WO2005020677A1 - Method of selecting animal models from animals which have been subject to mutagenesis, and the use of myb transcription factors for screening - Google Patents
Method of selecting animal models from animals which have been subject to mutagenesis, and the use of myb transcription factors for screening Download PDFInfo
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- WO2005020677A1 WO2005020677A1 PCT/AU2004/001167 AU2004001167W WO2005020677A1 WO 2005020677 A1 WO2005020677 A1 WO 2005020677A1 AU 2004001167 W AU2004001167 W AU 2004001167W WO 2005020677 A1 WO2005020677 A1 WO 2005020677A1
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Classifications
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- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- A01K2217/03—Animals modified by random mutagenesis, e.g. using ENU, chemicals
-
- 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
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
Definitions
- the present invention relates generally to target molecules for potential therapeutic and/or prophylactic intervention. More particularly, the present invention provides a 10 physiological assessment system in the form of vertebrate animal models to identify genetic or proteinaceous drug targets therein associated with a disease or a particular condition or phenotype. In an illustrative embodiment, target molecules associated with modulating platelet levels and ameliorating the symptoms of thrombocytopenia are described. The target molecules are useful as therapeutic and/or prophylactic agents such 15 as in antisense or iRNA form. The present invention also relates to the application of these drug targets in the identification or development of therapeutic and/or prophylactic agents which modulate the functional activity of the drug target or its interacting network of molecules.
- the invention relates to vertebrate and particularly rodent animal models or human diseases, exhibiting physiological characteristics such as one or more 20 symptoms of a disease or of a condition, which may be used to screen for randomly or non-randomly induced mutations one or more of which effectively ameliorate/s symptoms of the disease or effectively ameliorates the condition. Such mutations are proposed to be within potential targets for therapeutic intervention.
- the vertebrate animal model may be used as a recipient for other potential 25 physiology modifying agents such as sense or antisense molecules and small chemical, nucleic acid or proteinaceous molecules.
- Target validation is an essential and often technically difficult task which must be completed prior to committing huge resources to drug development. While experimental results may indicate that a molecule is involved with a disease or condition, it seldom follows that the molecule is a good drug target with potential to beneficially influence the interaction because, for example, other molecules in the subject may compensate by performing the same function.
- Thrombocytopenia is one disease for which validated drug targets are required. Thrombocytopenia may occur as an inherited illness or may occur as a result of autoimmune disease, viral infection or as a side effect of chemotherapy. The current treatment for thrombocytopenia is inadequate (Demetri G. D., Oncologist, 6 (Suppl.5): ⁇ 5- 23, 2001).
- TPO Thrombopoietin
- c-Mpl Thrombopoietin
- Mice and humans lacking functional Tpo or Mpl genes are profoundly thrombocytopenic and have a corresponding reduction in the numbers of megakaryocytes, megakaryocyte progenitor cells and stem cells (Alexander, W. S.
- 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>1 (SEQ ID NO: 1), ⁇ 400>2 (SEQ ID NO: 2), etc.
- a summary of sequence identifiers is provided in Table 1.
- a sequence listing is provided after the claims.
- the present invention is predicated, in part, on the use of physiologically assessable animal models of a human disease or condition in a physiological assessment system to identify drug targets. It is proposed that the instant assessment system will be used as a method of doing business, for example, in contract testing to identify drug targets.
- mutations are introduced into an animal model of a disease or of a physiological state of interest (condition), and animals identified in which symptoms of the disease or condition are ameliorated (changed).
- the mutation may be a random or non- random change at a genetic locus or a change in its expression or activity.
- mutations may be introduced into a wild type precursor of the animal model which is subsequently further modified such as, for example, by the administration of an infectious or chemotherapeutic agent or cancerous cells in order to develop the physiological characteristics of the animal model.
- the present invention provides animal models comprising one or more mutations which further affect the assessable physiological characteristics of the animal model.
- the proposal is that a mutation has occurred in a drug target resulting in an expression pattern or activity profile which ameliorates the symptoms of the particular disease or changes the condition.
- the genetic molecules comprising the mutations are cloned positionally and they, or their encoded molecules including RNA or proteinaceous molecules, comprise validated drug candidates themselves or substrates for the development of agonists and antagonists useful in the treatment and/or prophylaxis of the disease or condition.
- mutations are introduced into an animal model of thrombocytopenia, and animals identified in which platelet levels are elevated and/or symptoms of thrombocytopenia ameliorated.
- mutations are introduced into wild type animals which are subsequently developed into the animal model.
- thrombocytopenia may be induced by chemotherapy.
- the animal model may be induced by downregulation or inhibition of Mpl by various agents such as antibodies, receptor antagonists or antisense/iRNA molecules.
- a mutation occurring in a drug target for the treatment of thrombocytopenia or for modulating platelet levels results in an expression pattern or activity profile in the animal model which effectively modulates platelet levels and/or overcomes the reduced platelet levels and ameliorates the symptoms of thrombocytopenia.
- Identification of the drug target facilitates identification of further drug targets selected from the group of molecules with which the initial drug target is known to interact.
- thrombocytopenia Animals with reduced Mpl activity exhibit inter alia low platelet counts and provide therefore a useful animal model for thrombocytopenia and other conditions associated with an over supply or under supply of platelets in a subject or tissue. Conveniently, this occurs by introducing a mutation into one or both Mpl alleles.
- a Mpl " A mouse is used as a model of thrombocytopenia and used in a physiological assessment system to identify drug targets for the treatment of thrombocytopenia.
- the present invention provides the use of a rodent model of thrombocytopenia, preferably with reduced Mpl levels, to screen for drug targets for use in the treatment or development of treatment for thrombocytopenia.
- the Plt3 and Plt4 mutations have been mapped to the Myb transcription factor gene and it is demonstrated herein that mutation in this gene can ameliorate thrombocytopenia caused by lack of TPO signalling through its receptor Mpl. In addition and importantly, it is also shown that reduction in the function of Myb ameliorates thrombocytopenia that occurs following administration of chemotherapeutic agents of the type that are used in treating human cancer patients. As demonstrated herein, down regulation of Myb is important in hematopoiesis and platelet production and has been revealed to be a validated candidate for the development of small molecule pharmaceuticals with which to treat thrombocytopenia (see, for example Figure 13 A, B and C).
- the present invention provides antagonists and agonists of the molecules identified in the subject physiological assessment system.
- antagonists and agonists may comprise all or part of the target molecules themselves, or their complementary sequences, chemical analogues, mimetics, sense or antisense molecules including iRNA-type agents, antibodies, or other molecules in the genetic network to which the target molecules identified by the instant methods belong or their derivatives.
- the antagonists or agonists may be synthetic chemicals or natural products identified by screens known in the art. Once the target molecules are identified, a wide range of screening strategies known in the art are available for the identification, production, design and development of antagonists or agonists.
- the rational design of molecules which interact with the active site of proteinaceous target molecules may be achieved using the solution structures of the target and/or target-ligand complexes. Spectroscopic and computer modelling techniques are generally used to determine a solution structure and subsequently the three dimensional structure can be displayed and manipulated using computer enhanced algorithms for the design of agonists or antagonists.
- the present invention presents pharmaceutical compositions comprising recombinant synthetic or isolated forms of the present drug targets and one or more pharmaceutically acceptable carriers, diluents or excipients and furthermore contemplates methods of their use in vitro or in vivo in methods for the treatment of subjects, and in particular humans.
- Figure 1A, B, C, D and E are photographic representations depicting the results of Automated Hematological analysis to determine platelet counts in (A) Mpl+/+ mice, (B) Mpl' ' mice, (C) GI progeny, (D) progeny of G2 mouse No. 985.34 with about half the progeny exhibiting very low platelet counts characteristic of Mpl* ' mice (pedigree Plt3), and (E) progeny of G2 mouse No. 1118.11 with about half progeny exhibiting very low platelet counts characteristic of Mpl ' ' ' mice (pedigree Ptl4).
- Figure 2A, B, C, D and E are photographic representations depicting the results of Automated Hematological analysis to determine platelet counts in homozygous Plt3 and PU4 mice compared with (A) Mpl+/+ mice and (B) Mpl 1' mice.
- Figure 2 (C) represents the platelet counts of individual mice resulting from crosses between Plt3/+ Mpl 1' and +/+Mpl' ' or Plt3/+ Mpl' ' and PH3/+ Mpl' ' .
- Figure 2 (D) represents the platelet counts of individual mice resulting from crosses between Plt4/+ Mpl' ' and +/+Mpl' ⁇ or PU4/+ Mpl' ' and Plt4/+ Mpl' ' .
- the right hand column in D shows the platelet counts of progeny of a cross between putative Plt4 homozygotes and +/+ Mpl' ' mice showing that all the progeny had mildly elevated platelet numbers expected of PU4I+ Mpl 1' mice.
- Figure 3A, B, C, D and E are graphical representations showing the increased production of platelets by precursors in mice harbouring the PU3 and Plt4 mutations.
