of FoxMlB protein in young CD-
- mice stimulated by growth hormone. Shown are micrographs (200 X, left panel and
- Figure 21 shows hepatocyte nuclear expression of FoxMlB protein in young TTR-FoxMlB transgenic mice stimulated by growth hormone. Shown are micrographs (200 X, left panel and 400X, right panel) of TTR-FoxMlB liver sections displayed FoxMlB nuclear staining (indicated by arrows) between 30 minutes (C-D), 2 hours (E-F) and 3 hours (G-H) following growth hormone administration but not in control transgenic mice (A-B).
- Figure 22 shows a time course of FoxMlB mRNA levels in regenerating liver of untreated 2-month old (young) and 12-month old Balb/c mice as well as 12-month old Balb/c mice treated with human growth hormone.
- Figure 23A shows a graph representing number of BrdU positive hepatocytes from regenerating livers in mice treated with growth hormone.
- Figure 23B shows a graph representing number of mitotic hepatocytes from regenerating livers in mice treated with growth hormone.
- Figure 24A-D depicts immunohistochemical staining with FoxMlB antibody showing localization of GFP-FoxMlB-NLS (B) and GFP-FoxMlB in the presence and absence of growth hormone (C and D).
- Panel A is a control.
- isolated polynucleotide as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” is found in nature, (2) is linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
- isolated protein means a protein encoded by genomic DNA, cDNA, recombinant DNA, recombinant RNA, or synthetic origin or some combination thereof, which (1) is free of at least some proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell, (5) is not linlced (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the "isolated protein" is linked in nature, (6) is operatively linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature, or (8) does not occur in nature.
- the isolated protein is substantially free from other contaminating proteins or polypeptides or
- polypeptide or “protein” is used herein to refer to native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or sequences that have deletions, additions, and/or substitutions of one or more amino acids of the native sequence.
- polypeptide and protein specifically encompasses FoxMlB, or species thereof that have deletions, additions, and/or substitutions of one or more amino acids of FoxMlB having at least one functional property of the FoxMlB protein.
- naturally-occu ⁇ ing refers to an object that can be found in nature, for example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature and which has not been intentionally modified by man.
- naturally occurring or “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man.
- “recombinant,” “non-naturally occuiring” or “non- native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.
- a conservative amino acid substitution does not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not disrupt secondary structure that characterizes the parent or native protein, such as a helix).
- a replacement amino acid should not disrupt secondary structure that characterizes the parent or native protein, such as a helix.
- Examples of art-recognized polypeptide secondary and tertiary structures are described in PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES (Creighton, Ed.), 1984, W. H. New York: Freeman and Company; INTRODUCTION TO PROTEIN STRUCTURE (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton et at., 1991, Nature 354: 105, which are each incorporated herein by reference.
- amino acid residues may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
- Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.
- non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
- substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.
- the hydropathic index of amino acids may be considered.
- Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); tlireonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5) (Kyte et al, 1982, J. Mol. Biol. 157:105-131).
- the substitution of like amino acids can be made effectively on the basis of hydropliilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case.
- the greatest local average hydrophilicity of a protein correlates with its immunogenicity and antigen-binding or immunogenicity, i.e., with a biological property of the protein.
- hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4).
- the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is included, in certain embodiments, those that are within ⁇ 1 are included, and in certain embodiments, those within ⁇ 0.5 are included.
- Exemplary amino acid substitutions are set forth in Table 1. Table 1
- a skilled artisan can determine suitable variants of the polypeptide as set forth herein using well-known techniques.
- one skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity.
- even areas that are important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
- one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins.
- One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.
- One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants.
- Stereoisomers e.g., D-amino acids of the twenty conventional amino acids, non- naturally occurring amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
- unconventional amino acids include but are not limited to: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-
- the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
- Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non- peptide compound are termed “peptide mimetics” or “peptidomimetics.” (See Fauchere,
- a paradigm polypeptide i.e., a polypeptide that has a biochemical property or pharmacological activity
- confo ⁇ nationally-constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61: 387), incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
- the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
- the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the franscription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
- polynucleotide as used herein means a polymeric form of nucleotides that are at least 10 bases in length.
- the bases may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
- the term includes single and double stranded forms of DNA.
- oligonucleotide as used herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and or non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising no more than 200 nucleotides. In certain embodiments, oligonucleotides are 10 to 60 nucleotides in length. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are single stranded, e.g. for use in the construction of a gene mutant using site directed mutagenesis techniques.
- Oligonucleotides of the invention may be sense or antisense oligonucleotides.
- naturally occurring nucleotides includes deoxyribonucleotides and ribonucleotides.
- modified nucleotides includes nucleotides with modified or substituted sugar groups and the like.
- oligonucleotide linkages includes oligonucleotides linkages such as phosphate, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like.
- An oligonucleotide can include a detectable label, such as a radiolabel, a fluorescent label, an antigenic label or a hapten.
- agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an exfract made from biological materials.
- label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
- marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods.
- the label or marker can also be therapeutic.
- Various methods of labeling polypeptides and glycoproteins can be used that are known in the art.
- labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, U 1 ln, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin, and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
- recombinant nucleic acid construct refers to a DNA or RNA sequence that comprises a coding sequence that is operatively linked to a control sequence.
- a recombinant nucleic acid construct of the invention is capable of expressing a protein that is encoded by the coding sequence when introduced into a cell.
- a recombinant nucleic acid construct of the invention preferably comprises the nucleic acid sequence that encodes a protein as set forth in SEQ ID NO: 2, such as the nucleic acid sequence as set forth in SEQ ID NO: 1, whereby a cell contacted with the recombinant nucleic acid construct expresses FoxMlB protein.
- operatively linked refers to components that are in a relationship pe ⁇ nitting them to function in their intended or conventional manner.
- a control sequence "operatively linlced" to a coding sequence is ligated thereto in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
- control sequence refers to polynucleotide sequences that can effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences may differ depending upon the host organism.
- control sequences for prokaryotes may include promoters, repressors, operators, ribosomal binding sites, and transcription termination sequences and antisense mRNA.
- control sequences for eukaryotes may include promoters, enhancers and transcription termination sequence, protein degradation , mRNA degradation, nuclear localization, nuclear export, cytoplasmic retention, protein phosphorylation, protein acetylation, protein sumolation, RNAi inhibition.
- control sequences can include leader sequences and/or fusion partner sequences.
- Control sequences are "operatively linked” to a coding sequence when the "control sequence” effects expression and processing of coding sequences to which they are ligated.
- liver specific promoters refers to nucleic acid sequences that are capable of directing transcription of a coding sequence and are activated specifically within a liver cell.
- Liver specific promoters suitable for the methods of the invention include, but are not limited to, human or mouse ⁇ l-antifrypsin, albumin promoter, serum amyloid A, transthyretin, and hepatocyte nuclear factor 6.
- vector is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.
- Viral vectors suitable for the methods of the invention include those derived from, for example, an adenovirus, an adeno-associated virus, a retrovirus, a herpes simplex virus, or a vaccinia virus.
- expression vector refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
- the term "host cell” is used to refer to a cell into which has been introduced, or that is capable of having introduced, a nucleic acid sequence and then of expressing a gene of interest.
- the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the gene is present.
- transduction is used to refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by viruses such as retroviruses. The term “transfection” is used to refer to the uptake of foreign or exogenous
- transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
- a cell is transformed where it is genetically modified from its native state.
- the fransfonriing DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
- a cell is stably transformed when the DNA is replicated with the division of the cell.
- pharmaceutical composition refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
- therapeutically effective amount refers to the amount of growth hormone or a compound identified in a screening method of the invention determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
- substantially pure means an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
- a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis or on a weight or number basis) of all macromolecular species present.
