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WO2006105602A1 - Modeles animaux et cellules dotees d’un gene modifie codant pour une proteine associee a la transthyretine et applications de ceux-ci - Google Patents

Modeles animaux et cellules dotees d’un gene modifie codant pour une proteine associee a la transthyretine et applications de ceux-ci Download PDF

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WO2006105602A1
WO2006105602A1 PCT/AU2006/000456 AU2006000456W WO2006105602A1 WO 2006105602 A1 WO2006105602 A1 WO 2006105602A1 AU 2006000456 W AU2006000456 W AU 2006000456W WO 2006105602 A1 WO2006105602 A1 WO 2006105602A1
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trp
cell
plt2
polypeptide
gene
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William S. Stevenson
Andrew Roberts
Douglas J. Hilton
Warren S. Alexander
Andrienne A. Hilton
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Walter and Eliza Hall Institute of Medical Research
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Walter and Eliza Hall Institute of Medical Research
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
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    • A01K67/0276—Knock-out vertebrates
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/524—Thrombopoietin, i.e. C-MPL ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/00—Animals characterised by purpose
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    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • the present invention relates generally to compositions comprising agents that modulate cellular activity and in particular agents that modulate cancer (tumor) development and the development of haemopoietic lineages such as platelet production.
  • the present invention also provides animal models, cellular models and agents, drug targets, and methods for screening for and testing agents useful in the modulation of cellular activity and treatment and prevention of cancer.
  • Cancer is one widespread example of a disease or condition which is associated with uncontrolled cellular proliferation.
  • the most widely applied treatment for primary and metastatic cancer is a combination of surgery, radiotherapy and chemotherapy.
  • Some cancers have a viral aetiology, for example, hepatitis B and C are causal agents in liver cancer.
  • Chemotherapy affects rapidly dividing cells and a frequent side effect of chemotherapy is thrombocytopenia (low platelet numbers) due to destruction of cells including megakaryocytes and their progenitors in the bone marrow. Platelets are required for blood clotting and haemostasis.
  • Thrombocytopenia may also occur as an inherited disease, as a result of autoimmune disease or viral infection.
  • the steady state platelet count in humans is predominantly genetically determined (Buckley M. F. et al, Thromb. Haemost. , ⁇ 3:480-484, 2000).
  • the genes that are important in platelet production, release, circulation and clearance which collectively determine inter-individual variation in platelet counts are largely unknown.
  • Thrombopoietin is the principal growth factor that regulates steady state platelet production via the stimulation of megakaryocyte and megakaryocyte progenitor proliferation and differentiation through the cellular receptor c-Mpl (Bartley T. D. et al, Cell 77:1117-1124, 1994; de Sauvage F. J. et al., Nature 3(59:533-538, 1994; Kaushansky K. et al., Nature 3(59:568-571 1994; Lok S. et al., Nature 369:565-568, 1994).
  • TPO transcription appears to be constant, and the level of the cytokine in the body is thought to be regulated by the rate of receptor-mediated uptake and degradation by c-Mpl-expressing platelets and megakaryocytes (Stoffel R. et al., Blood ⁇ 7:567-573, 1996; Fielder P. J. et al, Blood 57:2154-2161, 1996).
  • c-Mpl-mediated TPO uptake by platelets results in an increased concentration of TPO available to the bone marrow to drive accelerated (or emergency) thrombopoiesis.
  • TPO regulation there is also some evidence that other physiological mechanisms may be important in TPO regulation.
  • thrombopoietin has been demonstrated to be normal or elevated in reactive thrombocytosis in some studies (Cerutti A. et al, Br. J. Haematol. 99:281-284, 1997) and there is evidence that TPO production may be differentially regulated in other sites such as the bone marrow stroma (Sungaran R. et al, Blood 59:101-107, 1997).
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>l (SEQ ID NO:1), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO:1 sequence identifiers ⁇ 400>l
  • SEQ ID NO:2 sequence identifiers
  • a summary of sequence identifiers is provided in Table 1.
  • a sequence listing is provided after the claims. Genes and other genetic material (eg mRNA, constructs etc) are represented in italics and their proteinaceous products are represented in non-italicised form. Thus, TRP polypeptide is the product of the TRP gene.
  • TRP or "TRP” is used to encompass all homologs, including orthologs and paralogs and variants in any species including, unless otherwise stated, TRP-PLT2.
  • the invention includes a human TRP homolog. Accordingly, homologous animal including avian and fish TRP (and TRP-PLT2 forms and their products are encompassed in the terms TRP and TRP. Mammalian TRP polypeptide is preferred.
  • TTR transthyretin
  • TRP transthyretin related protein
  • TTR family members are homotetrameric transport proteins which bind to and transport thyroxine and retinol- binding protein in the plasma. TTR is associated in man with amyloidosis which is a group of conditions characterized by amlyoid deposits in one or more tissues or organs of the body.
  • TRPs from various mammalian, amphibian, fish, plant, bacteria, parasitic, fungal and mycobacterial species have been described (see Figure 10)
  • the function and ligands of mammalian TRPs have not previously been elucidated and the present finding that a mammalian TRP/TRP is a tumor suppressor notably in the liver and modulates TPO-mediated cellular differentiation pathways provides new therapeutic and diagnostic applications inter alia for TRP and TRP, and variants, mimetics, analogues, binding partners, receptors, ligands, agonists and antagonists thereof.
  • the present invention pertains to the identification of a role for
  • TRP or TRP in modulating TPO activity are diseases and conditions that are associated with TPO activity.
  • a mutation in TRP leads to up regulation of TPO-dependent pathways, particularly in the liver or other tissues where
  • TPO/Mpl function to regulate cellular activity.
  • the present invention identifies and pertains to TRP as a tumor suppressor gene, notably in the liver.
  • genetically modified cells or non-human organisms comprising such cells are also provided by the present invention.
  • the cells comprise genetically modified TRP or produce modified TRP.
  • Such cells and animals are useful in in vivo or in vitro cellular model systems to identify and isolate, inter alia, modulators of TRP or TRP.
  • Such cells are also useful in cell therapy, including transplantation.
  • Genetically modified non-human organisms may be provided in the form of embryos for transplantation. Embryos are preferably maintained in a frozen state and may optionally be sold with instructions for use. Targeting constructs and genetically modified cells are also preferably maintained in a frozen state and may optionally be sold with instructions for use. All such cells are referred to herein as an in vivo or in vitro cellular model system.
  • the present invention provides a method for modulating cellular activity in a cell, tissue or subject comprising administering an agent which modulates the level or activity of TRP or TRP.
  • cellular activity is cancer development while in other embodiments, cellular activity is megakaryocyte differentiation or megakaryocyte progenitor proliferation or development and platelet production.
  • modulation is up regulation of the level or activity of TRP or TRP. In certain embodiments, down regulation of the level or activity of TRP or TRP will be undertaken.
  • the present invention provides compositions comprising agents, which modulate the level or activity of TRP or TRP.
  • agents are useful in modulating cellular activity, such as cell proliferation and differentiation.
  • down regulation of the level or activity of TRP causes megakaryocyte and megakaryocyte progenitor differentiation and platelet production.
  • agents which up regulate the level of TRP or TRP are proposed for lowering platelet levels or production in a subject, as required.
  • agents which modulate the level or activity of TRP or TRP comprise TRP or TRP or variants, derivatives, mimetics and analogs thereof.
  • the present invention contemplates administering TRP polypeptide or an agent from which TRP polypeptide is producible.
  • the agents are ligands, receptors, regulatory molecules and other binding partners, agonists or antagonists and variants, derivatives, mimetics and analogs thereof.
  • Such agents are identified inter alia through screening assays which are routinely performed by the skilled artisan using all or part of TRP or TRP.
  • the agents are used in the manufacture of medicaments for the treatment or prevention of cancer.
  • medicaments are suitable for the regulation of haematopoiesis. The agents may be used in conjunction with other cancer treatments to enhance their efficacy or reduce side-effects.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding all or a part of a TRP-PLT2 (also referred to as the long form of TRP) polypeptide having an amino acid sequence substantially as set out in SEQ ID NO: 4 ( Figure 9) or a sequence of amino acids having at least 60% sequence identity thereto.
  • the sequence of amino acids has at least 60% similarity to about 20 to 30 contiguous amino acids at the N-terminal end of the polypeptide.
  • the invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set out in SEQ ID NO: 3 or its complement or which has about 60% sequence identity to all or a part thereof or which hybridises thereto under conditions of low or medium stringency.
  • the nucleotide sequence has at least about 60% sequence identity in the 5' end portion having about 60 to 100 contiguous nucleotides and/or hybridises to this 5' portion under conditions of medium or high stringency.
  • Methods of risk assessment for cancer comprising screening for mutations in TRP or TRP. Any form of cancer is contemplated, although in one embodiment cancer in tissues such as the liver are particularly contemplated. BRIEF DESCRIPTION OF THE FIGURES
  • Figure 1 is a representation of data showing that plt2/plt2 animals display thrombocytosis and hepatomegaly.
  • A Mice homozygous for the plt2 mutation display thrombocytosis.
  • B Liver weight expressed as a proportion of total weight is increased in mice homozygous for the plt2 mutation.
  • C The F2 generation produced by intercrossing plt2/plt2 and MpI '1' animals produced a wide range of platelet counts that was bimodal in distribution. The platelet counts of wild-type mice (WT), mice with the c-Mpl knock-out allele (MpI) and mice inferred to be homozygous for the plt2 mutation based on liver size are included for comparison.
  • WT wild-type mice
  • MpI c-Mpl knock-out allele
  • Figure 2 is a representation of data showing that the pH2 Mutation acts extrinsically on the hemopoietic system.
  • A 8 week post-transplantation platelet counts from 9 wild-type and 9 plt2/plt2 recipient mice after they received bone marrow from either a plt2/plt2 (M) or wild- type donor (L " S).
  • B The high purity of the megakaryocyte suspension after purification is illustrated in this photomicrograph of a cytocentrifuge preparation stained with acetylcholinesterase and then counterstained with hematoxylin.