- Figure 3 (A) depicts numbers of Bone Marrow Blast-CFC in homozygous Plt4/Plt4 Mpl' ' mice, Plt4/+ Mpl ' ' mice, +/+Mpl' ⁇ mice and +/+Mpl + + mice.
- Figure 3 (B) depicts numbers of Bone Marrow Megakaryocyte-CFC in PU4/PU4 Mpl' ' mice, Plt4/+ Mpl' ' mice, +/+Mpl' ' mice and +/+Mpl+/+ mice.
- Figure 3 (C) depicts numbers of Spleen Megakaryocyte-CFC in homozygous Plt4/Plt4 Mpl' ' mice, PU4/+ Mpl 1' mice, +/+Mpl' ⁇ mice and +/+Mpl+/+ mice.
- Figure 3 (D) depicts numbers of Bone Marrow Megakaryocyte in homozygous Plt4/Plt4 Mpl' ' mice, PU4/+ Mpl' ' mice, +/+Mpr' ' mice and +/+Mpl+/+ mice.
- Figure 3 (E) depicts numbers of Spleen Megakaryocyte in homozygous Plt4/Plt4 Mpl' ' mice, PU4/+ Mpl' ' mice, +/+Mpl' ⁇ mice and +/+Mpl+/+ mice.
- Figure 4A, B and C are photographic representations depicting Automated Hematological results providing platelet counts in the Mpl +/+ mice (A), and Mpl " mice (B). To distinguish compound heterozygotes from double heterozygotes, mice with the highest platelet levels (Figure 4C left hand column) were mated with +/+Mpl' ⁇ .
- compound heterozygotes yield progeny, all of which will be heterozygote (PU3/+ Mpl' ' or Plt4/+ Mpl' ' ), where as double heterozygotes yield double heterozygotes (PU3/+ PU4/+ Mpl' ' ), single heterozygotes (PU3/+ +/+ Mpl' ' or +/+ Plt4/+ Mpl' ' ) or wild type (+/+ +/+ Mpl').
- Plt3 and Plt4 the mutations are clearly in the same gene or closely linked since the progeny of the above mating all have the phenotype of heterozygotes ( Figure 4C right hand column).
- Figure 5 is a representation showing the chromosomal location of Plt4.
- PU4/+ Mpl' ' mice on a C57BL/6 background were mated to +/+ Mpl' + on a 129Sv background and the PU4/+ Mpl' ' (C57BL/6xl29Sv) Fi progeny were selected because of the elevated platelet numbers compared with +/+ Mpl ' controls.
- FIG. 6 is a representation showing the chromosomal location of Plt3 on mouse chromosome 10.
- Pit 3/+ Mpl' ' mice on a C57BL/6 background were crossed with PU3/+ Mpf ' mice on a 129Sv background.
- Plt3/+ Mpl' ' FI animals were then identified at 7 weeks of age because of their elevated platelet counts and were backcrossed to +/+ Mpf ' mice on a 129Sv background to generate N2 mice.
- N2 mice that were Mpl' ' were identified and their platelet counts were determined at 7 weeks of age.
- Mice were then sacrificed and their livers were removed and genomic DNA prepared.
- SSLPs across chromosome 10 (Table 4) were then amplified and analysed as described for PU4 confirming the co-localisation of the two mutations in the Myb gene.
- Figure 7 is a representation of results of sequencing of genomic DNA from Plt3 and Plt4 mice showing the nature of the mutation in these mice.
- Primers (see Table 5) were designed to amplify the exons and flanking intronic DNA from genomic DNA of P 4/PU4 Mpl' ' , PU4/+ Mpl' ' , P 3/PU3 Mpl' ' , Plt3/+ Mpl'; +/+ Mpl' ' and +/+ Mpl+/+ mice all on a C57BL/6 background.
- Figure 7 (A) shows Plt3 mice in which nucleotide A455 is altered to T leading to a substitution of Val for Asp at amino acid 152.
- Figure 7 (B) shows the results for PU4 mice indicating a nucleotide alteration from A to T in the Plt4 mutation.
- Figure 8 is a representation showing the nucleotide and amino acid sequence of mouse Myb.
- the mutation in PU3 mice is shown at nucleotide A455 leading to a substitution of Asp for Val at amino acid position 152.
- the mutation in Plt4 mice is shown at nucleotide position Al 151 where an A is replaced with a T, leading to a substitution of Val for Asp at amino acid 384.
- Figure 9 is a representation showing the nucleotide and amino acid sequence of human Myb.
- Figure 11 A, B, C and D are representations of results of an analysis to determine linkage between mutants, their map position and position within Myb, and the effect of the mutation on the ability of expressed protein to activate transcription.
- Plt3 and Plt4 are tightly linked on chromosome 10 and are alleles of c-Myb. a, Pit 3/+, P 4/+ and Plt6/+
- Mpl' ' mice were inter-crossed to produce compound heterozygotes, single heterozygotes and mice wild type for both Pit mutations.
- the platelet counts of the progeny are shown and the genotype was inferred by analysis of the progeny derived from mating these animals to +/+ Mpl' ' mice. Note that the progeny derived from crossing PU3/PU4 Mpl' ' mice to +/+ Mpl' ' mice all have intermediate platelet counts typical of either PU3/+ or
- mice were bled at 7 weeks of age and categorized as having low platelets ( ⁇ 150 x 10 6 /ml) characteristic of +/+ Mpl' ' mice, moderate numbers of platelets (150xl0 5 - 2000 x 10 6 /ml) characteristic of Plt4/+ Mpl' ' mice or extremely high platelets (>2000 xl0 6 /ml) characteristic o ⁇ PU4/Plt4 Mpl' ' mice. Animals were then genotyped and markers found to be homozygous 129/Sv are shown in white, markers that were heterozygous are shown in grey and markers homozygous C57BL/6 are shown in black. The number of animals with each haplotype is shown below.
- Figure 12A, B and C are representations of results of an analysis to determine the effect of PU3 and PU4 mutations in cells and tissue of the hematopoietic system in homozygous and heterozygous form. Mutation of c-Myb results in an elevation in progenitor cells, megakaryocytes and platelets independent of Mpl.
- CFU-s colony-forming units-spleen
- Figure 13A, B and C are graphical representations showing that a reduction in the function of Myb ameliorates thrombocytopenia that occurs following administration of chemotherapeutic agents of the type used in treating cancer patients.
- c-Myb Plt4/Plt4 mice ( ⁇ ) treated with carboplatin are resistant to thrombocytopenia compared with their control c-Myb+/+ littermates (A).
- Carboplatin was administered at the beginning of the experiment and the platelet counts of animals were measured at the indicated times afterwards.
- the "animal models" of the present invention are selected from those vertebrate animals in which genetic studies are feasible.
- Murine animal model are preferred.
- Animal models express or are capable of expressing (i.e. they are used in precursor form) physiologically assessable symptoms of a disease or condition which occurs or which has substantial similarities to a disease or condition which occurs in man or other subjects of interest.
- ameliorate or “ameliorating” or “treating” or “therapy of or “treatment” are used in the broadest context and include any measurable or statistically significant change in one or more symptoms or frequency of symptoms of a disease or one or more assessable indications of a condition as well as complete recovery from the disease or elimination of an associated or other condition, its symptoms or its underlying cause.
- the present invention is applicable to a large range of diseases or conditions and the skilled addressee must determine the precise parameters of the assessment of phenotypes on a case by case basis. Conditions may be associated with one, or more than one, disease but there is no requirement for this.
- the amelioration of a condition encompasses any desirable physiological or behavioural change provided it is directly or indirectly assessable.
- thrombocytopenia assessment is conveniently made of the level of platelets in the animal model.
- the parameters of this assessment include measuring the levels of haematopoietic cells and their precursors which are affected in thrombocytopenia by, for example, automated haemotological analysis. Platelet levels are a readily assessable physiological aspect of thrombocytopenia. Thrombocytopenia may also be associated with reduced levels of megakaryocytes and committed progenitor cells and levels of markers for these cell types provide an alternative means for physiological assessment. Other methods of assessment of ameliorating the symptoms of thrombocytopenia will be well known to those skilled in the art and reference is made to Alexander W. S., Int. J. of Biol. & Cell Biol, 37(10):1027-1035, 1999 and to citations therein.
- administered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
- the subject agent may be administered together with an agonistic agent in order to enhance its effects.
- sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
- the oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
- the term “compound” refers to a chemical compound that induces a desired pharmacological and/or physiological effect.
- the term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
- the term “compound” is not to be construed narrowly but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof.
- a “derivative" of a polypeptide of the present invention is a modified form of the polypeptide and also encompasses a portion or a part of a full-length parent polypeptide which may or may not retain the functional activity of the parent molecule.
- functional and non-functional derivatives may be used in determining the three dimensional structure of the target molecule.
- the derivative retains function activity, for example in the case of Myb
- the derivative retains the transcription factor activity of the parent polypeptide which is important in enhancing megakaryocytopoiesis.
- Such "biologically-active fragments" include deletion mutants and small peptides, for example, of at least 10, preferably at least 20 and more preferably at least 30 contiguous amino acids, which exhibit the requisite activity.