- a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition.
- the object species is purified to essential homogeneity (wherein contaminating species cannot be detected in the composition by conventional detection methods) wherem the composition consists essentially of a single macromolecular species.
- patient includes human and animal subjects.
- autograft refers to removal of part of an organism and its replacement in the body of the same individual.
- An autograft can be the introduction of autologous organs, tissue, or cells in an individual.
- an allograft refers to the removal of part of one individual and its replacement in the body of a different individual.
- An allograft is also referred to as a xenograft, heterograft, or heterologous graft.
- AUografts can be obtained, for example, from organ donation.
- liver cells refers to the cells that make up a mammalian liver. Liver cells include, for example, hepatocytes, Kupffer cells, biliary epithelial cells, fenestrated endothelial cells, and cells of Ito.
- liver regeneration refers to the growth or proliferation of new liver tissue. Regenerated liver tissue of the invention will have cytological, histological, and functional characteristics of normal liver tissue. Such characteristics can be examined by any method known in the art. For example, regenerated liver tissue of the invention can be examined for expression of common markers indicative of liver function.
- liver function refers to one or more of the many physiological functions performed by the liver. Such functions include, but are not limited to, regulating blood sugar levels, endocrine regulation, enzyme systems, interconversion of metabolites (e.g., ketone bodies, sterols and steroids and amino acids); manufacturing blood proteins such as fibrinogen, serum albumin, and choliuesterase, erythropoietic function, detoxification, bile formation, and vitamin storage.
- Several tests to examine liver function are known in the art, including, for example, measuring alanine amino transferase (ALT), alkaline phosphatase, bilirubin, prothrombin, and albumin.
- liver disease refers to any condition that impairs liver function.
- Liver damage can occur in response to liver injury caused by any of a number of factors, including, for example, viral infections, parasitic infections, genetic predisposition, autoimmune diseases, exposure to radiation, exposure to hepatotoxic compounds, mechanical injuries, and various environmental toxins.
- Alcohol, acetominophen, a combination of alcohol and acetaminophen, inhalation anaesthetics, niacin, and the herbal supplement kava are some examples of compounds that can cause liver damage.
- Most forms of liver damage lead to cirrhosis. Cirrhosis is a pathological condition associated with chronic liver damage that includes extensive fibrosis and regenerative nodules.
- Fibrosis refers to the proliferation of fibroblasts and the formation of scar tissue in the liver.
- liver diseases include, but are not limited to, Reye's syndrome in young children, Wilson's disease, hemochromatosis, alpha- 1-antifrypsin deficiency, various parasitic infections, viral diseases, cirrhosis, and liver cancer.
- viral diseases include infection by hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, and hepatitis G.
- parasitic infections include Schistosoma mansoni, Schistosoma hematobiurn, and Schistosoma japonicum.
- growth hormone refers to growth hormone from any species, including bovine, ovine, porcine, equine, and preferably human, in native-sequence or in variant form, and from any source, whether natural, synthetic, or recombinant.
- Preferred herein for human use is human native-sequence, mature growth hormone with or without a methionine at its N-terminus.
- recombinant human growth hormone liGH
- liGH recombinant human growth hormone
- Somafrem Human growth hormone is commercially available and known as somafrem and somafropin.
- Somafrem is typically used to treat children with growth failure caused by hGH deficiency.
- the usual weekly dosage of somafel for children is 0.3 milligram (mg) per kilogram (kg) of body weight.
- Somafropin is used to treat growth failure caused by Turner's syndrome, kidney disease, or a lack of hGH.
- the usual weekly dosage of somafropin for children is 0.16 to 0.375 mg per kg of body weight. For adults, 0.006 mg per kg is usually taken daily and increased gradually as needed. AIDS patients experiencing dramatic weight loss are given up to 6 mg of somafropin per day depending on body weight.
- Somafropin and somafel are typically administered by injection under the skin or directly into a muscle. Forms of orally administered growth hormone are also known in the art (see, for example, U.S. Patent No. 6,239,105).
- GH growth hormone
- GH freated old aged mice exhibited increase in regenerating hepatocyte DNA replication and mitosis to levels found in young regenerating liver.
- increased expression and nuclear localization of FoxMlB is the mechanism by which GH restores hepatocyte proliferation in regenerating liver of old aged mice. This suggests that GH mediates increased hepatocyte proliferation by restoring FoxMlB expression in regenerating livers of old aged mice.
- short term GH administration can be used to stimulate FoxMlB expression and liver cell proliferation in diseased liver that exhibit defects in liver regeneration.
- short term GH administration can be effective in live donor fransplants of liver to recipient. These are donors that give recipient one of thefr liver lobes and require regeneration of liver in both donor and recipient.
- GH administration several days prior to donor and recipient with liver disease can stimulate liver regeneration in the liver of the live donor and in the recipient and allow better prognosis for both patients.
- the Examples herein demonstrate that GH administration is a useful therapeutic intervention that will enhance liver regeneration through increased expression and nuclear localization of FoxMlB.
- the invention provides methods for treating patients diagnosed with liver damage or disease.
- patients are freated with growtli hormone in a medically acute manner rather than a medically chronic manner, that is, the treatment has a duration that is limited by the nature and extent of the disease, injury or damage and terminates upon detection of positive response in the patient.
- the invention provides transient nuclear localization of FoxMlB protein in the patients freated with growth hormone in a medically acute manner.
- “fransient nuclear localization” refers to non-permanent localization of FoxMlB protein in the nucleus of a cell.
- FoxMlB protein can be induced to localize in the nucleus of a hepatocyte by exposure to growth hormone, while the FoxMlB protein is not detectable in the nucleus once exposure to growth hormone is discontinued.
- Patients are preferably screened for liver damage or disease using various assays known in the art.
- serum levels of liver aminotransferases enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
- ALT levels alanine aminotransferase
- AST aspartate aminotransferase
- AST levels increase less than ALT (i.e., the ratio of AST/ALT is less than 1). In liver injury caused by alcohol, however, the ratio is often > 2.
- liver damage in a patient involves measuring levels of bilirubin, prothrombin, and albumin.
- levels of bilirubin, prothrombin, and albumin For a review of various methods for screening and diagnosing liver damage and disease, see THE MERCK MANUAL, 17 TH Edition, (Beers and Berkow, Ed.), 1999, Whitehouse Station, N. J.
- patients with, for example, high serum levels of ALT, AST, and bilirubin and with low serum albumin levels advantageously would be administered growth hormone according to the methods of the invention.
- hGH human growth hormone
- a suitable dosage for human adminisfration ranges from 0.001 mg to about 0.2 mg per kg of body weight per day.
- therapeutically effective daily dosages of hGH will be from about 0.05 mg to about 0.2 mg per kg of bodyweight per day.
- doses of from 0.07 to 0.15 mg/kg, in one or more applications per day is effective to obtain the desired result.
- hGH may be administered less frequently, particularly where formulated in a timed-release form, e.g., every other day or every third day for certain indication.
- liver function is restored to a level that resembles that of a healthy liver, suggesting that liver regeneration process is sufficient, growth hormone adminisfration is discontinued.
- growth hormone adminisfration is discontinued.
- the methods of the invention are advantageously used with patients having, for example, traumatic liver damage, as well as those who are at high risk for obtaining liver damage, such as alcoholics and those with genetic disposition for liver disease, and those who are regularly exposed to environmental, commercial, and chemical toxins.
- the invention provides methods for treating liver damage or liver disease in mammals by inducing FoxMlB protein to translocate from the cytoplasm to the nucleus in liver cells, where it potentiates transcription of many cell cycle promotion genes and stimulates cellular proliferation.