  • a control female mouse that did not receive a marrow transplant (J) has no evidence of the Sry allele and a semi-quantitative control of 1 part male megakaryocyte suspension mixed with 9 parts female suspension (m/f) demonstrates the presence of a signal at the Si ⁇ allele.
  • Figure 3 is a representation of data showing that thrombopoietin is present in excess in plt2/plt2 mice.
  • B Tpo transcript expression as measured by quantitative real-time PCR of reverse-transcribed RNA extracted from the liver of 4 wild-type mice and 4 plt2/plt2 animals.
  • the relative Tpo expression is presented as fold change in gene expression normalised to two separate housekeeping genes (Hmbs and Polr2a) relative to one of the control liver samples.
  • C Quantitative real-time PCR of Tpo transcript in a panel of tissues normalised to Polr2a and relative to a control kidney sample for 3 wild-type mice (w) and 3 plt2/plt2 mice (p).
  • D Thrombopoietin content of liver lysates was measured by ELISA for 8 wild-type mice and 8 plt2/plt2 mice.
  • Figure 4 is a representation of data showing that the plt2 locus lies between D7Wehi28 and D7Mit46 on mouse chromosome 7.
  • A Genetic linkage for thrombocytosis was observed at D7Mitl89 after a genome wide scan was performed on 89 N2 backcross mice with 162 SSLP markers.
  • B The presence of a homozygous C57BL/6 allele at D7Mit46 separates the thrombocytosis phenotype from the range of platelet counts displayed by the 282 N2 backcross mice.
  • telomeric to D7Mit71 were homozygous C57BL/6 ( ⁇ ) in the 50 backcross mice with the highest platelet counts (>2217xlO 9 /L) and heterozygous for the C57BL/6 and the Balb/c allele (LJ) in the 50 mice with the lowest platelet counts ( ⁇ 1272xlO 9 /L).
  • 15 intercross mice with high (>2194xlO 9 /L) or low platelet counts ( ⁇ 1296xlO 9 /L) were observed to have a recombination event between D7Wehi28 and D7Mit46. Progeny testing was performed on 2 mice that defined the telomeric end and one mouse that defined the centromeric end of the interval in the low platelet group (*).
  • Figure 6 is a graphical representation of nucleotide sequencing data showing the A to G mutation in the genetic region encoding TRP and causing a tyrosine to cysteine modification in plt2 animals.
  • total cellular RNA was isolated from liver samples from a wild-type and plt2/plt2 animal after they were snap-frozen into liquid nitrogen and homogenised in TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was then purified using the RNeasy kit (Qiagen GmbH, Germany) according to the manufacturer's protocol. First strand cDNA synthesis was performed using Superscript II Reverse Transcriptase (Invitrogen).
  • the cDNA was the used in a 35 cycle PCR reaction using a PFU polymerase (Promega) and primers specific for the predicted short and long versions of the AK00470 (TRP) gene (short 5'- acggactggctgatcactct-3', 5'- caaagcccatgatttgtgtg-3' and long 5'- tgcacagaccagagcttcag-3', 5'- caggcagatagatggctttctt-3').
  • TRP AK00470
  • Figure 7 is a graphical representation of data showing allelic discrimination between plt2 homozygous, heterozygous and wild type genotypes.
  • Genomic DNA was prepared from a tail biopsy taken from experimental mice at approximately 3 weeks of age. DNA was amplified using primers specific for exon 2 of AK004470 (77??) (5'- GGCACCTATAAGCTGTTCTTCGA-3' and 5'-ACCCTGACACTCACCTCTACATAG-S'). The PCR product was then identified as mutant or wild type by measuring specific fluorescence associated with the mutant or wild-type fluorescent-tagged oligonucleotide probe (wild-type: VIC- CAGAGCGCTACTGGAAA, plt2 mutant: FAM- AGCGCTGCTGGAAA). The PCR reaction and alleleic discrimination detection were performed on an ABI Prism 7900HT Sequence Detection System.
  • Figure 8 is a diagrammatic representation of the exon structure of the gene affected by the plt2 mutation.
  • Figure 9 is a representation of data showing the nucleotide and predicted amino acid sequence of TRP family members identified herein, (a) the nucleotide sequence of short wild- type form of mouse TRP as set forth in SEQ ID NO:1. (b) the nucleotide sequence of the long wild-type form of mouse TRP as set forth in SEQ ID NO.3. (c) the nucleotide sequence (cDNA) NCBI Accession No. AK00447 as set forth in SEQ ID NO: 5. (d) amino acid sequence of short wild-type mouse TRP protein as set forth in SEQ ID NO: 2. (e) amino acid sequence of long wild-type mouse TRP protein as set forth in SEQ ID NO: 4.
  • Figure 10 is a multiple sequence alignment TRP and TTR family members extracted from Figure 1 of Eneqvist T. et al, 2003 ⁇ supra) incorporated herein in its entirety.
  • Amino acid sequences of TTR-related proteins from 47 species aligned and compared with TTR sequences from 20 species (reviewed by Eneqvist T. et al., Amyloid: Int. J. Exp CHn. Invest. 5:149-168, 2001). Similarity was defined as amino acid substitutions within one of the following groups: FYW, IVLM, RK, DE, GA, TS, and NQ. Positions that are more than 80% identical are red, and those more than 80% similar are pink.
  • Residues displaying an identity of 80% or higher within the TRP family are shown in dark green, while those more than 80% similar are light green. Similarly, positions displaying above 80% identity and 80% similarity in the TTR family are shown in dark and light blue, respectively. Confirmed or predicted signal peptides are indicated with yellow background colouring. Numbering and secondary structure elements are based on human TTR and are shown as green arrows ⁇ -strands) and a red box ⁇ -helix). Residues lining the hormone-binding channel in TTR are marked with blue stars. The N-terminal sequences of TRPs (residues preceding 10 according to human TTR numbering) were not aligned, whereas these residues in TTR were aligned manually.
  • FIG 11 is diagrammatic representation of data as a phylogenetic tree of TRP and TTR members extracted from Figure 9 of Eneqvist, T. et al., 2003 ⁇ supra) incorporated herein in its entirety by reference. The tree was based on the multiple sequence alignment comprising 49 TRP sequences and 20 TTR sequences presented in Figure 10. TRP sequences from species where it is unclear if a functional TRP gene exists and those with predicted signal peptides are marked with (?) and (SP), respectively. The TTR family branch represented by vertebrates is also indicated.
  • Figure 12 is a graphical representation of data showing that plt2/plt2 mice have an aberrant liver gene expression profile. Liver gene expression from four plt2/plt2 mice were compared to four wildtype mice. The Y axis scale is logarithmic fold change in gene expression between plt2/plt2 replicates and wild-type replicates. The X axis is logarithmic change in signal intensity. The three statistically most differentially expressed genes between plt2/plt2 and wild-type mice are listed (Sult2a2, Ig ⁇ p2, Scd2).
  • Figure 13 is a diagrammatic representation of the nucleotide and amino acid sequence of long and short forms of TRP cloned from mouse liver that confirmed the presence of a mutation at Y98C.
  • A Nucleotide sequence demonstrating the open reading frame from the short transcript cloned from wild-type mouse liver. In plt2/plt2 mice, the short transcript is identical except at nucleotide 224, where an A to G point mutation has occurred (underlined).
  • B Nucleotide sequence demonstrating the open reading frame from the long transcript cloned from wild-type mouse liver. In plt2/plt2 mice, the long transcript is identical except at nucleotide 293, where an A to G point mutation has occurred (underlined).
  • FIG 14 is a photographical representation of data showing TRP transcript are expressed in a range of tissues.
  • A Reverse transcriptase PCR was performed on cDNA from a panel of tissues from wild-type and plt2/plt2 mice. Primers specific for the 5' UTR of TRP short and TRP long were selected to allow differentiation of short and long transcripts to be determined. At relatively low PCR cycle number, short and long TRP cDNA was present in samples derived from liver from both wild-type and plt2/plt2 mice. At higher cycle number, TRP cDNA was apparent from a range of organs. The presence of amplified cDNA was determined by staining with ethidium bromide after the PCR product had been electrophoresed on an agarose gel.
  • FIG. 15 is a photographical representation of data showing TRP is markedly reduced in plt2/plt2 hepatocytes.
  • A Western blot performed on protein lysates derived from a panel of tissues from wild-type and plt2/plt2 mice. A polyclonal antisera raised against TRP ( ⁇ TRP) recognises a single 15 kDa band in wild-type liver lysate that was absent in plt2/plt2 liver. Adequate protein loading was determined by staining protein lysates for heat shock protein 70 ( ⁇ HSP70).
  • FIG 16 is a diagrammatical representation of an alignment of amino acid sequence showing protein homology and hydrophobicity comparison between TRP and transthyretin (TTR).
  • TRP transthyretin
  • TRP is related to transthyretin and the transthyretin protein for humans and mice is included for comparison.
  • the tyrosine that is mutated by the plt2 mutation is marked (*).
  • TRP sequence from lower organisms was identified by a BLAST search (www.ncbi.nlm.nih.gov) with transcript from murine TRP. Nucleotide sequence was then used to align predicted proteins with Clustal W software (www.ebi.ac.uk) and the amino acid output has been colour-coded based on protein hydrophobicity using ASAD software (bioinf.wehi.edu.au/software/ASAD/).
  • Figure 17 is a diagrammatical representation of the predicted protein structure of wild- type and modified TRP.
  • Tyrosine-98 blue
  • the structure of murine TRP was modelled on the crystal structure of transthyretin from Spams aurata (Folli et al., FEBS Lett., 555:279- 284, 2003). This was performed using Swiss model software (Guex et al, Electrophoresis, 75:2714-2723, 1997) and then images were generated using the pymol program (http://www.pymol.org).
  • Table 1 provides a description of the SEQ ID NOs provided herein.
  • Table 2 provides an amino acid sub-classification.
  • Table 3 provides exemplary amino acid substitutions.
  • Table 4 provides a list of non-natural amino acids contemplated in the present invention.
  • Table 5 (including supplemental Table 5) provides the hematological profile of plt2/plt2 mice.
  • Table 6 provides a list of the SSLP markers used for the genome wide scan for linkage described in Example 7.