- peptides can be produced by digestion of an amino acid sequence of the invention with proteinases such as endoLys-C, endoArg- C, endoGlu-C and staphylococcus V8-protease.
- the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Any such fragment, irrespective of its means of generation, is to be understood as being encompassed by the term "derivative" as used herein.
- Genetic forms of the subject target molecules may be DNA or RNA.
- DNA When the genetic form is DNA it may be genomic DNA or cDNA.
- RNA forms of the genetic molecules of the present invention are generally mRNA.
- the genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules.
- “hybridization” means the pairing of complementary strands of oligomeric compounds.
- the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
- nucleobases complementary nucleoside or nucleotide bases
- adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
- Hybridization can occur under varying circumstances.
- Modulation of a target molecule includes completely or partially inhibiting or reducing or down regulating all or part of its functional activity and enhancing or up regulating or potentiating all or part its functional activity.
- the target is a genetic sequence its functional activity may be modulated by, for example, modulating its binding capabilities or transcriptional or translational activity, or its half-life.
- the target is an encoded polypeptide
- 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.
- Modulators are agonists or antagonists which achieve modulation.
- 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. 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.
- polypeptide or "proteinaceous molecule” refer to a polymer of amino acids and its equivalent and does not refer to a specific length of the product, thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like. Soluble form of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as those given in Table 2b or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell.
- a physiologically assessable symptom includes a symptom (trait or phenotype) which is capable of being measured or detected using the level, activity or amount of any molecule or event which is associated with the symptom.
- the assessment system assesses the symptoms with respect to any reliable marker thereof.
- markers include, without limitation, genetic or proteinaceous molecules, cells, infectious agents, temperature, electrical conductance, levels of ions.
- reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of one or more different molecule or events. Detection may be either qualitative or quantitative.
- the most commonly used reporter molecules in detecting antigen bound antibody are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
- an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
- Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
- the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
- suitable enzymes include alkaline phosphatase and peroxidase.
- fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme- labeled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away.
- a solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody.
- the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
- Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
- fluorescent compounds such as fluorescein and rhodamine
- fluorescent compounds may be chemically coupled to antibodies without altering their binding capacity.
- the fluorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
- the fluorescent labeled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
- Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
- sequence similarity and “sequence identity” as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
- a “percentage of sequence identity” 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) or the identical amino acid residue (e.g.
- 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.
- the percentage similarity between a particular sequence and a reference sequence is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater.
- Percentage similarities or identities between 60% and 100% are also contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
- 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 25-30°C to about 42°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.
- 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
- high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
- 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., 4(5:83, 1974).
- Formamide is optional in these hybridization conditions.
- 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°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
- 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 and amino acid sequence comparisons are made at the level of identity rather than similarity.
- references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
- 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
- Altschul et al Nucl. Acids Res., 25:3389, 1997.
- a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994- 1998, Chapter 15).
- Subjects contemplated in the present invention refer to the treatment or prophylaxis of a disease or condition in any animal of commercial or humanitarian interest including plants, primates, livestock animals including fish and birds, laboratory test animals, companion animals, or captive wild animals. Man is a preferred subject.
- target refers herein to one or more genetic sequences or molecules within or derived from a subject vertebrate animal whose modulation in vivo or ex vivo effectively ameliorates a disease or condition.
- drug targets are targets for prophylactic and/or therapeutic intervention.
- the term includes the same or homologous or variant genetic sequences or molecules within or derived from subjects to be treated, such as man.
- Targets comprise the genetic sequences or their encoded products in the immediate vicinity of the mutation, or longer sequences encoding protein or nucleic acid molecules, complementary forms thereof and/or their regulatory/expression control regions. Targets also extend to functional homologs from other species or genera and functional derivatives having at least about 60% amino acid or nucleic acid similarity to all or an appropriately functional part or domain of the parent molecule after optimal alignment.
- targets include without limitation, positive and negative regulators of transcription, precursors, upstream and downstream molecules, or co-factors.
- the therapeutic compositions of the present invention interact with the target molecule.
- the therapeutic compositions interact directly with a target molecule which may constitute an interacting molecule in the genetic or biochemical network comprising the target molecule, including molecules with which it interacts directly.
- a target molecule which may constitute an interacting molecule in the genetic or biochemical network comprising the target molecule, including molecules with which it interacts directly.
- Myb binds to a region of substrate nucleic acid in order to promote transcription. Molecules which interfere with this protein:DNA interaction will antagonise Myb factor functional activity.
- antagonists may comprise DNA binding molecule including nucleic acid or proteinaceous molecules.
- variant refers to nucleotide sequences displaying substantial sequence identity with a reference nucleotide sequences or polynucleotides that hybridize with a reference sequence under stringency conditions that are defined hereinafter.
- nucleotide sequence polynucleotide
- nucleic acid molecule may be used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
- certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference nucleotide sequence whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.
- variant also includes naturally-occurring allelic variants.
- “Functional derivatives” of a target molecule include active portions of the target molecule whose modification in a subject ameliorates a disease or condition and which may be further modified to enhance this affect.
- a functional derivative of a target molecule in the form of a protein or peptide comprises a sequence of amino acids having at least 60% similarity to the target molecule or portion thereof.
- a "portion" in peptide form may be as small as an epitope comprising less than 5 amino acids or as large as several hundred kilodaltons.
- the length of the polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues and preferably more than about 35 residues.
- a functional derivative When in nucleic acid form, a functional derivative comprises a sequence of nucleotides having at least 60% similarity to the target molecule or portion thereof.
- a "portion" of a nucleic acid molecule 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 10-35 nucleotides including 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 or 35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600 nucleotides or nucleic acid molecules having any number of nucleotides within these values.
- the present invention provides, inter alia, the use of physiologically assessable vertebrate animal models of human diseases or conditions in a physiological assessment system to identify genetic or proteinaceous drug target molecules associated with the amelioration of symptoms of a disease or of a condition.
- humans are a preferred subject, other subjects are clearly contemplated and encompassed.
- Target molecules are particularly useful as therapeutic compounds themselves or in the development of agonists or antagonists as therapeutic compounds which ameliorate symptoms of a particular disease or condition.
- the present invention provides both animal models and methods for their use in a physiological assessment system which will be particularly useful for pharmaceutical development, in business methods such as contract testing and for the sale of embryos and progeny of their animal models, including gametes and stem cells therefrom.
- one aspect of the present invention provides a physiological assessment system to identify a pedigree of a vertebrate animal model of a disease or condition which exhibits ameliorated symptoms of the disease or condition comprising:
- the present invention provides a physiological assessment system to identify genetic or proteinaceous drug targets associated with amelioration of symptoms of a disease or condition comprising:-
- Progeny are preferably GI, G2 or G3 progeny and subsequent progeny generated by breeding GI, G2 or G3 progeny. Sequencing of target molecules may be required. However, if the target molecule has been previously sequenced it may not be necessary to sequence the entire target. In order to determine the precise nature of a mutation in the drug target, sequencing of this region is performed. The identification of genetic sequences includes recognising homologous or potentially hybridising sequences in a nucleotide database (for example by BLAST searching) and also includes pin pointing the location of the target genetic sequences between closely linked genetic markers.
- the identification of a genetic sequence also includes identity by name, for example when the drug target molecule is a known molecules, or by sequence which is generally confirmed or derived directly by sequencing.
- the encoded products of the instant genetic sequences include nucleic acid molecules such as RNA such as miRNAs and siRNA or polypeptide molecules such as short peptides or larger proteins.
- Mutagenesis with an alkylating agent such as ethylnitrosourea (ENU) is preferred for the production of mutants in rodents however other efficient mutagens may be used such as, for example, chlorambucil.
- ENU mutagenesis is mainly point mutations. Many of the mutations caused by ENU will therefore be hypomorphic partial loss of function mutations although gain of function and complete loss of function mutations are also contemplated. Protocols are established which allow very efficient mutagenesis rates in several mice strains. An advantage of ENU treatment is that it results in random mutations being introduced into premeiotic spermatogonial stem cells and eventually sperm cells.
- mice are mated to untreated females to produce first generation (GI) progeny which are heterozygous for a set of random mutations inherited from their father. These GI progeny are scored phenotypically and a large number of progeny are analysed in order to review the effects of the induced mutations on the mouse model.
- the progeny analysed in accordance with the present invention may be GI, G2, G3 or subsequent progeny generated by breeding GI, G2 or G3 mice.
- mice When mice are identified which exhibit an ameliorated disease or condition, they are bred further to determine whether the phenotype/mutation is inheritable. Further breeding experiments may be conducted to compare the phenotype of the homozygous and heterozygous form of the mutation against various genetic backgrounds.
- Positional cloning of the target molecule in confirmed candidates may be achieved following a number of alternative routes as known to those of skill in the art.
- Linkage studies employing sequence length polymorphisms such as SSLP markers is a standard preferred strategy for genetic mapping of mouse loci via an outcrossing/backcross mating system between the candidate mouse strain and a marker strain.