- the mammal is treated with growth hormone to induce nuclear localization of FoxMlB protein.
- the invention provides methods of screening for compounds that induce expression of FoxMlB protein, induce nuclear localization of
- FoxMlB protein or induce both expression and nuclear localization of FoxMlB protein.
- Compounds identified in these screens can be used in the methods of treating liver damage and liver disease as discussed herein.
- Screening for compounds the induce expression of FoxMlB protein can be accomplished, for example, with cells that comprise the FoxMlB gene but do not express FoxMlB protein under normal culture conditions.
- Such cells can include, for example, hepatocytes from aged individuals, host cells comprising the FoxMlB gene as discussed below, or quiescent cells that do not express FoxMlB protein.
- the method of screening for compounds that induce expression of FoxMlB in mammalian cells can be accomplished as follows: (a) contacting a plurality of cells that comprise the FoxMlB gene, wherein the FoxMlB protein is not expressed under normal culture conditions, with a candidate compound in the presence of human growth hormone; (b) contacting a plurality of cells that comprise the FoxMlB gene, wherein the FoxMlB protein is not expressed under normal culture conditions, with the candidate compound in the absence of human growth hormone; and (c) assaying FoxMlB expression and localization in the cells from step (a) and step (b); wherein a candidate compound is selected if FoxMlB is localized in the nuclei of cells from step (a) and in the cytoplasm of cells from step (b).
- Said assay can be a direct assay for nuclear localization of FoxMlB, or can be an indirect assay for the presence or activity of a gene product expressed as a consequence of FoxMlB translocation into the nucleus from the cyto
- the method of screening for compounds that induce nuclear localization of FoxMlB protein can be accomplished by contacting a cell with a candidate compound, wherein the cell expresses FoxMlB protein, and examining localization of FoxMlB protein in the cell.
- the candidate compound is selected if FoxMlB protein is localized in the nucleus of the cell.
- the Fox M1B is endogenous, i.e., it comprises the genomic DNA complement of the cell.
- the FoxMlB is exogenous and is experimentally introduced, most preferably as a recombinant nucleic acid construct of the invention encoding most preferably a heterologous Fox M1B gene, i.e., from a mammalian species different from the host cell species.
- the method of screening for compounds that induce both expression and nuclear localization of FoxMlB protein in a manner similar to that of growth hormone can be accomplished as follows: (a) contacting a plurality of cells that comprise the FoxMlB gene, wherein the FoxMlB protein is not expressed under normal culture conditions, with a candidate compound; y and (b) assaying FoxMlB expression and localization in the cells from step (a); wherein a candidate compound is selected if FoxMlB is expressed and localized in the nuclei of cells contacted with the compound in a manner similar to the pattern observed in cells contacted with growth hormone.
- the cells of step (a) can be contacted with growth hormone prior to assay in step (b).
- Recombinant nucleic acid constructs of the invention typically comprise a nucleic acid molecule encoding the amino acid sequence of FoxMlB protein that is inserted into an appropriate expression vector using standard ligation techniques.
- the recombinant nucleic acid construct of the invention comprises the nucleic acid sequence that encodes a protein as set forth in SEQ ID NO: 2.
- the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene can occur).
- expression vectors used in any of the host cells contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
- sequences collectively referred to as “flanking sequences” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete infron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
- Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (ie., from a species other than the host cell species or strain), hybrid (t.e., a combination of flanking sequences from more than one source), synthetic or native.
- the source of a flanking sequence may be any prokaryotic
- Assaying for nuclear localization and expression of FoxMlB protein can be accomplished by any method known the art.
- immunohistochemisfry using anti-FoxMlB antibodies and secondary antibodies labeled with fluorescent markers such as fluorescein isothiocyanate (FITC)
- FITC fluorescein isothiocyanate
- the primary antibody can be labeled, with a fluorescent label or otherwise.
- Alternative labels, such as radioactive, enzymatic and hapten labels, are within the scope of this invention.
- methods of the invention comprise expressing FoxMlB protein in a host cell by introducing into the cell a recombinant nucleic acid construct of the invention.
- the cells are fransformed with the recombinant nucleic acid construct using any known method for infroducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos.
- the transformation procedure used may depend upon the host to be fransformed.
- Methods for introduction of heterologous polynucleotides into mammalian cells include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, elecfroporation, encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acid with positively-charged lipids, and direct microinjection of the DNA into nuclei.
- flanking sequence is functional in, and can be activated by, the host cell machinery.
- Flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.
- flanking sequence may be obtained using in vitro amplification methods such as polymerase chain reaction (PCR) and/or by screening a genomic library with a suitable oligonucleotide and/or flanking sequence fragment from the same or another species.
- PCR polymerase chain reaction
- flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen ® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose is readily apparent to one of ordinary skill in the art.
- the vector may contain a "tag"-encoding sequence, t.e., an oligonucleotide molecule located at the 5' or 3' end of the FoxMlB polypeptide coding sequence, the oligonucleotide sequence encoding polyHis (such as hexaHis), or another "tag” for which commercially available antibodies exist, such as FLAG, HA (hemaglutinin influenza virus), or myc.
- This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification of the FoxMlB polypeptide from the host cell. Affinity purification can be accomplished, . for example, by column chromatography using antibodies against the tag as an affinity matrix.
- the tag can subsequently be removed from the purified FoxMlB polypeptide by various means such as using certain peptidases for cleavage.
- An origin of replication is typically a part of prokaryotic expression vectors, particularly those that are commercially available, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
- the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.
- SV40 polyoma
- adenovirus vesicular stomatitus virus
- papillomaviruses such as HPV or BPV
- a transcription termination sequence is typically located 3' of the end of a polypeptide-coding region and serves to terminate transcription.
- a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein. In eukaryotes, the sequence AAUAAA functions both as a transcription te ⁇ nination signal and as a poly A signal required for endonuclease cleavage and followed by the addition of poly A residues (200 A residues).
- a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
- Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
- Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
- a bacterial neomycin resistance gene can also be used most advantageously for selection in both prokaryotic and eukaryotic host cells.
- a ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
- the element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
- various presequences can be manipulated to improve glycosylation or yield.
- the peptidase cleavage site of a particular signal peptide can be altered, or pro-sequences added, which also may affect glycosylation.
- the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed.
- the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the amino- temiinus.
- use of some enzyme cleavage sites may result in a slightly truncated yet active form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.
- the expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operatively linked to nucleic acid encoding the FoxMlB protein.
- Promoters are unfranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that confrol franscription of the structural gene.
- Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of franscription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.
- Constitutive promoters initiate continual gene product production; that is, there is little or no experimental confrol over gene expression.
- a large number of promoters, recognized by a variety of potential host cells, are well known.
- a suitable promoter is operatively linked to the DNA encoding FoxMlB protein by removing the promoter from the source DNA by restriction enzyme digestion or amplifying the promoter by polymerase chain reaction and inserting the desired promoter sequence into the vector.
- Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
- Oilier suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.
- promoters useful in the practice of the recombinant expression vectors of the invention include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22: 787- 97); the herpes mymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1444-45); the regulatory sequences of the metallothionine gene (Brinster et al, 1982, Nature 296: 39-42).
- elastase I gene control region that is active in pancreatic acinar cells (Swift et al, 1984, Cell 38: 639-46; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
- the promoter of a recombinant nucleic acid construct of the invention is active in the liver.
- the albumin gene confrol region is active in liver (Pinkert et al, 1987, Genes and Devel 268-76); the alpha-feto-protein gene confrol region is active in liver (Krumlauf et al, 1985, Mol. Cell Biol.
- Enhancers may be inserted into the vector to increase the transcription of a nucleic acid encoding a FoxMlB protein by higher eukaryotes.