  • Table 7 tabulates the details of mice informative for further fine mapping of plt2 mutation described in Example 10.
  • Table 8 tabulates the details of primers and the method used (SSLP or SNP) for further fine mapping described in Example 10.
  • Table 9 provides the details of individual genes in the genetic interval between
  • CeleraSNP12 and Celera SNP17 based on the prediction by UCSC Genome Browser (May 2004 genome assembly). Exon and exon-intron boundaries were sequenced in two animals that were homozygous for C57BL/6 markers across the region of interest, one intercross animal that was Balb/C across the region of interest and one control C57BL/6 mouse as described in Example 11.
  • Table 10 provides nucleotide sequences of primers used in PCR and sequencing reactions for each of the genes sequences as described in Example 11.
  • Table 11 provides twenty most differentially expressed genes between plt2/plt2 and wild-type forms.
  • Table 12 provides the gene ontology groups for the most differentially expressed genes between plt2/plt2 and wild-type forms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention is predicated, in part, upon the identification and analysis of a pedigree of mice, called plt2, which displays recessive thrombocytosis associated with increased thrombopoietin production. Furthermore, homozygous plt2/plt2 mice develop tumors at increased frequency after natural aging or after radiation, compared to wild-type mice, identifying the encoding region as comprising a tumor suppressor. Using markers polymorphic between different inbred mouse strains the locus bearing this mutation was in accordance with the present invention mapped to an 8.6 Mb region of the telomeric end of chromosome 7.
  • TRP-PLT2 Transthyretin related protein
  • the present invention provides an isolated cell, or a non- human animal comprising such cells, wherein TRP or TRP is modified to effectively modulate its functional activity in the cell or animal compared to a non-modified animal of the same species.
  • Cells may be derived from human or non-human animal sources.
  • the term "derived” does not necessarily mean that the cells are directly obtained from a particular source.
  • Reference to a "cell” includes a system of cells such as a particular tissue or organ.
  • the modified cells are bacterial yeast or insect cells.
  • Viral constructs comprising modified TRP are also contemplated including bacteriophage.
  • modified includes genetically modified but encompasses non-genetic or epigenetic modifications to affect TRP or TRP activity by, for example, the administration of an agent such as, without limitation, an organic or inorganic chemical agents, antibody, enzyme, peptide, genetic, oligosaccharide, lipid or proteinaceous molecule to effectively modulate the functional activity of TRP or TRP.
  • an agent such as, without limitation, an organic or inorganic chemical agents, antibody, enzyme, peptide, genetic, oligosaccharide, lipid or proteinaceous molecule to effectively modulate the functional activity of TRP or TRP.
  • modulate and modulation includes completely or partially inhibiting or reducing or down regulating all or part of TRP or TRP functional activity and enhancing or up regulating all or part its functional activity. Functional activity may be modulated by, for example, modulating TRP or TRP binding capabilities or TRP transcriptional or translational activity, or its half-life.
  • TRP its functional activity may be modulated by, for example, modulating its binding capabilities, its half-life, location in a cell or membrane or its enzymatic capability.
  • TRP level or activity may be modulated by modulating TRP expression, transcript stability, post translational modification, and the activity of regulatory molecules such as promoters, enhancers and such like.
  • Reference to the "activity" or “functional activity” of TRP or TRP encompasses any relevant, measurable activity or characteristic of the molecule in proteinaceous or genetic form. Binding activity is a preferred activity, which may conveniently be assessed as described herein.
  • Such assays may be conveniently adapted for high throughput monitoring using, for example, chromatographic methods such as HPLC or thin layer chromatography.
  • TRP also binds to TPO-pathway members and binding assays are performed to determine whether this activity of TRP is modulated. Binding is conveniently assayed using antibodies to TRP or to other heterologous epitopes associated with the expressed polypeptide. Antibodies or antigen binding molecules specific to TRP are expressly contemplated. TRP is also required in some embodiments for platelet homeostasis. Accordingly, in vitro or in vivo assays may employ these outcomes as markers of TRP activity using, for example, the methods exemplified herein. For example, platelet levels or turnover may be measured using automated haemological analysis as described in Example 1.
  • TRP or TRP may be monitored using antibodies or other proteinaceous or genetic agents in a number of assays which are well known to those of skill in the art.
  • Antibodies may be used to detect TRP by Western Blotting, histochemical or ELISA procedures. As discussed herein below, such agents may also distinguish between active and inactive forms of the TRP or between long and short forms of TRP or TRP.
  • mutant forms of TRP or TRP are forms of TRP (found in a population of subjects) associated or linked with aberrant haematopoiesis, such as thrombocytopenia or thrombocytosis or a risk or presence of tumor development.
  • TRP may also be conveniently be detected using nucleic acid based assays well known in the art and as described herein.
  • low levels of active TRP may be produced as a result of mutations in TRP leading to altered expression levels, altered transcript stability or altered post-transcriptional or post-translational processing.
  • TRP activity may be monitored indirectly by monitoring RNA production and/or stability, or the levels of regulatory molecules such as enhancers and repressors.
  • genetically modified refers to changes at the genome level and refers herein to a cell or animal that contains within its genome a specific gene which has been altered. Alternations may be single base changes such as a point mutation or may comprise deletion of the entire gene such as by homologous recombination. Genetic modifications includes alterations to regulatory regions, insertions of further copies of endogenous or heterologous genes, insertions or substitutions with heterologous genes or genetic regions etc. Alterations include, therefore, single of multiple nucleic acid insertions, deletions, substitutions or combinations thereof.
  • Cells and animals which carry a mutant TRP allele or where one or both alleles are mutated or deleted can be used as model systems to study the effects of TRP in megakarocytopoiesis and/or to test for substances which have potential as therapeutic agents when these function are impaired.
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant TRP alleles (including those carrying loxP flanking sequences), usually from a second animal of the same species, as well as insertion of disrupted homologous genes.
  • the endogenous TRP gene of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques.
  • the cells may be isolated from individuals with TRP mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the TRP allele, as described above. After a test substance is applied to the cells, the phenotype of the cell is determined. Any trait of the cells can be assessed.
  • a genetically modified animal or cell includes animals or cells from a transgenic animal, a "knock in” or knock out” animal, conditional variants or other mutants or cells or animals susceptible to co-suppression, gene silencing or induction of RNAi.
  • targeting genetic constructs are initially used to generate the modified genetic sequences in the cell or organism.
  • Targeting constructs generally but not exclusively modify a target sequence by homologous recombination.
  • a modified genetic sequence may be introduced using artificial chromosomes.
  • Targeting or other constructs are produced and introduced into target cells using methods well known in the art which are described in molecular biology laboratory manuals such as, for example, in Sambrook, Molecular Cloning: A Laboratory Manual, 3 rd Edition, CSHLP, CSH, NY, 2001; Ausubel (Ed) Current Protocols in Molecular Biology, 5 th Edition, John Wiley & Sons, Inc, NY, 2002.
  • Targeting constructs may be introduced into cells by any method such as electroporation, viral mediated transfer or microinjection. Selection markers are generally employed to initially identify cells which have successfully incorporated the targeting construct.
  • ES cells embryonic stem cells
  • ES cells are conveniently obtained from pre-implantation embryos maintained in vitro (Robertson et ah, Nature, 322:445-448, 1986). Once correct targeting has been verified, modified cells are injected into the blastocyst or morula or other suitable developmental stage, to generate a chimeric organism. Alternatively, modified cells are allowed to aggregate with dissociated embryonic cells to form aggregation chimera. The chimeric organism is then implanted into a suitable female foster organism and the embryo allowed to develop to term. Chimeric progeny are bred to obtain offspring in which the genome of each cell contains the nucleotide sequences conferred by the targeting construct. Genetically modified organism may comprise a heterozygous modification or alternatively both alleles may be affected.
  • Another aspect of the present invention provides cells or animal comprising one, two or more genes or regions which are modified.
  • the genetically modified cells or animals may comprise a gene capable of functioning as a marker for detection of modified cells.
  • the instant animals may be bred with other transgenic or mutant non- human animals to provide progeny some of which exhibit one or both traits or a modified trait/s. Chimeric animals are also contemplated.
  • polynucleotide include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g.
  • phosphorothioates phosphorodithioates, etc.
  • pendent moieties e.g. polypeptides
  • intercalators e.g. acridine, psoralen, etc.
  • chelators e.g. acridine, psoralen, etc.
  • alkylators e.g. ⁇ - anomeric nucleic acids, etc.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the present invention further contemplates recombinant nucleic acids including a recombinant construct comprising all or part of TRP.
  • the recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosonal DNA of the host cell.
  • Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic or synthetic origin which, by virtue of its origin or manipulation: (i) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (ii) is linked to a polynucleotide other than that to which it is linked in nature; or (iii) does not occur in nature.
  • nucleic acids according to the invention include RNA, reference to the sequence shown should be construed as reference to the RNA equivalent with U substituted for T.
  • Such constructs are useful to elevate TRP levels or to down-regulate TRP levels such as via antisense means or RNAi- mediated gene silencing. As will be well known to those of skill in the art, such constructs are also useful in generating animal models carrying a modified TRP allele. Genetically modified cells or non-human organisms may be provided in the form of cells or embryos for transplantation. Cells and embryos are preferably maintained in a frozen state and may optionally be distributed or sold with instructions for use.
  • the present invention provides a genetically modified cell, or non- human animal comprising such cells, wherein a TRP gene is modified and the cell or animal produces a substantially enhanced level or activity of TRP, or substantially reduced level or activity of TRP compared to a non-modified animal of the same species, or is substantially incapable of producing TRP.
  • the genetically modified cells and non-human animals may be a non-human primate, livestock animal, companion animal, laboratory test animal, captive wild animal, reptile, amphibian, fish, bird or other organism.
  • the genetically modified non-human animal is a murine animal.
  • the modified cell or non-human animal is genetically modified and produces a substantially reduced level of TRP, or is substantially incapable of producing TRP, or produces TRP having substantially reduced or no activity.
  • a TRP gene is modified. Modification may be in one or both alleles and may optionally be within a regulatory region of the gene.