- genetic markers not linked to the mutation exhibit no correlation between genotype and phenotype.
- Further markers may be identified by standard procedures (Sambrook et al, Molecular Cloning, A Laboratory Manual CSH Press, Cold Spring Harbour USA, 1989) to further focus on the genetic sequences encompassing the mutation of interest.
- flanking markers are amplified from DNA samples and sequenced in order to confirm the sequence of the region and identify the mutated sequences.
- a candidate gene approach may be available which simplifies the identification of the responsible mutation. Specifically, genes or sequences considered to be involved in a particular disease or condition and which are detected in a candidate region identified by positional cloning are preferentially sequenced or assessed geneotypically for mutations. Homologous sequences may be identified in the available databases or by hybridisation- based strategies.
- Diseases and conditions to which the present invention can be best applied relate to those diseases for which current treatments are inadequate and for which new targets for pharmaceutical development are required, and for which there are good model animals.
- the present invention is described using mouse models of human disease however, any laboratory animal can be employed which is commonly used in genetic studies and for which a desired therapeutic or physiological effect can be assessed. Rodents are preferred model animals.
- the mouse is a most preferred model animal because of the large number of available mouse models of human disease. Table 2 and 2a provides non-exhaustive list of examples of suitable mouse models of human disease.
- the present invention extends to the use of vertebrate animal models in which genetic studies are feasible and to the use of random or non-random mutagenesis.
- the following description concentrates on describing embodiments of the invention which use murine animal models with random point mutation created by infecting male mice with ENU.
- the disease or condition is caused by a single transgene or an engineered or random mutation in a single gene.
- the disease may be manifest in a recessive manner or a dominant manner.
- mice with the disease remain viable and fertile; where as in more complicated situations the disease may be lethal or render mice infertile.
- the situation may require mutations in two or more genes or a combination of a transgene and an engineered or random mutation before the disease is manifest.
- the physiological assessment system can be employed by one skilled in the art to enable the identification of targets for therapeutic intervention for these many classes of heritable disease.
- Some of the breeding strategies also lead to gains of efficiency, in that the proportion of mice that is informative is increased over more conventional approaches.
- mice that are homozygous for the disease causing mutation are injected with ENU and bred to females also homozygous for the disease causing mutation.
- the first generation (GI) progeny are then screened to determine whether the disease is ameliorated relative to non-mutated mice homozygous for the disease causing mutation.
- This scenario is applicable wherein, for example, a recessive disease model leaves animals fertile and viable.
- mice with ameliorated disease are then tested to determine whether the phenotype is heritable by crossing them to non-mutagenised mice homozygous for the disease-causing mutation. If approximately half of the resultant progeny have the course of the disease ameliorated then the disease suppression is likely to be heritable and to be caused by an ENU-induced- mutation that may be mapped and identified by conventional genetic and genomic methods, such as those described herein.
- GI mice are intercrossed or crossed to non-mutagenised mice homozygous for the disease-causing mutation.
- the resultant G2 mice are then either intercrossed or backcrossed to their GI parent to yield a cohort of G3 progeny that are screened to determine whether the disease is ameliorated relative to non-mutated mice homozygous for the disease causing mutation.
- mice with a suppressed phenotype are found among the G3 progeny, they are then tested for example by one or more of the following crosses to determine whether the phenotype is heritable, (i) Suppressed G3 mice will be crossed to non-mutagenised mice homozygous for the disease-causing mutation, (ii) G3 mice with suppressed disease will be intercrossed, (iii) Non-suppressed siblings will be crossed to suppressed G3 mice, (iv) Non-suppressed siblings will be intercrossed.
- mice with a suppressed disease phenotype By monitoring the proportion of mice with a suppressed disease phenotype among the various test crosses one skilled in the art should be able to determine whether phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner. Where disease suppression is found to be heritable, the relevant ENU-induced mutation is mapped and identified by conventional genetic and genomic methods, such as those described herein.
- mice with ameliorated disease in any particular cross will vary. However, a reduction in the penetrance of the suppression of disease does not preclude identification of the causative ENU induced mutation.
- Many mouse models of human disease can be utilized using the methods revealed and summarized in Scenario 1 , including Mpl " " thrombocytopenia described herein and others listed in Table 2 and 2a.
- Mpl " thrombocytopenia described herein and others listed in Table 2 and 2a.
- One skilled in the art will recognise that many others can be found in publicly available databases (e.g. http://research.bmn.com/mkmd, http://informatics.jax.org).
- animal models are used in homozygous form. This embodiment is applicable for dominant disease animal models that leave animals fertile and viable (Scenario 2). If mice homozygous for the disease causing mutation are viable and fertile then one can essentially follow Scenario 1, even though the disease is dominant.
- the advantage of this is strategy compared with injecting a male mouse that is heterozygous and mating it to an unaffected female or injecting an unaffected male and mating it to a heterozygous female is that all, rather than half of the GI progeny are informative, since they carry the disease causing mutation and hence can be screened for suppression of disease.
- homozygous male mice are treated with ENU and mates to animals that do not carry the disease causing- mutation.
- male mice that do not carry the disease-causing mutation are treated with ENU and mated to females that are homozygous for the disease-causing mutation.
- all of GI progeny will be heterozygous for the disease causing mutation and hence all can be screened for ENU-induced mutations that suppress disease.
- mice homozygous for the disease-mutation throughout every stage of the breeding program is even more advantageous if G3 progeny are to be screened, since again, all of the G3 animals may then be screened for ENU-induced mutations that suppress disease.
- mice with two copies of the disease-causing mutation are healthy and fertile, this presents no difficulties (Scenarios 1 and 2); however, often modelling a disease in mice results in animals that succumb to the effect of the disease or have reduced fertility and fecundity because of the disease.
- mice that carry the disease-causing mutation but do not manifest disease are used in some embodiments.
- the mice are heterozygous for the disease-causing mutation.
- male mice heterozygous for the disease causing mutation are injected with ENU and mated to female mice also heterozygous for the mutation to generate GI pups. 75% of the pups are not useful since they carry no copies or one copy of the disease-causing mutation, will not get the disease and hence cannot be screened to determine whether an inherited ENU-induced mutation is capable of suppressing the disease.
- these non-diseased mice must be discriminated from the rare cases among the remaining 25% of animals which carry two copies of the disease-causing mutation but which have the onset or progression of the disease ameliorated because of an ENU-induced mutation.
- discrimination may be achieved by genotyping the GI mice to allow the number of disease-causing mutations in each individual to be measured.
- genotyping may be achieved by genotyping the GI mice to allow the number of disease-causing mutations in each individual to be measured.
- One skilled in the art would appreciate that there are many well-established methods of detecting disease-causing mutations whether they are large or small insertions and/or deletions of nucleotides or point mutations. These methods include Southern blotting following restriction enzyme digestion of genomic DNA and a range of proprietary and non-proprietary fluorescent-based techniques for discriminating single nucleotide polymorphisms (SNPs).
- SNPs single nucleotide polymorphisms
- An alternative means of discriminating between animals that carry zero and one copy of the disease-causing mutation from those that carry two copies is by genetically linking the disease-causing mutation to another mutation or polymorphism that alters an easily measured trait.
- a semi-dominant coat-colour marker such as a transgene expressing the agouti protein may be introduced as part of the gene targeting process.
- the coat-colour of the parental strain is black, this may allow mice with two copies of the disease causing mutation (and hence two copies of the agouti transgene) to be identified because of their light brown or yellow coat, mice with one copy of the disease causing mutation (and hence one copy of the agouti transgene) to be identified because of their agouti/dark brown coat and mice with no copies of the disease causing mutation (and hence no copies of the agouti transgene) to be identified by their black coat.
- mice that were heterozygous for this pair of linked mutations were treated with ENU and bred to females also heterozygous for this pair of linked mutations.
- the resultant progeny with non or one copy of the disease-causing Socsl mutation are agouti in colour and discarded. It is then possible to monitor the black mice to determine whether their disease is suppressed. If a black animal appears healthy they can be genotyped to determine whether they carry two copies of the disease-causing Socsl mutation or whether there has been a recombination between the Socsl mutation and the coat-colour marker mahoganoid. The closer the disease-causing mutation and the coat-colour mutation are, the less frequently that this recombination will occur.
- the general idea of this method is to generate strains of animals which carry the disease causing mutation in a latent form and are healthy and fertile; but which when mated yield offspring that all carry the disease-causing mutation and exhibit the disease unless its course is modified by a disease-suppressing ENU induced mutation.
- the creation of latent disease-causing alleles may be achieved using site-specific DNA recombinases such as cre-recombinase, which recognizes lox sites and flp recombinase, which recognizes frt sites.
- site-specific DNA recombinases such as cre-recombinase, which recognizes lox sites and flp recombinase, which recognizes frt sites.
- cre/lox and flp/frt to create conditional disease- causing mutations. These include creation of loss of function alleles through deletion of all or part of a gene or through insertion of foreign DNA into a gene or through expression of a transgene from an exogenous promoter. In each the principle is similar; one animal is created in which lox sites or frt sites flank the salient piece of DNA.