- Enhancers are cis- acting elements of DNA, usually about 10-300 bp in length, that act on promoters to increase transcription. Enhancers are relatively orientation and position independent. They have been found within introns as well as both within several kilobases 5' and 3 ' to the transcription unit.
- enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein, insulin, transthyretin, and HNF-6). An enhancer from a virus can be used if increased expression of gene is desired.
- the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically located at a site 5' from the promoter.
- Expression vectors of the invention may be constructed from a convenient starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtainmg each of the flanking sequences are well known to one skilled in the art.
- the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
- the fransformation of an expression vector for a FoxMlB protein into a selected host cell may be accomplished by well-known methods including methods such as transfection, infection, calcium chloride, elecfroporation, microinjection, lipofection, DEAE-dextran method, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al , ibid.
- the host cell when cultured under appropriate conditions, synthesizes a FoxMlB protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
- the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
- Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
- ATCC American Type Culture Collection
- CHO Chinese hamster ovary
- HeLa cells HeLa cells
- BHK baby hamster kidney
- COS monkey kidney cells
- human hepatocellular carcinoma cells e.g., Hep G2
- cell lines may be selected through deterrmning which cell lines have high expression levels of FoxMlB protein.
- the invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound that induces FoxMlB expression, nuclear localization or expression and or nuclear localization in mammalian liver cells together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
- the invention provides pharmaceutical compositions that comprise a therapeutically effective amount of a compound that induces FoxMlB expression in mammalian liver cells and also induces FoxMlB protein to translocate into the nucleus of mammalian liver cells together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
- Such compounds are preferably identified in screening methods of the invention.
- Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
- the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
- Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine,' asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tefraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dexfrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying
- compositions can be determined by one skilled in the art depending upon, for example, the intended route of adminisfration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, ibid. Such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.
- the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
- a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral adminisfration.
- Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
- Pharmaceutical compositions can comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
- compositions of the invention may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, ibid.) in the form of a lyophilized cake or an aqueous solution. Further, the FoxMlB-inducing product may be formulated as a lyophilizate using appropriate excipients such as sucrose.
- optional formulation agents REMINGTON'S PHARMACEUTICAL SCIENCES, ibid.
- Formulation components are present in concentrations that are acceptable to the site of administration. Buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
- compositions of the invention can be delivered parenterally.
- the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising FoxMlB protein or the desired compound identified in a screening metiiod of the invention in a pharmaceutically acceptable vehicle.
- a particularly suitable vehicle for parenteral injection is sterile distilled water in which the compound identified in a screening method of the invention or FoxMlB protein is formulated as a sterile, isotonic solution, properly preserved.
- Preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which may then be delivered via a depot injection.
- an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which may then be delivered via a depot injection.
- Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation.
- Implantable drug delivery devices may be used to introduce the desired molecule.
- Administering FoxMlB protein to a patient can be used for short-term stimulation of liver cell proliferation, for example, in a recipient of a liver transplant.
- FoxMlB protein can be administered to a liver donor after the liver or a portion thereof is removed to stimulate liver regeneration to reestablish organ function.
- compositions may be selected for inhalation.
- a compound identified in a screening method of the invention or FoxMlB protein is formulated as a dry powder for inhalation, or inhalation solutions may also be formulated with a propellant for aerosol delivery, such as by nebulization.
- Pulmonary adminisfration is further described in PCT Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins .
- compositions of the invention can be delivered through the digestive tract, such as orally.
- the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
- FoxMlB protein or compounds of the invention that are administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
- a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
- Additional agents can be included to facilitate absorption of the FoxMlB protein or compound identified in a screening method of the invention. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
- a pharmaceutical composition may involve an effective quantity of FoxMlB protein or a compound identified in a screening method of the invention in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
- excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
- sustained- or confrolled-delivery formulations include formulations involving FoxMlB protein or compounds of the invention in sustained- or confrolled-delivery formulations.
- Techniques for formulating a variety of other sustained- or confrolled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
- Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g.
- films, or microcapsules polyesters, hydrogels, polylactides (U.S. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, 1983, Biopolymers 22: 547-556), poly (2- hydiOxyethyl-methacrylate) (Langer et al, 1981, J. Biomed. Mater. Res. 15: 167-277) and Langer, 1982, Chem. Tech.
- Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Eppstern et al, 1985, Proc. Natl. Acad. Sci USA 82: 3688-3692; EP 036,676; EP 088,046 andEP 143,949.
- the pharmaceutical composition to be used for in vivo adminisfration typically is sterile. In certain embodiments, this may be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral adminisfration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- composition of the invention may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
- Such formulations may be stored either in a ready-to- use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
- kits for producing a single-dose administration unit may each contain both a first container having a dried protein compound identified in a screening method of the invention and a second container having an aqueous formulation, including for example single and multi-chambered pre-filled syringes (e.g., liquid syringes, lyosyringes or needle-free syringes).
- syringes e.g., liquid syringes, lyosyringes or needle-free syringes.
- a pharmaceutical composition of the invention to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
- the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the pharmaceutical composition is being used, the route of adminisfration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
- a clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
- Typical dosages range from about 0.1 ⁇ g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above.
- the dosage may range from 0.1 ⁇ g/kg up to about 100 mg/kg; or 1 ⁇ g/kg up to about 100 mg/kg; or 5
- ⁇ g/kg up to about 100 mg/kg.
- the dosing frequency will depend upon the pharmacokinetic parameters of the FoxMlB protein or compound identified in a screening metiiod of the invention in the formulation. For example, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
- the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
- Administration routes for the pharmaceutical compositions of the invention include orally, through injection by intravenous, intraperitoneal, infracerebral (intra- parenchymal), infracerebroventricular, mfrarhuscular, infra-ocular, infraarterial, infraportal, or infralesional routes; by sustained release systems or by implantation devices.
- the pharmaceutical compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
- the pharmaceutical composition also can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous adminisfration.
- cells, tissues or organs that have been removed from the patient are exposed to pharmaceutical compositions of the invention or a recombinant nucleic acid construct of the invention comprising the FoxMlB gene after which the cells, tissues and/or organs are subsequently implanted back into the patient.
- FoxMlB protein, FoxMlB encoding recombinant nucleic acid constructs or pharmaceutical compositions of compounds identified in a screening method of the invention can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide.
- Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic, or may be immortalized.
- the cells may be encapsulated to avoid infiltration of surrounding tissues.
- Encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
- compositions of the invention can be administered alone or in combination with other therapeutic agents, in particular, in combination with other cancer therapy agents.
- agents generally include radiation therapy or chemotherapy.
- Chemotherapy for example, can involve treatment with one or more of the following: anthracyclines, taxol, tamoxifene, doxorubicin, 5-fluorouracil, and other drugs known to one skilled in the art.
- One approach for increasing, or causing, the expression of FoxMlB polypeptide from a cell's endogenous FoxMlB gene involves increasing, or causing, the expression of a gene or genes (e.g., transcription factors) and/or decreasing the expression of a gene or genes (e.g., transcriptional repressors) in a manner which results in de novo or increased FoxMlB polypeptide production from the cell's endogenous FoxMlB gene.
- a gene or genes e.g., transcription factors
- a gene or genes e.g., transcriptional repressors
- This metiiod includes the introduction of a non-naturally occurring polypeptide (e.g., a polypeptide comprising a site specific DNA binding domain fused to a transcriptional factor domain) into the cell such that de novo or increased FoxMlB polypeptide production from the cell's endogenous FoxMlB gene results.
- a non-naturally occurring polypeptide e.g., a polypeptide comprising a site specific DNA binding domain fused to a transcriptional factor domain
- the present invention further relates to DNA constructs useful in the method of altering expression of a target gene.