  • the genetic modification resulting in a cell or animal capable of exhibiting a modified level or activity of TRP comprises genetic modification outside the TRP gene to cause expression of genetic or proteinaceous molecules which effectively modulate the activity of TRP or TRP.
  • the modified cell or non-human animal is genetically modified and substantially overproduces TRP having normal or altered activity relative to an unmodified cell or animal of the same species.
  • the invention provides a method of screening for or testing an agent capable of complementing a phenotype shown by a cell or non-human animal comprising a modified TRP or TRP and exhibiting a substantially modified level or activity of TRP.
  • the cell or animal is contacted with the agent and its effect on the phenotype of the cell or animal determined.
  • the method comprises screening for mutants which exhibit a complementing phenotype and then mapping and identifying the modifying gene.
  • the method comprises screening for agents which enhance the level or activity of TRP in a normal or modified cell.
  • the subject invention provides a use of a cell or non-human animal comprising a modified TRP or TRP and exhibiting a substantially reduced level or activity of TRP in screening for or testing agents for use in the treatment or prophylaxis of haematological disorders such as thrombocytopenia and/or cancer.
  • substantially refers to a statistically significant change having a phenotypic or physiological effect.
  • substantially enhanced level or activity refers to significantly greater amounts having a phenotype or physiological effect.
  • substantially reduced level or activity refers to zero amounts to about 90% lower amounts compared to amounts detectable in a non- modified animal or cell.
  • a substantially reduced level or activity of TRP or TRP is conveniently assessed in terms of a percent reduction relative to normal cells or animals or pre-treatment/pre- administration.
  • a substantial reduction is one which results in detectable thrombocytosis in a subject or aberrant megakaryocytosis or cancer development.
  • the reduction is at least 20% compared to normal animals, more preferably about 25%, still more preferably at least about 30% reduction, more preferably at least about 40% reduction in TRP or TRP level or activity.
  • the reduction may of course be complete loss of TRP activity in a cell or animal.
  • a "modified" level or activity includes enhanced levels of TRP activity relative to pre- treatment levels and may equate to or exceed the level or activity of TRP or TRP detectable in healthy subjects or subjects unlikely to develop thrombocytopenia or cancer.
  • the present invention further provides a method for identifying agents useful in the treatment or prophylaxis of cancer or haematological disorders such as thrombocytopenia comprising screening compounds for their ability to modulate the functional activity of TRP or TRP.
  • the present invention provides a composition comprising an agent which down regulates the level or activity of TRP or TRP in a cell for use in modulating platelet production.
  • the present invention provides a composition comprising an agent which down regulates the level or activity of TRP in a subject for use in modulating platelet numbers in circulation.
  • the modulatory agents of the present invention may be chemical agents such as a synthetic or recombinant molecules, polypeptides, peptides or proteins, lipids, glycoproteins or other naturally or non-naturally occurring molecules or analogs thereof.
  • genetic agents such as DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs )) ribozymes, aptamers, DNAzymes or other ribonuclease- type complexes may be employed.
  • DNA gDNA, cDNA
  • RNA sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), micro RNAs (miRNAs), small nucleolar
  • TRP Treactive protein
  • antibodies or peptides, oligosaccharides, peptidomimetics or analogs and other such biomolecules may be conveniently employed.
  • genetic mechanism are used to indirectly modulate the activity of TRP.
  • various strategies are well documented and include mechanisms for pre or post-transcriptional silencing. The expression of antisense molecules or co-suppression or RNAi or siRNA or DNA strategies are particularly contemplated.
  • RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, Clin. Chem., 45(9): 1628- 1650, 1999; Morris et al, Proc. Natl. Acad. ScI, USA, 95(6):2902-2907, 1998).
  • Aptamers are nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing. Aptamers are described, for example, in U.S. Pat. Nos. 5,475,096; 5,270,163; 5,589,332; 5,589,332; and 5,741,679.
  • agents which modulate the level or activity of TRP or TRP may be derived from TRP or TRP or be variants thereof. Alternatively, they may be identified in in vitro or in vivo screens. Natural products, combinatorial, synthetic/peptide/polypeptide or protein libraries or phage display technology are all available to screening for such agents. Natural products include those from coral, soil, plant, or the ocean or antarctic environments.
  • the agent to be tested is contacted with a system comprising TRP or TRP. Then, the following may be assayed for: the presence of a complex between the agent and TRP or TRP, a change in the activity of the target, or a change in the level of activity of an indicator of the activity of the target.
  • Competitive binding assays and other high throughput screening methods are well known in the art and are described for example in International Publication Nos. WO 84/03564 and WO 97/02048).
  • Antisense or other inhibitory or gene silencing polynucleotide sequences are useful agents in preventing or reducing the expression of TRP.
  • morpholines may be used as described by Summerton and Weller (Antisense and Nucleic acid Drug Development:! 187-195, 1997).
  • Antisense molecules may interfere with any function of nucleic acid molecule.
  • the functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise.
  • RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
  • One preferred result of such interference with target nucleic acid function is modulation of the expression of the TRP gene.
  • antisense compound is a single-stranded antisense oligonucleotide
  • double-stranded RNA (dsRNA) molecules has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals. Double stranded DNA molecules are also usefully employed.
  • oligomeric compound refers to a polymer or oligomer comprising a plurality of monomeric units.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • mimetics chimeras, analogs and homologs thereof.
  • This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly.
  • Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
  • oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well,
  • High-throughput screening protocols are well used such as those described in Geysen (International Publication No. WO 84/03564). Briefly, large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods. A similar method involving peptide synthesis on beads, which forms a peptide library in which each bead is an individual library member, is described in U.S. Patent No. 4,631,211 and a related method is described in International Publication No. WO 92/00091.
  • a significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide or electrophoretic tag, so as to facilitate identification of the amino acid sequence of each library member.
  • a unique identifier tag such as an oligonucleotide or electrophoretic tag
  • Another chemical synthesis screening method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface wherein each unique peptide sequence is at a discrete, predefined location in the array. The identity of each library member is determined by its spatial location in the array.
  • Such systems in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage, are useful for creating libraries of antibody fragments (and the nucleotide sequences that encoding them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen.
  • the nucleotide sequences encoding the V H and V L regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E. coli and the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or p VIII).
  • antibody fragments are displayed externally on lambda phage capsids (phage bodies).
  • phage-based display systems An advantage of phage-based display systems is that selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold, Science, 249:505, 1990; Ellington and Szostak, Nature, 346:818, 1990).
  • Tuerk and Gold Science, 249:505, 1990; Ellington and Szostak, Nature, 346:818, 1990.
  • a similar technique may be used to identify DNA sequences which bind to carbohydrate, polysaccharide, proteoglycan, glucosaminoglycans and the like.
  • in vitro translation can be used to synthesize polypeptides as a method for generating large libraries.
  • These methods which generally comprise stabilized polysome complexes, are described further in International Publication No. WO88/08453.
  • Alternative display systems which are not phage- based, such as those disclosed in International Publication Nos. WO 95/22625 and WO 95/11922 (Affymax) use the polysomes to display polypeptides for selection.
  • the genetic agents or compositions in accordance with this invention preferably comprise from about 8 to about 80 nucleobases or greater (i.e. from about 8 to about 80 or greater linked nucleosides).
  • the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.
  • the agents or compositions of the present invention may be TRP or parts thereof, or TRP or parts thereof or complementary forms or molecules derived or designed from TRP or TRP.
  • the present invention provides a composition comprising TRP or TRP (ie the molecule in genetic or proteinaceous form) or a functional variant, functionally equivalent derivative, mimetic, analog or homolog thereof which substantially enhances the activity of TRP or TRP.
  • the composition effectively modulates megakaryocytopoiesis and/or cancer development.
  • the present invention provides a composition comprising TRP or TRP (ie the molecule in genetic or proteinaceous form) or a functional variant, functionally equivalent derivative, mimetic, analog or homolog thereof which substantially enhances the activity of TRP or TRP for use in modulating megakaryocytopoiesis and/or cancer development.
  • the present invention provides a composition comprising TRP or TRP or a functional variant, functionally equivalent derivative, mimetic, analog or homolog thereof which substantially enhances the activity of TRP or TRP in a subject for use in the treatment or prophylaxis of cancer.
  • Particularly preferred compositions are pharmaceutical compositions comprising TRP or TRP or a functional part or functionally equivalent derivative thereof capable of enhancing TRP level or activity suitable for use in the treatment or prophylaxis of cancer.
  • subject includes, without limitation, humans and non-human primates, livestock animals, companion animals, laboratory test animals, captive wild animals, reptiles and amphibians, fish, birds and any other organism.
  • a subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, subject, animal, host or recipient.
  • compositions and terms such as “agent”, “medicament”, “active” and “drug” are used interchangeably herein to refer to a chemical compound or cellular composition which induces a desired pharmacological and/or physiological effect.
  • the terms encompass pharmaceutically acceptable and pharmacologically active ingredients including but not limited to salts, esters, amides, pro-drugs, active metabolites, analogs and the like.
  • the term includes genetic and proteinaceous or lipid molecules or analogs thereof as well as cellular compositions as previously mentioned.
  • the instant compounds and compositions are for the manufacture of a medicament for the treatment and/or prevention of thrombocytopenia and/or cancer.
  • agents which modulate TRP polypeptide activity in a cell are useful reagents in vitro cell cultures or maintenance.
  • the present invention extends to cellular compositions including genetically modified stem cells which are capable of regenerating tissues and/or organs, such as the liver, of an animal subject in situ or in vivo.
  • Stem cells or stem cell-like cells are preferably multipotent or pluripotent.
  • Cells may be directly derived from humans however, totipotent embryonic stem cells from human embryos are not encompassed.
  • Other cellular compositions comprise vectors such as viral vectors for delivery of nucleic acid constructs as described later herein.
  • the terms functional form or variant, functionally equivalent derivative or homolog include molecules which hybridize to TRP or a complementary form thereof over all or part of the genetic molecule under conditions of low stringency at a defined temperature or range of conditions, or which have about 60% or greater sequence identity to the nucleotide sequence defining TRP.