- mice of these two strains are crossed the recombinase (ere or flp) can act to delete or rearrange the DNA flanked by the relevant sites (lox or frt) to create an active disease-causing allele.
- one strain is generated that carries two copies (i.e. is homozygous) for the latent (lox or frt flanked) disease causing mutation and an other strain is generated which has two copies (i.e. is homozygous) for the relevant transgenic recombinase construct.
- ENU is injected into male mice of one of the strains and they are mated to females of the other strain. All of the resultant pups will inherit the recombinase transgene and hence express the recombinase protein.
- GI animals can then be monitored to identify those individuals that do not succumb to the disease. These animals are then mated and by monitoring the proportion of mice with a suppressed disease phenotype among the various test crosses one skilled in the art should be able to determine whether phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner. Where disease suppression is found to be heritable, the relevant ENU- induced mutation may be mapped and identified by conventional genetic and genomic methods, such as those described herein.
- the disease is manifest in a recessive manner (i.e. requires two active copies of the disease-causing mutation) it is preferred to create one strain of mice that is homozygous for the latent-disease causing mutation flanked by lox sites and which is homozygous for the transgene that expresses flp recombinase and a second strain of mice which that is homozygous for the latent-disease causing mutation flanked by frt sites and which is homozygous for the transgene that expresses ere recombinase.
- males of one of these strains are then injected with ENU and mated to females of the other strain.
- This basic principle of creation of animals with latent disease-causing mutations and crossing them to animals with the required recombinase to activate the mutation can be extended to more complicated diseases that require two or more different types of mutation in order to manifest.
- mice in which the disease-causing mutation creates a mild reduction in health or fertility might be utilized using methods described in Scenario 1 or Scenario 3 or alternatively Scenario 2 or 3.
- strategies outlined as follows are contemplated in some embodiments in which the disease or condition is experimentally induced (Scenario 4).
- Some mouse models of human, disease rather than occurring spontaneously in mutant animals must be induced experimentally either in wild type mouse strains or genetically modified mice. Examples of induced diseases include without limitation ischaemic injury, inflammation and infection.
- animals of the salient genetic background are treated with ENU and mated to the required mice to generate GI animals of a genetic background known to be susceptible to the induction of the disease. Once GI animals have reached the required age, the disease is induced and its course is monitored to allow those mice in which the disease course has been ameliorated to be identified.
- mice with an ameliorated physiological assessable symptom suppressed disease phenotype
- one skilled in the art is able to determine whether the phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner.
- the responsible ENU-induced mutation is mapped and identified by conventional genetic and genomic methods, such as those described herein.
- the physiologically assessable animal model of a human disease is a mouse model of thrombocytopenia which lacks normal TPO receptor signalling through Mpl and exhibits subnormal platelet levels and/or qualitative changes in megakaryocytopoiesis.
- male c-Mp ' ' mice were subjected to ENU mutagenesis, bred with female c-Mpl' ' mice and their progeny assessed for platelet numbers.
- GI mice showing elevated platelet levels were mated to untreated mice of the same genetic background and their G2 progeny assessed for platelet levels to determine if the altered phenotype is heritable.
- thrombocytopenia include vertebrate animals in which c-Mpl is down regulated or inhibited. Such inhibition may be mediated for example using antibodies to the thrombopoietin (TPO) receptor. Mutagenesis may also be conducted on the wild type precursor of the animal model which is subsequently transformed or its progeny are transformed into a thrombocytopenia model by administration of an inhibitory anti-Mpl antibody or receptor antagonist. Inhibitory agents other than antibodies are contemplated such as antisense or co-suppression molecules or constructs which effectively down regulate Mpl activity including it's downstream signalling activity in the model.
- TPO thrombopoietin
- another embodiment of the present invention provides a physiological assessment system to identify a pedigree of a vertebrate animal model of thrombocytopenia which exhibits ameliorated symptoms of thrombocytopenia comprising:
- Progeny are preferably GI, G2 or G3 progeny and subsequent progeny generated by breeding GI, G2 or G3 animals.
- thrombocytopenia Various rodent models of thrombocytopenia are contemplated exhibiting altered platelet numbers compared to normal levels, or qualitative changes in megakaryocytopoiesis. Such pedigrees are useful for defining further members of the TPO/Myb pathway as defined herein and other pathways involved in megakaryocytopoiesis.
- models include mutants with, inter alia, recessive thrombocytopenia on a Mpf + background (Pltl, Figure 10a), a mutant with recessive thrombocytosis on a Mp /+ background (Plt2), a mutant with recessive exacerbation of thrombocytopenia (mldl), and a mutant with dominant alterations in platelet granularity.
- a preferred rodent model of thrombocytopenia is a Mpl' ' rodent or a rodent in which Mpl is down regulated or inhibited. Regulation may be achieved using inducible promoters to inhibit or knock out all or part of Mpl. Inhibition may be mediated for example using antibodies to the thrombopoietin (TPO) receptor.
- TPO thrombopoietin
- one or more of Plt3, Plt4 and Plt6 rodent pedigrees are disclosed each exhibiting elevated platelet levels.
- the animal models of the present invention may be in the form of the animals or may be, for example, in the form of embryos stem cells or gamete for transplantation.
- the embryos satem cells or gametes are preferably maintained in a frozen state and may optionally be sold with instructions for use.
- the present invention also provides an assessment system to identify genetic or proteinaceous drug targets associated with elevating platelet levels or ameliorating the symptoms of thrombocytopenia comprising:-
- step (ii) assessing platelet levels or symptoms of thrombocytopenia in the mutated rodent and its progeny wherein if a wild type rodent is used in step (i) these animals are subjected to inhibition or down regulation of Mpl activity prior to assessment;
- the present invention provides a rodent or mouse model with reduced Mpl levels when used to screen for drug targets to treat thrombocytopenia or other conditions characterised by low platelet levels.
- Progeny are preferably GI, G2 or G3 progeny and subsequent progeny generated by breeding GI, G2 or G3 mice.
- the present invention further provides an assessment system to identify genetic or proteinaceous targets associated with elevating platelet levels or ameliorating the symptoms of thrombocytopenia comprising:
- any convenient and well characterized rodent strain or intercross, backcross or outbred derivatives of such strains may be used to develop the model of thrombocytopenia such as, for example, C57B1/6 or C3H strains.
- a wide range of mouse strains is described in, for example, The Jackson Laboratory website.
- the present invention provides genetic or proteinaceous drug targets identified using the instant physiological assessment method, or derivatives, variants, functional derivatives, or homologs thereof as a drug target for use or when used, or to screen for or develop agonists or antagonist compounds useful, in the treatment of the symptoms of a particular disease of condition.
- agonists of inhibitory interacting molecules in the genetic net work of the target will also be therapeutic or targets for development of therapeutics and are expressly encompassed, where appropriate.
- Drug target are particularly contemplated for use, or when used, to screen for or develop interacting compounds useful in the treatment and/or prophylaxis of thrombocytopenia or other conditions characterized by low platelet levels.
- the present invention particularly provides a genetic or proteinaceous form of Myb transcription factor signaling pathway and other interacting members of the genetic network comprising Myb or derivatives thereof, variants, functional derivatives, homologs thereof as a drug target for use in screening or developing interacting compounds useful in the treatment of thrombocytopenia or other conditions characterized by low platelet levels.
- the inhibition or down regulation of Myb expression or activity may be a critical function of the TPO signalling pathway which is necessary for effective megakaryocytopoiesis. Specifically, mutations in Myb cause a myeloproliferative syndrome and supra- physiological expansion of megakaryocyte and platele production in ⁇ ⁇ ' mice.
- SEQ ID NO: 1 represents the nucleotide sequence encoding murine Myb.
- SEQ ID NO: 2 represents the amino acid sequence of murine Myb.
- SEQ ID NO: 3 represents the nucleotide sequence encoding human Myb.
- SEQ ID NO: 4 represents the amino acid sequence of human Myb.
- Myb is used herein to refer to nucleic acid and/or proteinaceous forms of Myb as the context in which the term is used makes clear. In its broadest form, the term encompasses Myb from any organism but preferably a vertebrate organism.
- Target molecules in nucleic acid form include nucleic acid molecules comprising a nucleotide sequence capable of hybridising to the target molecule or its complementary form under low stringency conditions.
- the present invention contemplates a drug target for use in the identification of antagonists or agonists which effectively up regulate platelet levels or ameliorate the symptoms of thrombocytopenia in a subject.
- the functional activity of a target molecule may be modulated by agonising or antagonising the target in genetic or proteinaceous form.
- agonists of inhibitory interacting molecules in the Myb genetic network will also be therapeutic or targets for the development of therapeutics and are expressly encompassed.
- the present invention also provides methods of screening for agonists or antagonists of the identified drug targets comprising contacting the drug target with a compound and assaying for (i) the presence of a complex between the drug target and a compound or (ii) for the presence of complex between the drug target and a ligand, by methods well known in the art. In such competitive binding assays the drug target or the ligand is labelled in order to assess the activity of the compound.