- the exemplary DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, and (d) an unpaired splice-donor site.
- the targeting sequence in the DNA construct directs the integration of elements (a) - (d) into a target gene in a cell such that the elements (b) - (d) are operatively linked to sequences of the endogenous target gene. .
- the DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a) - (f) such that the elements of (b) - (f) are operatively linked to the endogenous gene.
- the targeting sequence is homologous to the preselected site in the cellular chromosomal DNA with which homologous recombination is to occur.
- the exon is generally 3' of the regulatory sequence and the splice-donor site is 3' of the exon. If the sequence of a particular gene is known, such as the nucleic acid sequence of
- FoxMlB polypeptide presented herein a piece of DNA that is complementary to a selected region of the gene can be synthesized or otherwise obtained, such as by appropriate restriction of the native DNA at specific recognition sites bounding the region of interest. This piece serves as a targeting sequence upon insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence attached thereto, will act as an Okazaki fragment and will be incorporated into the newly synthesized daughter strand of DNA.
- the present invention therefore, includes nucleotides encoding a FoxMlB polypeptide, which nucleotides may be used as targeting sequences.
- FoxMlB polypeptide cell therapy e.g., the implantation of cells producing FoxMlB polypeptides
- This embodiment involves implanting cells capable of synthesizing and secreting a biologically active form of FoxMlB polypeptide.
- Such FoxMlB polypeptide-producing cells can be cells that are natural producers of FoxMlB polypeptides or may be recombinant cells whose ability to produce FoxMlB polypeptides has been augmented by fransformation with a gene encoding the desired FoxMlB polypeptide or with a gene augmenting the expression of FoxMlB polypeptide.
- Such a modification may be accomplished by means of a vector suitable for delivering the gene as well as promoting its expression and secretion.
- the natural cells producing FoxMlB polypeptide be of human origin and produce human FoxMlB polypeptide.
- the recombinant cells producing FoxMlB polypeptide be transformed with an expression vector containing a gene encoding a human FoxMlB polypeptide.
- Implanted cells may be encapsulated to avoid the infiltration of surrounding tissue.
- Human or non-human animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or membranes that allow the release of FoxMlB polypeptide, but that prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue.
- the patient's own cells, transformed to produce FoxMlB polypeptides ex vivo may be implanted directly into the patient without such encapsulation.
- FoxMlB polypeptides In vivo, ex vivo and in vitro gene therapy delivery of FoxMlB polypeptides is also provided herein.
- One example of a gene therapy technique is to use the FoxMlB gene (either genomic DNA, cDNA, and/or synthetic DNA) encoding a FoxMlB polypeptide that can be operatively linked to a constitutive or inducible promoter to form a "gene therapy DNA construct."
- the promoter may be homologous or heterologous to the endogenous FoxMlB gene, provided that it is active in the cell or tissue type into which the construct is inserted.
- Oilier components of the gene therapy DNA construct may optionally include DNA molecules designed for site-specific integration (e.g., endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers or silencers, DNA molecules capable of providing a selective advantage over the parent cell, DNA molecules useful as labels to identify transformed cells, negative selection systems, cell specific binding agents (as, for example, for cell targeting), cell-specific internalization factors, transcription factors enhancing expression from a vector, and factors enabling vector production.
- DNA molecules designed for site-specific integration e.g., endogenous sequences useful for homologous recombination
- tissue-specific promoters e.g., enhancers or silencers
- DNA molecules capable of providing a selective advantage over the parent cell DNA molecules useful as labels to identify transformed cells
- negative selection systems e.g., cell specific binding agents (as, for example, for cell targeting), cell- specific internalization factors, transcription factors enhancing expression from a vector, and factors enabling vector production.
- a gene therapy DNA construct can then be infroduced into cells (either ex vivo or in vivo) using viral or non-viral vectors.
- viral vectors as described herein.
- Certain vectors, such as retroviral vectors, will deliver the DNA construct to the chromosomal DNA of the cells, and the gene can integrate into the chromosomal DNA.
- Other vectors will function as episomes, and the gene therapy DNA construct will remain in the cytoplasm.
- regulatory elements can be included for the controlled expression of the FoxMlB gene in the target cell. Such elements are turned on in response to an appropriate effector. In this way, a therapeutic polypeptide can be expressed when desired.
- One conventional control means involves the use of small molecule dimerizers or rapalogs to dimerize chimeric proteins which contain a small molecule-binding domain and a domain capable of initiating a biological process, such as a DNA-binding protein or transcriptional activation protein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899).
- the dimerization of the proteins can be used to initiate franscription of the fransgene.
- In vivo gene therapy may be accomplished by introducing the gene encoding FoxMlB polypeptide into cells via local delivery of a FoxMlB nucleic acid molecule, by direct injection or by other appropriate viral or non- viral delivery vectors.
- a nucleic acid molecule encoding a FoxMlB polypeptide may be contained in an adeno-associated virus (AAV) vector for delivery to the targeted cells (see, e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No. PCT/US95/07178).
- AAV adeno-associated virus
- the recombinant AAV genome typically contains AAV inverted terminal repeats flanking a DNA sequence encoding a FoxMlB polypeptide operatively ' linked to functional promoter and polyadenylation sequences.
- Alternative suitable viral vectors include, but are not limited to, refrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma virus vectors.
- U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene fransfer system involving a recombinant neurotrophic HSV-1 vector.
- U.S. Patent No. 5,399,346 provides examples of a process for providing a patient with a therapeutic protein by the delivery of human cells that have been treated in vitro to insert a DNA segment encoding a therapeutic protein.
- Nonviral delivery methods include, but are not limited to, liposome-mediated fransfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complex), elecfroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene gun).
- Gene therapy materials and methods may also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific integration, DNA sequences capable of providing a selective advantage over the parent cell, labels to identify fransformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as methods of vector manufacture.
- inducible promoters tissue-specific enhancer-promoters
- DNA sequences designed for site-specific integration DNA sequences capable of providing a selective advantage over the parent cell
- labels to identify fransformed cells negative selection systems and expression control systems (safety measures)
- cell-specific binding agents for cell targeting
- cell-specific internalization factors cell-specific internalization factors
- transcription factors to enhance expression by a vector as well as methods of vector manufacture.
- FoxMlB gene therapy or cell therapy can further include the delivery of one or more additional polypeptide(s) in the same or a different cell(s).
- additional polypeptide(s) in the same or a different cell(s).
- Such cells may be separately introduced into the patient, or the cells may be contained in a single implantable device, such as the encapsulating membrane described above, or the cells may be separately modified by means of viral vectors.
- Another means of increasing endogenous FoxMlB polypeptide expression in a cell via gene therapy is to insert one or more enhancer elements into the FoxMlB polypeptide promoter, where the enhancer elements can serve to increase transcriptional activity of the FoxMlB gene.
- the enhancer elements used are selected based on the tissue in which one desires to activate the gene - enhancer elements known to confer promoter activation in that tissue are selected. For example, if a gene encoding a FoxMlB polypeptide is to be "turned on" in T-cells, the lck promoter enhancer element may be used.
- the functional portion of the transcriptional element to be added may be inserted into a fragment of DNA containing the FoxMlB polypeptide promoter (and optionally, inserted into a vector and/or 5' and or 3' flanking sequences) using standard cloning techniques. This construct, known as a "homologous recombination construct," can then be introduced into the desired cells either ex vivo or in vivo.
- Transgenic CD-I mice were generated using the -3 kb transthyretin (TTR) promoter to constitutively express the FoxMlB transgene (SEQ ID NO: 1 as shown in Figure 1) in hepatocytes as described (Ye et al, 1999, Mol Cell Biol, T9: 8570-8580). Twelve-month old wild type CD-I (WT) and TTR-FoxMlB (TG) mice were anesthetized with methoxyflurane (Metofane; Schering-Plough Animal Health Corp., Union, N.