  • the terms functional form or variant, functionally equivalent derivative or homolog include molecules which hybridize to TRP or a complementary form thereof over all or part of the genetic molecule under conditions of medium or high stringency at a defined temperature or range of conditions, or which have about 60% to 80% sequence identity to the nucleotide sequence defining TRP.
  • TRP nucleotide sequences include those comprising nucleotide sequences set forth in SEQ ID NO: 1 (mouse TRP mRNA short form) and SEQ ID NO: 3 (mouse TRP mRNA long form) or their complements.
  • the terms “functional form” or “variant”, “functionally equivalent derivatives” or “homologs” include polypeptides comprising a sequence of amino acids having about 60% sequence identity to the TRP polypeptide of SEQ ID NO: 2 or 4.
  • Functional or active forms or variants of TRP polypeptide are selected among variants which retain functional activity, for example, in regulating TPO levels, platelet levels or liver homeostasis.
  • functional forms retain functional domains such as a hormone binding domain, a retinol binding domain or structural domains such as are important in forming a barrel structure or a highly ordered helix structure as shown in Figure 17.
  • functional forms retain the ability to modulate levels of proteins or their encoding genetic sequences such as those involved in lipid metabolism, protein metabolism, biotransformation, other metabolism, cell cycle control, acute phase response and blood co- aggulation and proteolysis and peptidolysis.
  • TRP polypeptide are capable of regulating sulphotransferases, insulin-like growth factor binding proteins, stearoyl-Coenzyme A desaturase, galactose binding lectin, cytochrome P450 and other molecules such as those set forth in Table 11 and Table 12.
  • Exemplary TRP amino acid sequences include those comprising sequences set forth in
  • the present invention provides an isolated TRP-PLT2 polypeptide comprising an amino acid sequence substantially as set out in SEQ ID NO: 4 or a functional variant thereof.
  • the present invention provides an isolated nucleic acid molecule comprising or complementary to a nucleotide sequence encoding a TRP-PLT2 polypeptide having an amino acid sequence substantially as set out in SEQ ID NO: 4 or a functional variant thereof.
  • the amino acid sequence comprises about 60% or greter sequence identity to about 20 to 30 contiguous amino acid residues at the N- terminal region of the polypeptide.
  • a nucleic acid molecule comprising a sequence of nucleotides substantially as set out in SEQ ID NO: 3 or a functional variant thereof or their complementary forms.
  • the nucleic acid molecules have about 60% or greater sequence identity to SEQ ID NO: 3 or a complementary form thereof over at least a 5'-terminal portion comprising about 60 to 100 contiguous nucleotides.
  • low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-3O 0 C to about 42 0 C.
  • the temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as "medium stringency", which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner et al., Eur. J. Biochem. 46: 83, 1974).
  • Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20 0 C to 65 0 C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • the nucleic acid molecule encoding a TRP polypeptide comprise a sequence of nucleotides as set forth in SEQ ID NO: 3 or which hybridises thereto or to a complementary form thereof under medium or high stringency hybridisation conditions.
  • the hybridisation region is about 12 to about 80 nucleobases or greater in length.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity and amino acid sequence comparisons are made at the level of similarity.
  • the percent similarity between a particular amino sequence and a reference sequence is about 30% or about 65% or about 70% or about 80% or about 85% or more preferably about 90% similarity or greater as about 95%, 96%, 97%, 98%, 99% or greater. Percent similarities between 30% and 100% are encompassed.
  • the precent identity between a particular nucleotide sequence and a reference sequence is about 30%, or 65% or about 70% or about 80% or about 85% or more preferably about 90% similarity or greater as about 95%, 96%, 97%, 98%, 99% or greater. Percent identities between 60 and 100% are encompassed.
  • a "reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al
  • FASTA Altschul et al
  • TFASTA TFASTA
  • a percentage of sequence identity between nucleotide sequences is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g. A, T, C, G, I
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity for amino acid sequences.
  • the present invention contemplates the use of full-length TRP or biologically active portions of those polypeptides.
  • biologically active TRP portions comprise one or more binding domain .
  • a biologically active portion of a full-length polypeptide can be a polypeptide which is, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, or more amino acid residues in length.
  • the TRP polypeptide of the present invention includes all biologically active or functionally naturally occurring forms of TRP as well as biologically active portions thereof and variants or derivatives of these.
  • variants include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein (e.g., wound- treating activity). Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native TRP polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a TRP polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • a TRP polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a TRP polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA, 52:488-492, 1985), Kunkel et al, ⁇ Methods in Enzymol, 754:367-382, 1987), U.S. Pat. No. 4,873,192, Watson et al.
  • Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify TRP polypeptide variants (Arkin et al., Proc. Natl. Acad. Sci. USA, 59:7811-7815, 1992; Delgrave et al., Protein Engineering, (5:327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.
  • Variant TRP polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to the parent TRP amino acid sequence.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:
  • Acidic The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having an acidic side chain include glutamic acid and aspartic acid.
  • the residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having a basic side chain include arginine, lysine and histidine.
  • the residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
  • amino acids having acidic or basic side chains i.e., glutamic acid, aspartic acid, arginine, lysine and histidine.
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan. As shown herein, loss of tyrosine from the ⁇ -helix of TRP polypeptide profoundly alters its ability to be active in vivo.
  • Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
  • proline This description also characterises certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity.
  • "small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not.
  • Amino acids having a small side chain include glycine, serine, alanine and threonine.
  • the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains.
  • the structure of proline differs from all the other naturally- occurring amino acids in that its side chain is bonded to the nitrogen of the ⁇ -amino group, as well as the ⁇ -carbon.
  • amino acid similarity matrices include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.
  • the degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.
  • Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large.
  • the residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not.
  • Small residues are, of course, always nonaromatic.
  • amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 2.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
  • the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
  • the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
  • the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).
  • a predicted non-essential amino acid residue in a TRP polypeptide is typically replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a TRP polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.
  • the present invention also contemplates variants of the naturally- occurring TRP polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues.
  • variants will display at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identity to a reference TRP polypeptide sequence as, for example, set forth in any one of SEQ ID NO: 2 or 4.
  • TRP polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to TRP polynucleotide sequences, or the non-coding strand thereof.
  • variant polypeptides differ from an TRP sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s).
  • variant polypeptides differ from the corresponding sequence in any one of SEQ ID NO: 2 or 4 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. If this comparison requires alignment the sequences should be aligned for maximum similarity. (“Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities.
  • the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type.
  • An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of an TRP polypeptide of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.
  • a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a TRP polypeptide as, for example, set forth in SEQ ID NO: 2 or 4, and has the activity of that TRP polypeptide.
  • TRP polypeptides may be prepared by any suitable procedure known to those of skill in the art.
  • the polypeptides may be prepared by a procedure including the steps of: (a) preparing a chimeric construct comprising a nucleotide sequence that encodes at least a portion of a TRP polypeptide and that is operably linked to one or more regulatory elements; (b) introducing the chimeric construct into a host cell; (c) culturing the host cell to express the TRP polypeptide; and (d) isolating the TRP polypeptide from the host cell.
  • the nucleotide sequence encodes at least a portion of the sequence set forth in SEQ ID NO: 2 or 4, or a variant thereof.
  • TRP polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook, et al., (1989, supra), in particular Sections 16 and 17; Ausubel et al., (1994, supra), in particular Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
  • the TRP polypeptides may be synthesised by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et al., (Science, 269:202, 1995).
  • derivatives or the plural “derivatives” and “variant” or “variants” are used interchangeable and, whether in relation to genetic or proteinaceous molecules, include as appropriate parts, mutants, fragments, and analogues as well as hybrid, chimeric or fusion molecules and glycosylation variants.
  • Particularly useful derivatives retain the functional activity of the parent molecule and comprise single or multiple amino acid substitutions, deletions and/or additions to the TRP amino acid sequence.
  • the derivatives Preferably, have functional activity or alternatively, modulate TRP functional activity.
  • modulate includes up modulate or up regulate and down modulate or down regulate.
  • TRP is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides.
  • This definition includes all sizes in the range of 10 to 35 as well as greater than 35 nucleotides.
  • this definition includes nucleic acids of 12,15, 20, 25, 40, 60, 100, 200, 500 nucleotides of nucleic acid molecules having any number of nucleotides between 500 and the number shown in SEQ ID NO: 1 or SEQ ID NO:3 or a complementary form thereof.
  • SEQ ID NO: 1 or SEQ ID NO:3 or a complementary form thereof.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage without the loss of other functions or properties.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved.
  • Preferred substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and tyrosine, phenylalanine.
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with the TRP polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence and its underlying DNA coding sequence and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. MoI. Biol. 157: 105-132, 1982).
  • homolog or “homologs” refers herein broadly to functionally or structurally related molecules including those from other species.
  • peptide mimetic includes carbohydrate, nucleic acid or peptide mimetics and it intended to refer to a substance which has conformational features allowing the substance to perform as a functional analog.
  • a peptide mimetic may be a peptide containing molecule that mimic elements of protein secondary structure (Johnson et al, "Peptide Turn Mimetics” in Biotechnology and Pharmacy, Pezzuto et ah, eds Chapman and Hall, New York, 1993).
  • Peptide mimetics may be identified by screening random peptides libraries such as phage display libraries for peptide molecules which mimic the functional activity of TRP. Alternatively, mimetic design, synthesis and testing is employed.
  • Nucleic acid mimetics include, for example, RNA analogs containing N3' ⁇ P5' phosphoramidate internucleotide linkages which replace the naturally occurring RNA O3' ⁇ P5' phosphodiester groups.
  • Enzyme mimetics include catalytic antibodies or their encoding sequences, which may also be humanised.
  • Peptide or non-peptide mimetics can be developed as functional analogues of TRP by identifying those residues of the target molecule which are important for function. Modelling can be used to design molecules which interact with the target molecule and which have improved pharmacological properties. Rational drug design permits the production of structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g.
  • agonists, antagonists, inhibitors or enhancers in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson (Bio/Technology 9: 19-21, 1991).