- the present invention also provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target to a compound and assaying for:-
- Target molecule may be expressed recombinantly or occur naturally or be upregulated in cells or cell lines which are useful in in vitro screens for agonists or antagonists.
- Natural products include those from coral, soil, plant or the ocean or antarctic environments.
- Two-hybrid screening is another useful method for identifying other members of a genetic network associated with or comprising a drug target.
- Target interactions and screens for inhibitors can be carried out using the yeast two-hybrid system, which takes advantage of transcriptional factors that are composed of two physically separable, functional domains.
- the most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA binding domain and a transcriptional activation domain.
- Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and if interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable phenotype.
- a reporter gene e.g. lacZ
- S. cerevisiae is co-transformed with a library or vector expressing a cDNA GAL4 activation domain fusion and a vector expressing a target pathway component fused to GAL4.
- lacZ is used as the reporter gene, co- expression of the fusion proteins will produce a blue colour.
- Small molecules or other candidate compounds which interact with a target will result in loss of colour of the cells.
- the present invention provides a method for the treatment or prophylaxis of a disease or condition comprising administering a therapeutic amount of a compound which modulates the activity of a herein described target molecule in genetic or proteinaceous form.
- the present invention provides a method for the treatment or prophylaxis of thrombocytopenia comprising administering a therapeutic amount of a compound which modulates the activity of herein described target molecule.
- the present invention provides a method for identifying compounds useful in the treatment or prophylaxis of thrombocytopenia comprising screening and/or developing compounds for their ability to modulate the functional activity of the herein disclosed target molecules.
- a drug target comprising a sequence of amino acids set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or a functional derivative or homolog thereof having at least 60% similarity thereto or a sequence of nucleotides encoding SEQ ID NO: 2 or SEQ ID NO: 4 or as set forth in SEQ ID NO:l or SEQ ID NO: 3 or a functional derivative or homolog having at least 60% similarity thereto or a sequence of nucleotides capable of hybridising to SEQ ID NO: 1 or SEQ ID NO: 3 its complement under conditions of low stringency hybridisation when used to screen for antagonists or antagonists which elevate platelet levels or ameliorates the symptoms of thrombocytopenia.
- domains of Myb which contribute to its functional effect on megakaryocytopoiesis are specifically targeted.
- the Plt3 mutation occurs in the sequence of nucleotides encoding the DNA binding domain and the Plt4 mutation occurs in the sequence of nucleotides encoding the leucine zipper domain.
- the subject antagonists or agonists compounds elevate platelet levels in vivo and/or in vitro.
- the present invention provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to modulate the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.
- the present invention provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to antagonise the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.
- Loss of Myb is lethal in fetal life resulting in a dyplastic state in which platelet formation is excessive but red cell and B-lymphocytic formation is reduced.
- the phenotypic abnormalities associated with antagonising the functional activities of Myb specifically in the DNA binding domain and leucine zipper domain are recessive except those in the platelet/megakaryocyte lineage.
- the present invention provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to agonise the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.
- the present invention also provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to antagonise the functional activity of genetic or proteinaceous forms of c-Myb.
- the present invention also provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to agonise the functional activity of genetic or proteinaceous forms of c-Myb.
- the target molecules are usefully in isolated or recombinant form for screening purposes. Accordingly, the present invention further provides recombinant nucleic acids including a recombinant construct comprising all or part of the drug target in nucleic acid form.
- 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. Reporter constructs are useful where the target molecule comprises promoters or enhancers.
- the isolated or recombinant drug targets of the instant invention are used to identify or engineer agonists and antagonists.
- modulators may be used directly or they may be further modified by methods well known in the art in order to improve their effectiveness as pharmaceutical, diagnostic or other reagents.
- Other considerations for an active compound include formulation and method of delivery.
- An agonist or antagonist includes molecules determined by all or part of the drug target or a variant of the drug target such as antibodies, mimetics or antisense molecules.
- Antibodies including anti-idiotypic antibodies, chaemeric antibodies and humanised antibodies are useful in this regard and their generation is now routine to those of skill in the art.
- Peptide or non-peptide mimetics can be developed as agonists of the drug targets 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.
- Antisense polynucleotide sequences are another useful example of a therapeutic agent which can modulate the activity of target molecules, as will be appreciated by those skilled in the art.
- Polynucleotide vectors for example, containing all or a portion of Myb sequences or other sequences from an Myb region (particularly those flanking a Myb gene locus) may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with gene transcription and/or translation.
- co-suppression and mechanisms to induce RNAi i.e. siRNA
- Such techniques may be useful to inhibit genes which positively promote target molecule gene expression and particularly Myb gene expression.
- antisense or sense molecules may be directly administered. In this latter embodiment, the antisense or sense molecules may be formulated in a composition and then administered by any number of means to target cells.
- morpholinos are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development, 7:187-195, 1997). Such compounds are injected into embryos and the effect of interference with mRNA is observed.
- the present invention employs compounds such as oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding a target molecule, i.e. the oligonucleotides induce transcriptional or post- transcriptional gene silencing. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding the transcription factor.
- target nucleic acid and “nucleic acid molecule encoding a transcription factor” have been used for convenience to encompass DNA encoding a target proteinaceous molecule, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
- RNA including pre-mRNA and mRNA or portions thereof
- cDNA derived from such RNA.
- antisense The hybridization of a compound of the subject invention with its target nucleic acid is generally referred to as "antisense”.
- antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
- 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.
- the functions of 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 a target gene.
- modulation and modulation of expression mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.
- An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
- compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
- these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops.
- the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
- RNAse H a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
- antisense compound is a single-stranded antisense oligonucleotide
- dsRNA double-stranded RNA
- oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.
- the compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).
- nucleobases i.e. from about 8 to about 80 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.
- nucleoside is a base-sugar combination.
- the base portion of the nucleoside is normally a heterocychc base.
- the two most common classes of such heterocychc bases are the purines and the pyrimidines.
- Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
- the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
- the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
- linear compounds are generally preferred.
- linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
- the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
- the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
- oligonucleotides containing modified backbones or non-natural internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
- modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
- Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphoro-thioates, phosphoro-dithioates, phosphotri-esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5
- Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
- Various salts, mixed salts and free acid forms are also included.
- the goal of rational drug design is to produce 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.
- 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 .
- 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
- 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 2b.
- peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
- the present invention provides methods for screening for agonists or antagonists of the identified drug targets comprising contacting the drug target with a compound and assaying for (i) the presence of a complex between the drug target and a compound or (ii) for the presence of complex between the drug target and a ligand, by methods well known in the art. In such competitive binding assays the drug target or the ligand is labelled in order to assess the activity of the compound.
- the present invention also provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target to a compound and assaying fo ⁇ -
- the present invention further provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target comprising all or part of Myb or a nucleotide sequence encoding same to a compound and assaying for:-
- the present invention furthermore provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target comprising all or part of the DNA binding domain of Myb or a nucleotide sequence encoding same to a compound and assaying for:-
- the present invention provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target comprising all or part of the leucine zipper domain of Myb or a nucleotide sequence encoding same to a compound and assaying for:-
- Target molecule may be expressed recombinantly or occur naturally or be upregulated in cells or cell lines which are useful in in vitro screens for agonists or antagonists.
- Natural products, combinatorial, synthetic/peptide polypeptide or protein libraries or phage display technologies are all available for screening for modulators. A huge choice of high through put screening methods are available. Natural products include those from coral, soil, plant or the ocean or antarctic environments.
- Two-hybrid screening is also useful in identifying other members of a genetic network associated with or comprising a drug target.
- Target interactions and screens for inhibitors can be carried out using the yeast two-hybrid system, which takes advantage of transcriptional factors that are composed of two physically separable, functional domains.
- the most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA binding domain and a transcriptional activation domain.
- Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and if interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable phenotype.
- a reporter gene e.g. lacZ
- S. cerevisiae is co-transformed with a library or vector expressing a cDNA GAL4 activation domain fusion and a vector expressing a Myb pathway component fused to GAL4.
- lacZ is used as the reporter gene, co- expression of the fusion proteins will produce a blue colour.
- Small molecules or other candidate compounds which interact with a target will result in loss of colour of the cells.
- Another aspect of the subject invention provides a method for the treatment or prophylaxis of thrombocytopenia comprising administering a therapeutic amount of a compound which modulates a drug target identified by the herein disclosed physiological assessment system.
- the present invention provides a method for the treatment or prophylaxis of thrombocytopenia comprising administering a therapeutic amount of a compound which modulates one or more components of the Myb transcription factor signalling pathway to effectively modulate the activity of Myb.
- a compound which modulates one or more components of the Myb transcription factor signalling pathway to effectively modulate the activity of Myb.
- TPO appears to be a part of the Myb signally pathway and is already proposed for use in ameliorating thrombocytopenia this use of TPO is not proposed herein to part of the present invention.