- TTR transthyretin
- mice Two mice were sacrificed by CO 2 asphyxiation at 24, 32, 36, 40, 44, and 48 hours after partial hepatectomy (PHx) surgery and their livers were removed. The dissected livers were divided into three portions: one for paraffin embedding, one for total RNA isolation, and one for total protein isolation.
- Liver portions for paraffin embedding were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Tissues were cut into 5 ⁇ m sections with a microtome and fixed onto slides. Sections were dewaxed with xylenes, rehydrated with decreasing graded ethanol washes, and placed in PBS with 0.25% Triton X-100 (PBT). A microwave antigen-retrieval method was used to enhance antigenic reactivity of the antibodies as previously described (Zhou et al, 1996, J. Histochem. Cytochem. 44:1183- 1193). Sections were immunohistochemically stained with anti-BrdU monoclonal antibodies according to the manufacturer's instructions (Boehringer Mannheim).
- the number of BrdU positive nuclei per 1000 hepatocytes was counted and the mean BrdU positive cells and standard deviation (SD) were calculated using two regenerating liver samples from each time point.
- Regenerating livers from 2 month old (young) CD-I mice were examined and included as a comparison.
- the 2 month old livers display an S-phase peak at 40 hours after PHx ( Figure 2).
- a much smaller 40-hour S-phase peak was observed in the regenerating livers from 12 month old WT mice ( Figure 2).
- the regenerating livers of 12 month old TG mice exhibited a sharp S-phase peak at 40 hours similar to that observed in the 2 month old livers ( Figure 2).
- RNA from regenerating livers of wild type (WT) and transgenic (TG) mice was extracted 24, 32, 36, 40, and 44 hours post partial hepatectomy (PHx) by an acid guanidium thiocyanate-phenol-chloroform extraction method with RNA-STAT-60 (Tel- Test "B” Inc., Friendswood, TX).
- Antisense RNase protection probes for the human and mouse FoxMlB transgene and for mouse cyclophilin were generated as described (Ye et al, 1997, Mol Cell Biol 17:1626-1641; Wang et al, 2001, Hepatology 33:1404-1414).
- RNase protection assays were performed by hybridizing 20 to 40 ⁇ g of total liver RNA with ⁇ 32 P ⁇ UTP-labeled probes followed by digestion with RNase One, electrophoresis, and autoradiography as described previously (Ye et al, 1997, Mol. Cell Biol. Y ⁇ 626- 1641; Wang et al, 2001, Hepatology 33:1404-1414; Rausa et al, 2000, Mol. Cell Biol. 20:8264-8282). The X-ray films were scanned and the BioMax ID program (Eastman Kodak Co) was used to quantify expression levels, which were normalized to cyclophilin RNA levels.
- RNase protection probes for Cyclin Dl, Cyclin D3, Cyclin E, Cyclin Al, Cyclin A2, Cyclin Bl, Cyclin B2, and Cyclin F were purchased from Pharmingen (San Diego, CA) and probes for Cdc25B and p55Cdc were purchased from Clontech.
- RNase protection assays were performed for Cyclin genes using procedures described by the manufacturer and for other genes as described above on 20-40 ⁇ g of total liver RNA isolated from WT and TG mice 24, 32, 36, 40, and 44 hours after PHx.
- the expression of Cyclin Dl gene, which promotes S-phase was elevated in the aged TG mice at 36 to 40 hours post PHx, just before the initiation of hepatocyte DNA replication ( Figure 6).
- Cyclin F is essential for M-phase progression because it facilitates nuclear franslocation of the Cyclin B complexes (Kong et al, 2000, EMBO J. 19: 1378- 1388). M-phase progression is also mediated by Cdc25B, which activates the mitotic kinase cdkl/cyclin B (Sebastian et al, 1993, Proc. Natl. Acad. Sci. USA 90: 3521-3524; Trembley et al, 1996, Cell Growth Differ. 7: 903-916; Nilsson et al, 2000, Prog. Cell Cycle Res. 4: 107-114). Degradation of Cyclin proteins, a process necessary for completion of mitosis, is regulated by p55Cdc (Zachariae et al, 1999, Genes Dev. 13: 2039-2058).
- An RNase protection probe for p21 was received as a gift from Dr. Guy Adami (University of Illinois at Chicago). As above, approximately 2 x 10 5 cpm of each probe was hybridized at 45°C or 55°C to 20 ⁇ g of total RNA in a solution containing 20mM PIPES (pH6.4), 400mM NaCI, lmM EDTA and 80% formarnide overnight. After hybridization, samples were digested for lhr at 37°C by using 10 units per sample of RNase One enzyme according to the manufacturer's protocol (Promega, Madison, Wl).
- the RNase One protected fragments were electrophoresed on an 8% polyacrylamide-8M urea gel, followed by autoradiography. Quantitation of expression levels was determined with scanned X-ray films by using the BioMax ID program (Eastman Kodak, Rochester, NY). The cyclophilin hybridization signal was used for normalization control between different liver RNA samples. p21 mRNA levels were decreased during the Gl/S transition of the cell cycle in the old TG animals ( Figure 7, 32 to 40 hours post PHx).
- Paraffin embedded tissue samples from regenerating livers of 12 month old WT and TG mice dissected 24, 32, and 40 hours post PHx were sectioned with a microtome and prepared for immunohistochemical staining as described above. Sections were incubated with anti-p21 antibodies (Oncogene Science, Cambridge, MA) or anti- FoxMlB antibodies and detected using the ABC kit and DAB peroxidase substrate according to manufacturer's instructions (Vector Laboratories, Burlingame, CA).
- the number of p21 positive and FoxMlB positive hepatocytes per 1000 nuclei for each mouse liver was determined, and data from two mice for each time point were used to calculate the mean + standard deviation (SD) using the Analysis ToolPak i Macintosh Microsoft Excel 98.
- SD standard deviation
- p21 protein levels in the nuclei of regenerating liver of old TG mice were reduced compared with levels observed in the WT liver at 32 hours after PHx ( Figure 8).
- p21 nuclear protein levels in liver of TG mice were similar to those in WT liver ( Figure 8), which is consistent with the role of p21 in assembling the Cyclin D/cdk4/6 complex necessary for progression into S-phase (Cheng, et al, 1999, Embo J. 18:1571-1583).
- liver regeneration studies indicate that maintaining FoxMlB levels caused diminished expression of p53 and p21 Cipl proteins during the GI to S-phase transition in old-aged TTR FoxMlB TG mice, which is consistent with preventing reduced proliferating associated with an aging phenotype.
- Example 6 The effects of carbon tetrachloride induced liver injury on p21 levels in FoxMlB transgenic mice
- livers of WT and TG mice were removed 16, 20, 24, 28, 32, 36, and 40 hours after CC1 4 induced liver injury and examined by immunohistochemical staining as described above with anti-p21 antibodies.
- the number of p21 staining periportal hepatocytes present in regenerating TG hepatocytes was significantly decreased between 16 and 36 hours post CC1 4 liver injury compared with regenerating WT hepatocytes ( Figure 12A).
- RNA protection probes As described above, RNase protection assays were performed with Cyclin genes using RNA protection probes and a kit made by Pharmingen (San Diego, CA) following procedures recommended by the manufacturer.
- the ribosomal large subunit protein L32 and glyceraldehyde-3-phosphate dehydrogenase GAPDH signals were used to normalize Cyclin expression at the different time points during CC1 4 liver regeneration.