  • one first determines the three-dimensional structure of a protein of interest by x-ray crystallography, by computer modeling or most typically, by a combination of approaches.
  • Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins.
  • target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol. 202: 2699-2705, 1991).
  • an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide. It is also possible to isolate a target-specific antibody, selected by a functional assay and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based.
  • anti-idiotypic antibodies anti-ids
  • the binding site of the anti-ids would be expected to be an analog of the original receptor.
  • the anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.
  • Analogues preferably have enhanced stability and activity. They may also be designed in order to have an enhanced ability to cross biological membranes or to interact with only specific substrates. Thus, analogs may retain some functional attributes of the parent molecule but may posses a modified specificity or be able to perform new functions useful in the present context i.e., for administration to a subject.
  • Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 4.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids and the introduction of double bonds between Q x and C ⁇ atoms of amino acids.
  • the small or large chemicals, polypeptides, nucleic acids, antibodies, peptides, chemical analogs, or mimetics of the present invention can be formulated in pharmaceutic compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18 th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.
  • the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being administered intrathecal Iy, they may also be dissolved in cerebrospinal fluid.
  • the active agent is preferably administered in a therapeutically effective amount.
  • the actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, (supra).
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.
  • these agents could be produced in the target cell, e.g. in a viral vector such as those described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.
  • the vector could be targeted to the target cells or expression of expression products could be limited to specific cells, stages of development or cell cycle stages.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent.
  • the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 73 IA and International Patent Publication No. WO 90/07936.
  • the cells of a subject exhibiting modified TRP genetic sequences may be treated with a genetic composition comprising TRP.
  • TRP wild type or enhanced TRP function to a cell which carries a mutant or altered form of TRP should in this situation complement the deficiency and result in reduced cancer or hepatomegaly development in the subject.
  • the TRP allele may be introduced into a cell in a vector such that the gene remains extrachromosomally. Alternatively, artificial chromosomes may be used.
  • the vector may combine with the host genome and be expressed therefrom.
  • Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (In: Therapy for Genetic Disease, T. Friedman, Ed., Oxford University Press, pp. 105-121, 1991) or Culver ⁇ Gene Therapy: A Primer for Physicians, 2 nd Ed., Mary Ann Liebert, 1996).
  • Suitable vectors are known, such as disclosed in U.S. Patent No. 5,252,479, International Patent Publication No. WO 93/07282 and U.S. Patent No. 5,691,198.
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non-viral transfer methods.
  • viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses (e.g. SV40, Madzak et al, J, Gen. Virol, 73:1533-1536, 1992), adenovirus (Berkner, Curr. Top. Microbiol. Immunol, 158:39- 66, 1992; Berkner et al, BioTechniques, 6:616-629, 1988; Gorziglia and Kapikian, J. Virol, 66:4407-4412, 1992; Quantin et al, Proc. Natl. Acad. Sci.
  • papovaviruses e.g. SV40, Madzak et al, J, Gen. Virol, 73:1533-1536, 1992
  • adenovirus e.g. SV40, Madzak et al, J, Gen. Virol, 73:1533-1536, 1992
  • adenovirus e.g
  • Non-viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer.
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery.
  • plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged.
  • Liposome/DNA complexes are also capable of mediating direct in vivo gene transfer.
  • Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein.
  • the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes TRP, expression will produce TRP. If the polynucleotide encodes a sense or antisense polynucleotide or a ribozyme or DNAzyme, expression will produce the sense or antisense polynucleotide or ribozyme or DNAzyme. Thus, in this context, expression does not require that a protein product be synthesized.
  • the vector also contains a promoter functional in eukaryotic cells.
  • the cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters are routinely determined.
  • Receptor-mediated gene transfer may be achieved by conjugation of DNA to a protein ligand via polylysine.
  • Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. Receptors on the surface of liver cells may be advantageously targeted.
  • These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs.
  • co-infection with adenovirus can be included to disrupt endosome function.
  • patients who carry an aberrant TRP allele are treated with a gene delivery vehicle such that some or all of their cells receive at least one additional copy of a functional normal TRP allele.
  • a gene delivery vehicle such that some or all of their cells receive at least one additional copy of a functional normal TRP allele.
  • peptides or mimetics or other functional analogues which have TRP activity can be supplied to cells which carry aberrant TRP alleles.
  • Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • synthetic chemistry techniques can be employed to synthesize the instant active molecules. Active molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Supply of molecules with TRP activity should lead to platelet homeostasis and a reduced risk of developing cancer and particularly liver cancer.
  • Scanning methods include sequencing, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP and rSSCP, REF-SSCP), chemical cleavage methods such as CCM, ECM, DHPLC and MALDI-TOF MS and DNA chip technology.
  • DGGE denaturing gradient gel electrophoresis
  • SSCP and rSSCP single-stranded conformational polymorphism
  • REF-SSCP REF-SSCP
  • chemical cleavage methods such as CCM, ECM, DHPLC and MALDI-TOF MS and DNA chip technology.
  • Specific methods to screen for pre-determined mutations include allele specific oligonucleotides (ASO), allele specific amplification, competitive oligonucleotide priming, oligonucleotide ligation assay, base- specific primer extension, dot blot assays and RFLP-PCR.
  • ASO allele specific oligonucleotides
  • competitive oligonucleotide priming oligonucleotide ligation assay
  • base- specific primer extension oligonucleotide ligation assay
  • dot blot assays and RFLP-PCR.
  • the diagnostic and prognostic methods of the present invention detect or assess an aberration in the wild-type TRP gene or locus to determine if TRP will be produced or if it will be over-produced or under-produced.
  • the term "aberration" in the TRP gene or locus encompasses all forms of mutations including deletions, insertions, point mutations and substitutions in the coding and non-coding regions of TRP. It also includes changes in methylation patterns of TRP or of an allele of TRP. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g.
  • Germline mutations can be found in any of a body's tissues and are inherited.
  • a TRP allele which is not deleted e.g. that found on the sister chromosome to a chromosome carrying a TRP deletion
  • can be screened for other mutations such as insertions, small deletions, point mutations and changes in methylation pattern. It is considered in accordance with the present invention that many mutations found in cells such as hepatic cells are those leading to decreased or increased expression of the TRP gene.
  • Useful diagnostic techniques to detect aberrations in the TRP gene include but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded coformational analysis (SSCA), Rnase protection assay, allele-specific oligonucleotide (ASO hybridization), dot blot analysis and PCR-SSCP (see below). Also useful is DNA microchip technology.
  • Predisposition to cancer can be ascertained by testing any tissue of a human or other mammal for mutations in a TRP gene. This can be determined by testing DNA from any tissue of a subject's body. In addition, pre-natal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic fluid for mutations of the TRP gene. Alteration of a wild-type allele whether, for example, by point mutation or by deletion or by methylation, can be detected by any number of means.
  • SSCP single-stranded conformation polymorphism assay
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • Other methods which might detect mutations in regulatory regions or which might comprise large deletions, duplications or insertions include the protein truncation assay or the asymmetric assay.
  • SSCA single-stranded conformation analysis
  • DGGE denaturing gradient gel electrophoresis
  • primers are used which hybridize at their 3' ends to a particular TRP mutation or to junctions of DNA caused by a deletion of TRP. If the particular TRP mutation is not present, an amplification product is not observed.
  • Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Publication No. 0 332 435 and in Newtown et al (Nucl. Acids. Res. 17: 2503-2516, 1989). Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. DNA sequences of the TRP gene which have been amplified by use of PCR or other amplification reactions may also be screened using allele-specific probes.
  • probes are nucleic acid oligomers, each of which contains a region of the TRP gene sequence harboring a known mutation.
  • one oligomer may be about 20-40 nucleotides in length, corresponding to a portion of the TRP gene sequence as described in Example 11.
  • PCR amplification products can be screened to identify the presence of a previously identified mutation in the TRP gene.
  • Hybridization of allele- specific probes with amplified TRP sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tissue as in the allele-specific probe.
  • Microchip technology is also applicable to the present invention.
  • thousands of distinct oligonucleotide or cDNA probes are built up in an array on a silicon chip or other solid support such as polymer films and glass slides.
  • Nucleic acid to be analyzed is labelled with a reporter molecule (e.g. fluorescent label) and hybridized to the probes on the chip.
  • reporter molecule e.g. fluorescent label
  • the technique is described in a range of publications including Hacia et al.
  • Antibodies specific for products of mutant alleles could also be used to detect mutant TRP gene product.
  • Such immunological assays can be done in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA and RAPID assays.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production is derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation (i.e. comprising TRP) or can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature, 256:495-499, 1975; Kohler and Milstein, European Journal of Immunology, (5/511-519, 1976). Examples of primers used to amplify regions of TRP are set forth in the Examples. The present invention is further described by the following non-limiting Examples.
  • plt2/plt2 male mice were mated with MpI ' ' " females on a C57BL/6 background (Alexander, W. S. et ⁇ l, Blood, ⁇ 7:2162-2170, 1996) to produce offspring that were obligate heterozygotes for the plt2 mutation and the MpI knock-out allele. These mice were then brother-sister mated and the platelet count of their offspring (the F2 generation) determined.
  • the MpI genotype of the F2s was determined by Southern blot as previously described (Alexander, W. S. et ⁇ l., 1996, supra).
  • the platelet count was determined at 7 weeks of age by collection of peripheral blood from the retroorbital plexus and deposition into tubes containing potassium EDTA (Sarstedt Nuembrecht, Germany). The platelet count was determined by using an Advia 120 automated hematological analyser (Bayer, Tarrytown, NY). All hematological data are presented as mean+1 standard deviation and all wild-type experimental animals were on a C57BL/6 genetic background. Spleen colony-forming units (CFU-S) were enumerated by intravenous (iv) injection of 7.5x10 4 bone marrow cells from either a plt2/pU2 or C57BL/6 donor into five C57BL/6 recipients after they had received 11 Gy in two equal doses given three hours apart.
  • CFU-S Spleen colony-forming units
  • the cell suspension was stained with FITC conjugated CD41 monoclonal antibody or FITC IgGl kappa as the isotype control (BD Pharmingen, San Diego CA) and then incubated in a hypotonic propidium iodide solution (0.05 mg/ml PI in 0.1% sodium citrate) for a minimum of 2 hours on ice.