- the subject invention presents a method for the treatment of thrombocytopenia or conditions characterized by low platelet numbers comprising administering a therapeutic amount of a compound which antagonises or agonises one or more components of genetic or proteinaceous forms of a Myb transcription factor signalling pathway to effectively down regulate the activity of Myb.
- the subject invention further presents a method for the treatment of thrombocytopenia or conditions characterized by low platelet numbers comprising administering a therapeutic amount of a compound which antagonises or agonises one or more components of the Myb transcription factor signalling pathway to effectively up-regulate the activity of Myb.
- the present invention provides pharmaceutical compositions comprising recombinant synthetic or isolated forms of the present drug targets and one or more pharmaceutically acceptable carriers, diluents or excipients and furthermore contemplates methods of their use in vitro or in vivo in methods for the treatment or prophylaxis of subjects and in particular humans with or likely to develop the symptoms of thrombocytopenia.
- the present invention contemplates the use of a modulator of the Myb transcription factor signaling pathway in the manufacture of a pharmaceutical composition for the treatment of thrombocytopenia.
- the present invention provides the use of an antagonist of Myb in the manufacture of a pharmaceutical composition for the treatment of thrombocytopenia.
- the present invention is directed to the treatment of the symptoms of thrombocytopenia or the elevation of platelets in any organism of commercial or humanitarian interest including, primates, livestock animals including fish and birds, laboratory test animals, companion animals, or captive wild animals.
- the present invention extends to the elevation of platelet levels in vitro or ex vivo.
- the polypeptides, nucleic acids, antibodies, peptides, chemical analogs, agonists, antagonists 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.
- 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 intrathecally, 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.
- the modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about O.lmg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg. 0.9mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
- the modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules).
- the modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application).
- acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
- the tablet may contain a binder such as tragacanth, com starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
- a binder such as tragacanth, com starch or gelatin
- a disintegrating agent such as alginic acid
- a lubricant such as magnesium stearate.
- Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant.
- 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 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. 0 425 731 A and International Patent Publication No. WO 90/07936.
- the genetic or proteinaceous targets of the present invention may also have a range of diagnostic utilities.
- the detection or level of the target molecule or an aberrant form thereof is indicative a subjects propensity to develop thombocytopenia or response to therapy.
- the present invention extends to antibodies and other immunological agents directed to or preferably specific for the mammalian transcription factors or a fragment thereof.
- the antibodies may be monoclonal or polyclonal or may comprise Fab fragments or synthetic forms.
- Specific antibodies can be used to screen for the subject target molecules and/or their fragments.
- Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
- second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies referred to above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti- immunoglobulin antibody.
- An antibody as contemplated herein includes any antibody specific to any region of the mammalian transcription factors.
- Both polyclonal and monoclonal antibodies are obtainable by immunization with the subject target molecules or antigenic fragments thereof and either type is utilizable for immunoassays.
- the methods of obtaining both types of sera are well known in the art.
- Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of subject polypeptide, or antigenic parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techniques.
- antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
- 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 derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
- Another aspect of the present invention contemplates, therefore, a method for detecting a target molecule such as Myb or a member of the Myb transcription factor signalling pathway or fragment thereof in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for said Myb or a member of the Myb transcription factor signalling pathway or fragment thereof or its derivatives or homologs for a time and under conditions sufficient for an antibody-polypeptide complex to form, and then detecting said complex.
- a target molecule such as Myb or a member of the Myb transcription factor signalling pathway or fragment thereof in a biological sample from a subject
- the presence of the instant target molecules or their fragments may be detected in a number of ways such as by Western blotting and ELISA procedures.
- a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653.
- Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
- an unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
- a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody.
- the sample is one which might contain a subject target molecule including blood, bone marrow, spleen .
- the sample is, therefore, generally a biological sample comprising biological fluid.
- the Myb transcription factor for example is likely to be in blood.
- a first antibody having specificity for the instant polypeptide or antigenic parts thereof is either covalently or passively bound to a solid surface.
- the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
- the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
- the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
- an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the antibody.
- the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
- the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
- An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
- the present invention also contemplates genetic assays such as involving PCR analysis to detect RNA expression products of a genetic sequence encoding a target molecule.
- the genetic assays may also be able to detect nucleotide polymorphisms or other substitutions, additions and/or deletions in the nucleotide sequence of a mammalian transcription factor. Changes in levels of target molecule expression such as following mutations in the promoter or regulatory regions or loss of target activity is proposed to be indicative of a disease condition or a propensity for a disease condition to develop. For example, a cell biopsy could be obtained and DNA or RNA extracted.
- SSCP single stranded conformation polymorphoms analysis
- FNC first nucleotide change
- the present invention extends to polymorphisms in Myb genetic sequence genes which leads to alleviation of the symptoms of thrombocytopenia. For example a mutation at about nucleotide A455 or Al 151 in the Myb gene leads to amelioration of the symptoms of thrombocytopenia.
- kits to facilitate the rapid detection of the target molecules or their fragments in a subject's biological fluid.
- a biological fluid includes a cell extract such as a DNA/RNA extract.
- Non-conventional Code Non-conventional Code amino acid amino acid
- Non-conventional Code Non-conventional Code amino acid amino acid
- Non-conventional Code Non-conventional Code amino acid amino acid
- Non-conventional Code Non-conventional Code amino acid amino acid
- Mpl 1' mouse model for thrombocytopenia has a deletion of the receptor, termed Mpl, for thrombopoietin which is the major platelet producing hormone.
- mice Male Mpl' ' mice were treated with N-Ethyl-N-Nitrosourea (ENU) according to the method of Bode (Bode, 1984). Briefly, ENU (N3385, Sigma Chemical Company) was dissolved in 5 ml of ethanol and diluted with 50 mM sodium citrate pH 5.0 and was used within four hours. The concentration of the ENU was determined spectrophotometrically at 395 nm.
- ENU N-Ethyl-N-Nitrosourea
- mice Male mice were then injected intraperitoneally with one dose of 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg or 400 mg/kg, two weekly doses of 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg or 200 mg/kg or three weekly doses of 66 mg/kg, 83 mg/kg, 100 mg/kg, 116 mg/kg or 133 mg/kg.
- ENU-treated mice were mated with one or two female Mpl' ' mice. Following a period of sterility, the length of which increases as the total dose of ENU increases, first-generation (GI) progeny were produced.
- GI first-generation
- GI mice which had platelet numbers that were greater than 3.00x10 5 / ⁇ l (three standard deviation or more the mean value) were tested to determine whether their phenotype is heritable.
- GI mice were mated to untreated mice of the same genetic background and their G2 progeny were bled at 7 weeks of age and circulating platelet numbers were determined using the Advia Automated Analyser. Initially 10 G2 mice were analysed from each GI mouse. For a fully penetrant dominant phenotype one would expect approximately half of the mice to be affected. Given the likelihood of erroneously discarding a pedigree is less than 1 in 1000 (i.e.
- Mpl' ' mice were treated with the ENU as described above, which results in random mutations being introduced into the DNA of the spermatogonial stem cells and ultimately the sperm (Ranchik Trends in Genetics 7:15-21, 1991). These mice were mated to untreated female Mpl' ' mice to produce first generation (Gi) progeny, which are heterozygous for a set of ENU-induced mutations inherited from their father. At 7 weeks of age GI mice were bled and their peripheral blood platelet count was determined.
- GI mice (2037 mice) were examined for their platelet levels and of these, 7 exhibited platelet counts of more than 3.00xl0 5 / ⁇ l, that is they had platelet counts of more than 3 standard deviations from the mean of untreated Mpl' ' mice and were hence candidates which might carry an ENU-induced mutation that ameliorated thrombocytopenia (Figure IC).
- mice The remaining two mice (985.34 and 1118.11) did however appear to have heritable suppression of thrombocytopenia since approximately half of their progeny exhibited platelet counts of more than 3.00x10 5 / ⁇ l, while the remaining mice exhibited the very low platelet counts characteristic of Mpl' ' mice ( Figure ID); the resulting pedigrees were named Plt3 and Plt4.
- Mature megakaryocyte numbers were determined by microscopic examination of hematoxylin and eosin-stained histological sections of sternal bone marrow and spleen. Megakaryocytes were readily recognisable by their large size and distinctive morphology.
- DMEM Dulbecco's modified Eagle's medium
- EPO human erythropoietin
- Cytokines were obtained from the commercial sources indicated or produced in our own laboratories by expression of recombinant proteins in Pichia pastoris or E.coli and purified prior to use. Agar cultures were fixed in 2.5% glutaraldehyde, sequentially stained for acetylcholinesterase, Luxol fast Blue and hematoxylin, and the cellular composition of each colony determined by microscopic examination at 100 to 400- fold magnification. These conditions allowed optimal stimulation of neutrophil, neutrophil- macrophage, macrophage, eosinophil, megakaryocyte, erythroid, multilineage and blast cell colony-forming cells (CFC).
- CFC blast cell colony-forming cells
- hematopoiesis Analysis of general effects on hematopoiesis were conducted by measurements of hematocrits as well as total peripheral blood white cell counts, the latter by performing manual counts using hemocytometer chambers and via automated analysis.