- Antisense RNA probes for mouse Cdc25a and Cdc25b were generated from Atlas cDNA plasmids purchased from Clontech (Paolo Alto, CA).
- RNase protection assays were performed in duplicate to examine the temporal expression patterns of the Cyclin genes in CC1 4 regenerating TG and WT livers.
- regenerating TG liver displayed early increases in expression of S-phase promoting Cyclin Dl and E genes between 24 to 36 hours after CC1 4 injury, corresponding to the Gl/S transition of the cell cycle.
- the CC1 regenerating TG livers displayed a more significant peak in CyclinDl expression compared with the regenerating WT livers ( Figure 13 A), suggesting that premature FoxMlB can induce Cyclin Dl expression and accelerate hepatocyte entry into S-phase.
- Cdc25a expression was sustained through the peak of TG hepatocyte DNA replication allowing for progression into S-phase tlirough activation of the CyclinDl/CDK4 complex.
- cdc25M2 Cdc25b
- Figure 13G Early activation f Cdc25b mRNA levels was seen in regenerating TG liver at 36 hours post CC1 4 injury, whereas its expression did not increase in WT regenerating liver until the 40 hour time point ( Figure 13G).
- Cdc25b regulated M-phase progression by activating the mitotic kinase Cdkl/cyclin B via dephosphorylation (Nilsson et al, 2000, Prog. Cell Cycle Res. 4: 107-114; Sebastian et al, 1993, Proc. Natl. Acad. Sci USA. 90: 3521-3524; Trembley et al, 1996, Cell Growth Differ. 7: 903-916).
- Early expression of Cdc25b promotes entry into mitosis by activating cdkl-cyclinB kinase activity, which is required to initiate and execute mitosis (division of duplicated chromosomes to daughter cells).
- adenovirus vectors expressing FoxMlB AdFoxMlB
- AdCon adenovirus as a control
- the adenovirus expressing FoxMlB was generated by subcloning the 2.7 lcB EcoKL-HindUI fragment of the human FoxMlB cDNA into the adenovirus shuttle vector pGEMCMV NEW (gift from J. R. Nevins, Duke University).
- adenovirus infects the liver after tail vein injection with minimal infection of other organs. Adenovirus is efficiently delivered to most cells throughout the liver parenchyma.
- Mouse tail vein injection of AdFoxMlB effectively increases in vivo hepatic expression of FoxMlB.
- PHx operation was performed two days after adenovirus infection to avoid the initial acute phase response to viral infection, which is completed within the first 36 hours following adenovirus infection.
- IP infraperitoneal injection of a phosphate buffered saline (PBS) solution containing 10 mg/mL BrdU (Sigma; 50 ⁇ g/g body weight) was administered two hours prior to harvesting the remnant regenerating liver, which were harvested at different intervals between 24 and 48 hours following surgery as previously described (Ye et al, 1999, Mol. Cell Biol. 19:8570-8580).
- PBS phosphate buffered saline
- the liver tissue was used to prepare total RNA or paraffin embedded for immunohistochemical staining of BrdU incorporation into DNA to monitor hepatocyte DNA replication as described previously.
- RNase protection assays were performed with the FoxMlB RNase protection probe as described above, and demonstrated that AdFoxMlB infection elicited a large increase in FoxMlB mRNA ( Figure 14A).
- RNase protection assays were performed on liver RNA isolated from regenerating livers of 2 month-old (young) mice. Significant increases in FoxMlB expression were observed in these samples between 36 and 44 hours following PHx high expression ' levels and were sustained for the duration of the liver regeneration experiment (Figure 14A).
- the adenovirus mediated increase in FoxMlB expression stimulated an earlier peak in hepatocyte DNA replication at 32 hours post PHx ( Figure 14B), which normally occurs at 40 hours post PHx in young Balb/c mice. Consistent with the role of FoxMlB in mediating progression into S-phase, regenerating liver infected with AdCon or mock infected lacked significant increase in hepatocyte DNA replication ( Figure 14B). Hepatocyte mitotic figures were examined and are represented graphically in Figure 14C. Adenovirus mediated increase in FoxMlB expression stimulated hepatocyte mitosis between 36 to 44 hours post PHx compared to regenerating livers of old mice infected with either control adenovirus or uninfected (Figure 14C).
- FoxMlB knockout mice die immediately after birth. Therefore, to examine the role of FoxMlB in adult liver regeneration conditional FoxMlB knockout mice were generated using a triple-LoxP FoxMlB targeting vector to create a "Floxed" FoxMlB targeted locus (see Figure 17 for schematic of vector). Cre recombinase mediated deletion of the FoxMl genomic sequences spanning the two LoxP sites removes the entire winged helix DNA binding domain and the C-terminal transcriptional activation domain, thereby preventing expression of functional FoxMl isoforms.
- homologous recombinants were identified by Southern blotting of ES cell genomic DNA.
- mice were injected with the ES cells comprising the "Floxed" (fl/+) FoxMlB targeted allele, and chimeric mice with germ line transmission were selected. Viable mice homozygous for the "Floxed" (fl/fl) FoxMlB targeted allele were generated. Mice either homozygous (fl/fl) or heterozygous (fl +) for the FoxMlB (fl) allele were verified by PCR amplification of mouse genomic DNA with primers that flanked the LoxP site.
- the FoxMlB fl/fl mice exhibited an 8-hour earlier expression of FoxMlB (at 32- hrs post PHx) in comparison to regenerating WT liver (Id). Because FoxMlB is predominantly regulated at the post-franscriptional level, the LoxP neo construct at the 3' end of the FoxMlB gene is presumably stabilizing its mRNA and thus enhancing induced FoxMlB levels. FoxMlB (fl/fl) mice exhibited a bifunctional S-phase peak in BrdU incorporation post PHx ( Figure 18A), while a significant reduction in DNA replication was observed in FoxMlB (-/-) regenerating livers ( Figure 18A). In addition, progression into mitosis was significantly reduced in regenerating hepatocytes of FoxMlB (-/-) mice as evidenced by the paucity of mitotic figures between 36 to 52 hours post PHx ( Figure 18B).
- mice Two-month old WT and TG CD-I mice were subjected to intraperitoneal (IP) injection of human growth hormone (Somafropin (Nordifropin), Novo Nordisk Pharmaceuticals Inc., Princeton, New Jersey; 5 ⁇ g per gram body weight) in vehicle buffer (2.2 mg glycine, 0.325 mg Disodium Phosphate Dihydrate (Na 2 HPO 4 , 2H 2 O), 0.275 mg Sodium Phosphate Dihydrate (NaH 2 PO 4 , 2H 2 O), and 11 mg Mannitol per mL of solution). Liver tissue was harvested at various time intervals (from 0 to 3 hours) following growth hormone adminisfration.
- IP intraperitoneal
- mice were injected with BrdU as described above and their livers were harvested at various time intervals between 24 and 48 hours post-PHx. Portions of the liver tissues were used to prepare total RNA for RNase protection assays. Liver tissues were processed and liver sections were stained with anti-BrdU antibodies as described above. BrdU-stained hepatocytes and visible mitotic figures were counted as previously described (Wang et al, 2001, Proc. Natl. Acad. Sci U.S.A. 98: 11468-11473). Regenerating hepatocyte DNA replication as measured by BrdU incorporation was similar to levels observed in regenerating livers of young (2 month-old) mice ( Figure 23A). Also, mitosis in the regenerating livers of old-aged mice was similar to mitosis in regenerating livers of young mice ( Figure 23B).
- FoxMlB expression measured by RNase protection assays was elevated in the regenerating livers of old mice that received periodic HGH injections during the regeneration process ( Figure 22).
- HGH treatments restored expression of the FoxMlB target gene Cdc25B phosphatase to levels found in young regenerating livers.