  • the stained cell suspension was then washed with CATCH solution filtered through a lOO ⁇ m cell filter to remove cell aggregates and added to TruCOUNT tubes (BD Biosciences) containing a specified number of beads.
  • RNase was added to the suspension and the sample was analyzed on a Becton Dickinson FACScan.
  • bone marrow cells from a plt2/plt2 or C57BL/6 male donor were injected into plt2/plt2 and C57BL/6 recipient mice after they received a myeloablative dose of radiation (2 doses of 5.5 Gy, 3 hours apart).
  • Megakaryocyte engraftment after this procedure was studied by identifying the presence of sex-mismatch between the male bone marrow donor and female recipient mice. Specifically, bone marrow from recipient mice was harvested and cultured in IMDM supplemented with 1% Nutridoma-SP (Roche Diagnostics, Indianapolis, IN) and 50ng/mL of thrombopoietin for 5 days at 37°C.
  • Megakaryocytes were then purified on an albumin density gradient and an aliquot cytocentrifuged onto glass slides and stained with acetylcholinesterase (Jackson C. W., Blood 42:413-421, 1973). The remaining megakaryocyte suspension was lysed in non-ionic detergent and PCR was performed using primers specific for the murine sex-determining region of the Y chromosome (Sry) (Gubbay J. et ah, Nature 346:245-250, 1990) (5'-CTCTGCCTGTGCTGGTTG-S', and 5'-
  • TTGTGCTTTTTGTCCTCTTGT-3' TTGTGCTTTTTGTCCTCTTGT-3'
  • platelet derived growth factor receptor Pdgfr
  • Sry 5'-TCCAGTGCAGTGCTTTATGC-S'
  • Pdgfr 5'- CACTGACCAATGTCACTGGG-3'
  • ELISA Thrombopoietin Enzyme-linked immunosorbent assay
  • Peripheral blood was collected from the retroorbital plexus and allowed to clot at room temperature for 2 hours before centrifugation for 20 minutes at 200Og. The supernatant was removed and then stored at -20°C until the ELISA was performed. Serum samples were prepared in a similar manner from mice 11 days after 5FU injection (0.15 g/kg 5FU iv). Protein lysates were prepared from whole liver specimens that were weighed and then snap- frozen in liquid nitrogen.
  • KALB lysis buffer 150 mM NaCl, 50 mM Tris [pH 7.5], 1% [vol/vol] Triton X-100, 1 mM EDTA
  • protease inhibitors Complete cocktail tablets, Roche
  • the protein content of the lysate was calculated using the BCA protein assay kit (Pierce, Rockford IL).
  • the thrombopoietin concentration of the serum and liver lysates was then determined by Quantikine murine thrombopoietin ELISA (R&D Systems, Minneapolis, MN).
  • Real-time PCR reactions were set up for Thrombopoietin (Tpo), Hydroxymethylbilane synthase (Hmbs) and RNA polymerase II ⁇ Polr2d) using the Taqman gene expression assay protocols. Specifically, 1 ⁇ L of cDNA was used in a 20 ⁇ L PCR reaction with the pre-developed Taqman assay for Tpo, Hmbs or Polr2a, 10 ⁇ L Taqman universal master mix and water. Cycle conditions were 95°C for 10 min then 15 sec at 95°C and 1 min at 6O 0 C repeated for 40 cycles.
  • Tpo Thrombopoietin
  • Hmbs Hydroxymethylbilane synthase
  • RNA polymerase II ⁇ Polr2d RNA polymerase II
  • SSLPs (Table 6) were amplified by PCR using fluorescent dye labeled oligonucleotides and the sequence length of the PCR product determined on an ABI 3700 DNA sequence analyser as per the manufacturers instructions. Linkage was assessed using quantitative trait analysis in the statistical package R/qtl (Broman K. W. et al, Bioinformatics 7P:889-890, 2003). A normal model was used for the log transformed platelet count and the sex of the mice was included in the analysis. Once the region of interest was established, informative recombinants were sought among 353 F2 intercross mice.
  • the founders of the plt2 pedigree were recognised in the third generation of an ENU mutagenesis screen by virtue of a sustained thrombocytosis.
  • a plt2/plt2 homozygote pedigree was then established by breeding animals with the highest platelet counts and their plt2/plt2 genotype was confirmed by examining the platelet count distribution of their offspring (progeny testing).
  • the animals designated plt2/plt2 in this cohort were bred from parents that both displayed thrombocytosis with platelet counts greater than 2000 xlO 9 /L (a level of thrombocytosis not observed in the wild type population in this series) and animals designated heterozygous (plt2/+) had one parent with a platelet count greater than 2000 xlO 9 /L bred with a wild-type animal.
  • Platelet counts in the obligate heterozygotes produced from this breeding strategy were indistinguishable from the wild-type population demonstrating the recessive nature of this phenotype.
  • Other platelet parameters, such as Mean Platelet Volume and Platelet Distribution Width were similar between the groups (Table 5).
  • plt2/plt2 mice appeared overtly normal and were fertile. When observed for up to one year, they remained healthy with no observable complications related to the thrombocytosis.
  • No histological basis for the enlarged plt2/plt2 liver was detected. Specifically, there was no cellular infiltration, no degeneration of hepatocytes, no abnormal frequency of mitoses and no suggestion of enlargement of plt2/plt2 hepatocytes. Hepatic architecture was normal.
  • plt2/plt2 animals were intercrossed with Mp ⁇ f ⁇ animals.
  • the platelet counts of the F2 generation arising from the intercross of plt2/plt2 animals with the MpT* ' animals displayed a bimodal distribution (Figure 1C).
  • One cluster of platelet counts were present around the Mp ⁇ ' ⁇ mean of 150.
  • the second cluster covered the normal platelet range for a C57BL/6 mouse, but with a tail extending towards higher platelet counts.
  • CFU-S Colony forming units in the spleen
  • megakaryocyte engraftment after transplantation was studied in 10 female recipient mice that received bone marrow from one of the two male donor animals. Megakaryocytes were grown in culture from the bone marrow of the recipient animals and then purified. DNA from these purified megakaryocytes was amplified by PCR and a Southern blot performed to identify the presence of Y chromosome DNA (the Sry allele) in a semi-quantitative fashion. A control blot was also performed to identify the presence of DNA from an autosome (Pdgfr).
  • serum TPO was measured in wild-type mice with rebound thrombocytosis following 5FU injection (Radley J. M.
  • TPO production might be increased in these mice.
  • Tpo PvNA extracted from liver samples of 4 plt2/plt2 and 4 wild-type mice was quantitated in quadruplicate.
  • Tpo transcripts from a panel of tissues from 3 wild-type and 3 plt2/plt2 mice were also quantified in duplicate. A wide range of Tpo transcription across the organs sampled was observed, with kidney demonstrating the most prominent levels of transcription after the liver. However, no physiologically significant up-regulation of Tpo transcript was demonstrated in the kidneys of plt2/plt2 mice (Fig 3C), or in any organ examined. Gene expression normalised to Polr2a relative to a control kidney sample are presented in Figure 3C and similar results were found when Tpo expression was normalised to Hmbs (data not shown).
  • hepatic thrombopoietin protein was examined in whole liver lysates by ELISA.
  • Platelet counts from 89 N2 mice ranged from 1039 to 2424x10 9 /L and displayed a bimodal distribution consistent with the autosomal recessive inheritance of the plt2 allele (data not shown). Genetic linkage for thrombocytosis was observed on chromosome 7 at D7Mitl89 with a peak LOD score of 22.85 ( Figure 4A).
  • ENU mutagenesis has been utilized to identify a novel mouse pedigree with heritable thrombocytosis and hepatomegaly. Using well-characterised genetic polymorphisms between two inbred strains of mice this mutated locus has been mapped to an 8.6Mb region on chromosome 7 which contains at least 80 recognised genes. There is no specific gene in the defined interval previously implicated in platelet homeostasis, suggesting that the plt2 mutation is acting on a novel gene, or acting via a novel function of a known gene.
  • the plt2/plt2 mutant mice display a relatively mild thrombocytosis that appears to be driven by excessive thrombopoietin production.
  • the serum thrombopoietin level is elevated in these mice and the mutation is unable to rescue any of the thrombocytopenia displayed by MpY A mice indicating that the action of the plt2 mutation is dependent upon signalling through the c-Mpl receptor.
  • Thrombopoietin is primarily produced in the liver and in one embodiment the hepatomegaly alone displayed by the plt2/plt2 mice may cause the observed thrombocytosis. However, in another embodiment this mutation specifically up regulates thrombopoietin production. While no specific increase in TPO transcription was observed, TPO protein content per gram of liver weight was increased. These results may reflect either a subtle increase in TPO transcription in the liver below the level of detection using current technology or an alteration of TPO at the translational level. The translation of human TPO mRNA is reduced by the presence of inhibitory elements in the 5 '-untranslated region and these inhibitory elements are conserved between humans and mice (Ghilardi N.
  • mice heterozygous for the c-Mpl knock-out allele displayed a mild thrombocytosis compared to wild-type C57B1/6 animals with platelets elevated by 18% compared to wild-type.
  • the highest platelet counts of the F2 intercross were observed in plt2/plt2 Mpt' ⁇ mice which have increased serum TPO and decreased c-Mpl.
  • This effect of c-Mpl gene copy on platelet count is somewhat counter-intuitive, but there is precedent for reduced c-Mpl expression being associated with increased platelet counts in humans.
  • c-Mpl having two independent but related functions in regulating circulating platelet mass.
  • TPO signals through the c-Mpl receptor to stimulate megakaryocyte and megakaryocyte progenitor proliferation and maturation, driving platelet production.
  • c-Mpl also acts as the primary mechanism by which circulating TPO is removed from the circulation.
  • reduced platelet-mediated clearance leads to increased serum TPO concentration and enhanced megakaryopoiesis. This requires that intra-cellular signalling through the c-Mpl receptor is achieved at relatively low levels of receptor density, but that increasing levels of c-Mpl receptor above this level can still incrementally participate in TPO clearance.
  • the gene affected by theplt2 mutation is an organ specific tumor suppressor gene
  • plt2/plt2 mice which are derived from C57BL/6
  • Table 8 provides the primers and detection method used for each marker in the further fine mapping study.
  • Genomic DNA was prepared from the liver of experimental mice and amplified by PCR with the oligonucleotide primers listed in Table 8. The PCR product was then sequenced in a reaction using Big Dye Terminator and analysed on an Applied Biosystems automatic sequencer according to the manufacturers instructions. A set of nested primers were used to sequence JaxSNP4 set forth in Table 8. As a result, the interval was reduced to 0.66Mb between Celera SNP 12 and Celera SNP 17.
  • the known genes in the interval between Celera SNP 12 and Celera SNP 17 were sequenced to identify the plt2 mutation. Specifically, exon and exon-intron boundries were sequenced in two animals that were homozygous for C57BL/6 markers across the region of interest, one intercross animal that was Balb/C across the region of interest and one control C57BL/6 mouse. Genomic DNA corresponding to gene exons identified in Table 9 was prepared from the liver of experimental mice and amplified by PCR with the oligonucleotide primers listed in Table 10. The PCR product was then sequenced in a reaction using Big Dye Terminator and analysed on an Applied Biosystems automatic sequencer according to the manufacturers instructions.
  • Direct sequencing of PCR products identified a point mutation in Exon 2 of AK00447 (1190003J15 Riken cDNA).
  • the point mutation was found in both Backcross animals that were C57BL/6 in the region of interest that was absent in the intercross animal that was Balb/C in the region of interest and the C57BL/6 control.
  • the sequencing profiles in the region of the mutation are shown in Figure 6.
  • This mutation causes a predicted amino acid change from Tyrosine in the wild-type (encoded by nucleotides denoted TAC) to Cysteine (encoded by nucleotides denoted TGC) in the plt2/pH2 animals.
  • Genomic DNA was prepared from a tail biopsy taken from experimental mice at approximately 3 weeks of age. DNA was amplified using primers specific for exon 2 of AK004470 (5'- GGCACCTATAAGCTGTTCTTCGA-3 ' and 5'-ACCCTGACACTCACCTCTACATAG-S'). The PCR product was then identified as mutant or wild type by measuring specific fluorescence associated with the mutant or wild- type fluorescent-tagged oligonucleotide probe (wild-type: VIC- CAGAGCGCTACTGGAAA, plt2 mutant: FAM- AGCGCTGCTGGAAA). The PCR reaction and alleleic discrimination detection were performed on an ABI Prism 7900HT Sequence Detection System according to the manufacturers instructions.
  • Genomic DNA comprising regions encoding TRP (short form) and TRP-2 (long form)
  • the nucleotide sequence of NCBI Accession No. AK00447 contains three exons as shown in Figure 8.
  • the plt2 mutation is contained in the second exon of the structure.
  • Sequencing of the cDNA from the liver of both wild-type and plt2/plt2 animals identified a gene structure as predicted in AK00447 (herein referred to as the short form) as well as a larger gene containing an additional previously unidentified 5' exon (herein referred to as the long form or TRP-PLT2/77?P-Pir2).
  • the long form is also represented in Figure 8 which also indicates the position of the mutation in the penultimate exon.
  • microarray chips were used to measure the liver gene transcription profile from 4 plt2/plt2 and 4 sex-matched wild-type mice at 5 weeks of age (Figure 12). Microarray data were normalised between individual gene chips with robust multiarray averaging. Linear modelling was then performed to examine transcriptional differences between replicate arrays using software designed by the Bioinformatics Division at the Walter and Eliza Hall Institute (http://bioinf.wehi.edu.au/affylmGUI). Using this approach, there was no significant differential gene expression within the 4 wild-type biological replicates, nor within the 4 plt2/plt2 biological replicates indicating relatively homogeneous gene transcription profiles for each genotype. However, analysis comparing wild-type and plt2/plt2 arrays demonstrated significant differential gene expression between the two genotypes.
  • Amplified PCR products derived from wild-type and plt2/plt2 mutant liver were ligated into the pEF-BOS expression vector (Mizushima et al, Nucleic Acids Res., 18:5322, 1990) containing a FLAG epitope tag sequence and then cloned in the DHlOB strain of E. coli. Sequencing of these cloned nucleotide products confirmed that the predicted TRP sequence from the UCSC database was correct and verified the presence of the A to G point mutation in liver cDNA from plt2/plt2 mice ( Figure 13).
  • TRP transcript is expressed in a variety of organs inplt2/pH2 and wild-type mice
  • TRP mRNA expression was measured in a variety of tissues by semi-quantitative reverse- transcriptase (RT) PCR. Using primers specific for the 5' untranslated region of TRP short and long, mRNA expression was examined in a tissue panel from wildtype and plt2/plt2 mice ( Figure 14A). In both wild-type and mutant mice, the liver was the most abundant source of both TRP short and long transcript. At high PCR cycle number, transcript was also detected in lung, kidney, spleen, thymus and brain, identifying these as sites of low TRP transcript expression.
  • RT reverse- transcriptase
  • Transthyretin-related protein is expressed in hepatocytes from wild-type mice but is markedly reduced or absent in plt2/plt2 liver
  • hepatocytes were isolated from wild-type and plt2/plt2 mutant mice and examined for TRP expression by confocal microscopy (Figure 15B).
  • TRP protein was clearly present in isolated hepatocytes, distributed predominantly in the cytoplasm.
  • hepatocytes isolated from plt2/plt2 mice demonstrated no fluorescent signal, indicating the translated product of the mutant TRP gene was either markedly reduced or absent in these cells.
  • Plt2/plt2 mice transcribe the product of the TRP gene in liver, but transthyretin-related protein is not detected in hepatocytes suggesting the plt2 mutation is responsible for a defect that alters the normal expression of this protein in liver cells.
  • TRP amino acid sequence is highly conserved throughout evolution.
  • TRP is also closely related to transthyretin, with 29% of amino acids identical between mouse TRP and transthyretin. Conservation of the tyrosine mutated by the plt2 mutation in TRP, from bacteria to mouse shows that this residue is important for normal protein function. This tyrosine is also conserved between TRP and transthyretin ( Figure 16).
  • transthyretin The protein structure of transthyretin is well characterised (Blake et ah, J. MoI. Biol, 88:1-12, 1974; Hamilton et ah, J. Biol. Chem., 268:2416-2424, 1993). Given the high degree of amino acid homology between TRP and transthyretin, a model of mouse TRP was generated from the structural information determined from fish transthyretin (Sparus aurata) using Swiss model software (Guex et al, 1997 (supra)). This modelling predicts that murine TRP adopts a barrel structure similar to transthyretin.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • n 107 for each genotype.
  • n 3-4 for CFU-S and progenitor assays and 6-7 per genotype for megakaryocyte assays.
  • Supplementary table 5 contains the complete data set to a wide range of stimuli.
  • Supplementary Table 5 Colony-forming progenitor cells to a wide range of stimuli.
  • GM-CSF wild-type 16 ⁇ 2 4 ⁇ 2 24 + 5 3 ⁇ 1 plt2/plt2 17 ⁇ 3 9+ 5 22 + 7 3 ⁇ 2
  • JaxSNP4 GCTGCTTGGTCCTTAGGTTG ACTCAGGCCATGGATTGCT 7 127438668 to 127438868 SNP
  • CeleraSNP33 CTAACCTGGGCCGACTAATG ATTAATGGGTAGGGGGAGCA 7 128522680 to 128522876 SSLP
  • CeIeraSNP17 AGGGCAGTCCAGGCTACATA GGCCCTTTTCGTCTTTGAG 7 128991556 SSLP
  • JaxSNP4 Mouse Phenome Database (SNP ID WI_WGS_7_129989877) CATAGAGCTTCCTCGCATGT CTCAAGCTCCATGAAACACA
  • CAACTACTGGCCCCAAGGTA CAGTGTCCATCAGCACTTGG TTGCTCAGCTCTTGGTGATG AGGGAGCATTTAGGGTGCTT TGAAGGTAGCTCCTGCCCTA TTTGGATAGGCCTGCAGAAG tcagacagctccaaccaaga TTGTGGGACTGAGGGAAGAC
  • IFITM 1 TGCTTAGCAACTTGACTTCATCTA TCTACCCCAAATCCTGACCC GCCCACTGCGCAGCAGGCTC CACCTCCTGGGATTCCCTC CTAGGAAGGTGATGGGGAGC AACTCTGGTTAATTACTGCCCAG
  • IFITM2 AAGGGCGGGTCTACAGAACC TGAGTAGATGGCGCTTCAGG CAGGGAGCAGTTGGGGAAAT GGAGACCAGAAGCCTGACAA
  • SuItIaZ (sulfotransferase family 2A, member 2) ⁇ 47.0 8.4xlCr s 8.4X10 '8
  • SuJtSaI (sulfotransferase family 5A, member 1) 1 2.3 0.0011 0.00010
  • Hgfac kepatocyte growth factor activator
  • EpIuI epoxide hydrolase 1, microsomal
  • RpU3a ribosomal protein LOa
  • Lamrl (laminin receptor 1) t
  • Nola2 (nucleolar protein family A, member 2) f
  • Gsttn (glutathione S -transferase, mu land 2)
  • Cyp2c40 (cytochrome P450 2c40) i
  • Ig ⁇ p2 insulin-like, gro ⁇ vth factor binding protein 2
  • Ephxl epoxide hydrolase 1, microsomal

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Abstract

La présente invention concerne, notamment, des cellules modifiées ou des animaux non humains comprenant celles-ci, présentant un gène TRP modifié, des constructions génétiques dérivées de celui-ci et des procédés de criblage d’agents utiles dans le traitement du cancer, en particulier du cancer du foie et des thrombocytoses.
PCT/AU2006/000456 2005-04-06 2006-04-06 Modeles animaux et cellules dotees d’un gene modifie codant pour une proteine associee a la transthyretine et applications de ceux-ci Ceased WO2006105602A1 (fr)

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