- Green fluorescent protein was fused in frame with FoxMlB amino acids 1 to 748 and the CMV promoter was used to drive the expression of the GFP -FoxMlB fusion protein.
- the CMV-GFP-FoxMlB expression vector was delivered in 2.5 mL of saline via mouse tail vein injection. The technique has previously demonstrated transduction of DNA expression plasmids in 10% of hepatocytes in vivo. Livers from one group of transduced animals were harvested and processed as described above. A second group of mice transduced with the CMV-GFP-FoxMlB expression vector were given IP injections of HGH 45 minutes before their livers were harvested. Liver sections from both groups were examined under fluorescent microscope.
- GFP-FoxMlB resided in the cytoplasm of quiescent hepatocytes from animals not treated with HGH (Figure 24C) while GFP- FoxMlB displayed nuclear localization in hepatocytes from the second group of mice ( Figure 24D).
- the pattern of nuclear localization of GFP-FoxMlB induced by HGH was similar to localization of the dysregulated GFP-FoxMlB-NLS.
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Application Number | Priority Date | Filing Date | Title |
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EP02769770A EP1499190A4 (en) | 2001-05-17 | 2002-05-17 | Methods for treating liver disease and liver damage with growth hormone and foxm1b |
JP2002588932A JP2005504010A (en) | 2001-05-17 | 2002-05-17 | Method for treating liver disease and liver injury using growth hormone and FOXM1B |
CA002447116A CA2447116A1 (en) | 2001-05-17 | 2002-05-17 | Methods for treating liver disease and liver damage with growth hormone and foxm1b |
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US29178901P | 2001-05-17 | 2001-05-17 | |
US60/291,789 | 2001-05-17 | ||
US30582101P | 2001-07-16 | 2001-07-16 | |
US60/305,821 | 2001-07-16 | ||
US31548401P | 2001-08-28 | 2001-08-28 | |
US60/315,484 | 2001-08-28 |
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Cited By (11)
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WO2004019761A2 (en) * | 2002-08-28 | 2004-03-11 | The Board Of Trustees Of The University Of Illinois | Methods of treating age-related defects and diseases |
WO2004100977A1 (en) * | 2003-03-25 | 2004-11-25 | The Board Of Trustees Of The University Of Illinois | Inhibition of tumor cell proliferation by foxm1b inhibitors |
EP1747284A2 (en) * | 2004-02-06 | 2007-01-31 | Wyeth | Diagnosis and therapeutics for cancer |
JP2007508019A (en) * | 2003-10-10 | 2007-04-05 | マルチセル テクノロジーズ、インコーポレイテッド | Use of cell lines to produce active therapeutic proteins |
EP1915904A1 (en) * | 2005-06-30 | 2008-04-30 | Hiroshima Industrial Promotion Organization | Method of treating mouse carrying human hepatocytes |
EP2233150A2 (en) | 2003-12-30 | 2010-09-29 | Augustinus Bader | Erythropoietin for use in the treatment of wounds or the transplantation of cells |
AU2005271723B2 (en) * | 2004-07-13 | 2010-12-16 | Asterias Biotherapeutics, Inc. | Medium for growing human embryonic stem cells |
AU2005289597B2 (en) * | 2004-09-28 | 2011-06-09 | Wicell Research Institute, Inc. | Cultivation of primate embryonic stem cells |
US8029980B2 (en) | 2006-09-29 | 2011-10-04 | The Board Of Trustees Of The University Of Illinois | Identification and use of agents that modulate oncogenic transcription agent activity |
CN103160575A (en) * | 2011-12-15 | 2013-06-19 | 王荣芳 | Application of SAA1 beta / beta homozygote genotype to prognosis and diagnosis of cirrhosis |
CN103160576A (en) * | 2011-12-15 | 2013-06-19 | 德赛诊断系统(上海)有限公司 | SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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ES2200646B1 (en) * | 2001-09-21 | 2005-05-01 | Fundacion Para La Investigacion Medica Aplicada | USE OF CARDIOTROPHIN IN HEPATIC DISEASES. |
EP1550715A1 (en) * | 2003-12-30 | 2005-07-06 | Bionethos Holding Gmbh | Method for the regeneration of tissue |
JP5025173B2 (en) * | 2005-06-30 | 2012-09-12 | 公益財団法人ひろしま産業振興機構 | Method for treating mice with human hepatocytes |
JP5555952B2 (en) | 2007-08-20 | 2014-07-23 | オンコセラピー・サイエンス株式会社 | FOXM1 peptide and drug containing the same |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5849686A (en) * | 1991-03-11 | 1998-12-15 | Creative Biomolecules, Inc. | Morphogen-induced liver regeneration |
-
2002
- 2002-05-17 EP EP02769770A patent/EP1499190A4/en not_active Withdrawn
- 2002-05-17 WO PCT/US2002/015873 patent/WO2002092013A2/en not_active Application Discontinuation
- 2002-05-17 US US10/151,587 patent/US20020187936A1/en not_active Abandoned
- 2002-05-17 CA CA002447116A patent/CA2447116A1/en not_active Abandoned
- 2002-05-17 JP JP2002588932A patent/JP2005504010A/en active Pending
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US5849686A (en) * | 1991-03-11 | 1998-12-15 | Creative Biomolecules, Inc. | Morphogen-induced liver regeneration |
Non-Patent Citations (2)
Title |
---|
ASAKAWA K. ET AL: 'Human growth hormone stimulates liver regeneration in rats' JOURNAL OF ENDOCRINOLOGY INVESTIGATIONS vol. 12, no. 5, May 1989, pages 343 - 347, XP008035348 * |
See also references of EP1499190A2 * |
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WO2004019761A2 (en) * | 2002-08-28 | 2004-03-11 | The Board Of Trustees Of The University Of Illinois | Methods of treating age-related defects and diseases |
WO2004019761A3 (en) * | 2002-08-28 | 2005-03-17 | Univ Illinois | Methods of treating age-related defects and diseases |
WO2004100977A1 (en) * | 2003-03-25 | 2004-11-25 | The Board Of Trustees Of The University Of Illinois | Inhibition of tumor cell proliferation by foxm1b inhibitors |
US8431522B2 (en) | 2003-03-25 | 2013-04-30 | The Board Of Trustees Of The University Of Illinois | Methods of inhibiting tumor cell proliferation |
US7799896B2 (en) | 2003-03-25 | 2010-09-21 | The Board Of Trustees Of The University Of Illinois | Methods of inhibiting tumor cell proliferation |
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US8029980B2 (en) | 2006-09-29 | 2011-10-04 | The Board Of Trustees Of The University Of Illinois | Identification and use of agents that modulate oncogenic transcription agent activity |
CN103160575A (en) * | 2011-12-15 | 2013-06-19 | 王荣芳 | Application of SAA1 beta / beta homozygote genotype to prognosis and diagnosis of cirrhosis |
CN103160576A (en) * | 2011-12-15 | 2013-06-19 | 德赛诊断系统(上海)有限公司 | SAA1 beta / beta homozygote genotype based detection method for prognosis and / or diagnosis of cirrhosis |
WO2013086809A1 (en) * | 2011-12-15 | 2013-06-20 | Wang Rongfang | Application of saa1β/β homozygote in liver cirrhosis prognosis diagnosis and liver cirrhosis diagnosis |
Also Published As
Publication number | Publication date |
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EP1499190A4 (en) | 2005-06-15 |
EP1499190A2 (en) | 2005-01-26 |
CA2447116A1 (en) | 2002-11-21 |
US20020187936A1 (en) | 2002-12-12 |
JP2005504010A (en) | 2005-02-10 |
WO2002092013A3 (en) | 2004-11-04 |
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