- the relative numbers of morphologically recognisable precursor cells in hematopoietic organs were assessed by manual 100 to 400 cell leukocyte differential counts of peripheral blood, bone marrow, liver and spleen following preparation of smears or cytocentrifuge preparations stained with May-Grunwald-Giemsa.
- the relative numbers of hematopoietic cells expressing lineage-specific cell-surface markers were measured.
- Single cell suspensions of bone marrow, spleen and thymus from adult mice of each genotype were incubated with saturating amounts of 2.4G2 anti-Fc8 receptor antibody to reduce background staining, then with specific monoclonal antibodies to murine cell surface antigens: anti CD4 and CD8, IgM, Ly5-B220, Mac-1, F4/80, Gr-1, Ter-119, and Thyl.2 (Pharmingen, Torrey Pines, CA).
- Antibodies may be directly coupled to fluorescein isothiocyanate (FITC) or biotin, the latter being visualised with R-phycoerythrin- streptavidin.
- FITC fluorescein isothiocyanate
- biotin biotin
- Flow cytometric analyses were performed on a FACScan analyser (Becton- Dickinson, Franklin Lakes, NJ) with dead cells and eiythrocytes excluded by propidium iodide (lmg/ml) staining and gating of forward angle and side scatter of light.
- Histological sections of all major organs were also prepared by standard techniques, stained with hematoxylin and eosin and examined by light microscopy for evidence of abnormality.
- CFU-s were enumerated by intravenous injection of bone marrow cells into recipient mice that had been irradiated with l lGy of ⁇ - irradiation given in two equal doses given three hours apart from a 137c s source (Atomic Energy, Ottawa, Canada). Transplanted mice were maintained on oral antibiotic (l .lg/L neomycin sulfate; Sigma, St. Louis, MO). Spleens were removed after 12 days, fixed in Carnoy's solution (60% ethanol, 30% chloroform, 10% acetic acid), and the numbers of macroscopic colonies were counted.
- the megakaryocytes, megakaryocyte, progenitor cells (Meg- CFCs) and primitive multipotential haemopoietc cells (blast-CFCs) in the bone marrow and spleen were measured. These cells are the precursors of platelets.
- the presence of the Plt3 and Plt4 mutations increased the numbers of all of these cells suggesting that the effect of these mutations is to elevate the output of platelets by increasing the production of their cellular precursors ( Figure 3 A and 3B).
- a key step in using the information gleaned from generation and analysis of Plt3 and Plt4 mice in the development of agents to treat thrombocytopenia is the discovery of the gene or genes that are mutated in these animals.
- Plt3 and Plt4 are linked, heterozygous Plt3/+ Mpl' ' and PU4/+ MpX 1' mice on a C57BL/6 background were crossed and the mice that were putatively heterozygous for both genes were identified at 7 weeks of age because of their extremely high platelet counts (> 2 x 10 6 / ⁇ l).
- mice were then bred with Mpl' ' mice on a C57BL/6 background and the platelet numbers of progeny were determined at 7 weeks.
- Mpl' ' mice mice were then bred with Mpl' ' mice on a C57BL/6 background and the platelet numbers of progeny were determined at 7 weeks.
- all of the progeny from this cross would be heterozygous for either PU3 or PU4 and therefore exhibit platelet counts that were elevated (>300xl0 5 // ⁇ l) compared with Mpl ' ' mice.
- the progeny will be either compound heterozygotes (PU3/PU4 Mpl' ' ), single heterozygotes
- the mutant gene responsible for the platelet phenotype in P 4 is identified by a process of genetic mapping and sequencing.
- SSLPs Simple sequence length polymorphisms throughout the genome (Table 3) were amplified by touchdown PCR (Don et al, Nucleic Acid Research 25:4008, 1991) using forward primers labelled with FAM, HEX, or NED fluorescent dye (Applied Biosystems Custom Oligo Synthesis Service, Foster City, CA).
- PCRs contained 5ng/ ⁇ l genomic DNA, Taq polymerase, rabbit antiserum to Taq polymerase, 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgC12, 0.125 mM deoxynucleoside triphosphates, and 0.1 ⁇ M of the forward and reverse oligonucleotide primers. Reaction products with compatible allele sizes were pooled, separated on an ABI 3700 and genotyped using the ABI Genotyper program.
- the mutant gene responsible for PU3 was shown to be closely linked to Plt4 using the breeding test described above. To confirm this, Plt3/+ Mpl' ' mice on a C57BL/6 background were crossed with PU3/+ Mpf ' mice on a 129Sv background. PU3/+ Mpl' ⁇ Fl animals were then identified at 7 weeks of age because of their elevated platelet counts and were backcrossed to +/+ Mpf ' mice on a 129Sv background to generate N2 mice. N2 mice that were Mpl' ' were identified and their platelet counts were determined at 7 weeks of age. Mice were then sacrificed and their livers were removed and genomic DNA was made as described (Laird et al, supra, 1991). SSLPs across chromosome 10 (Table 4) were then amplified and analysed as described for Plt4.
- FIG. 5B shows mice in the N2 generation with the highest platelet levels (PH3/+ Mpl' ' ) were most likely to be heterozygous in this genomic interval, whereas the mice with the lowest platelet numbers (usually +/+ Mpl' ' ) were more likely to be homozygous 129Sv, confirming the co- localization of the Plt3 and PU4 mutations (Figure 6).
- mice generated by intercrossing either PU3/+ Mpl' ' mice or Plt4/+ Mpl' ' mice were typed as Plt3/Plt3, Plt3/+, P 4/PU4, PU4/+ or +/+ at 7 weeks of age by determining their peripheral blood platelet number.
- DNA was prepared by incubation of the tissue sample with 750 ⁇ l extraction buffer (50 mM TrisHCl, 100 mM EDTA, lOOmM NaCl, 1% SDS) and 40 ⁇ l of 20 mg/ml proteinase K overnight at 55°C, with rocking.
- PCR reactions comprised 0.5 ⁇ g genomic DNA, 1 ⁇ l Taq polymerase (Roche, Mannheim, Germany), 2 ⁇ l 10 mM deoxynucleotide triphosphates (dNTPs), 10 ⁇ l lOxPCR buffer (Roche), 200 ng of each oligonucleotide (SigmaGenosys, Australia). The final volume was made up to 100 ⁇ l with H 2 O.
- the PCR reaction involved heating to 96°C for 2 minutes, 30 cycles of 96°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds followed by incubation at 72°C for 10 minutes.
- CFU-e colony-forming unit-erythroid
- An advantage of modifier screens is their capacity to order genes in a pathway using analyses of epistasis.
- the phenotype of homozygous Plt4 mutants was assessed on a wildtype background (c-Myb p " 4/p " 4 Mpt' + ) in comparison with that of wild-type mice (c-Myb +/+ Mpl +/+ ) mice and c-Myb p " 4/p " 4 Mpl' ' mice.
- PU4/PU4 Mpl' ' mice were mated to +/+ Mpl +/+ mice to produce Plt4/+ Mpf ' animals, which were then intercrossed.
- the Mpl genotype of the progeny was determined by Southern blot (Alexander, W. S.
- suppressor screens in vertebrates are of value for the identification of targets for drug discovery. Just as most ENU-induced mutations cause loss of function, most small molecule therapeutics also reduce the function of proteins to which they bind. Accordingly, screens for genes that, when mutated, lead to amelioration of disease, such as c-Myb or other genes that will emerge from our Mpl' " suppressor screen, should provide genome-wide access to novel in vivo validated targets for pharmaceutical discovery.
- EXAMPLE 17 Summary of the PU3 and Plt4 Phenotypes
- Platelet counts have been collected from mice of several different genotypes. These are presented graphically in Figure 12a and tabulated in Table 6. • Mice heterozygous (Mpl '/" Myb Plt3 + , Mpl " '' ' Myb plt4/+ ) or homozygous (Mpl "/" Myb Plt3/Plt3. Mp j-/- Myb ' Plt4 ) for pjg Qr p k4 Qn ft MpJ fc ⁇ ou,. background.
- mice when compared to Mpl " ' " Myb + + mice demonstrate that mutation in the Myb gene can ameliorate the thrombocytopenia caused by lack of TPO signalling through its receptor Mpl. • Mice homozygous for Plt4 on a wild type background (Mpl +/+ Myb plt Plt4 ). The data from these mice, when compared with wildtype mice (Mpl + + Myb + + ) and Mpl "7" Myb plt4 plt4 mice demonstrate that the expansion of megakaryocyte and platelet production cause by Myb mutation occurs independently of TPO signalling through Mpl and suggests that down- modulation of Myb may be an important action of Mpl signalling.
- megakaryocytopoiesis platelets are produced as the end-products of a process of commitment of multipotent hematopoietic stem cells to the megakaryocyte lineage, production and proliferation of megakaryocyte progenitor cells, differentiation and maturation of megakaryocytes and ultimate release of platelets from mature megakaryocytes into the circulation.
- megakaryocyte progenitor and megakaryocyte counts provide evidence that the increased platelet production caused by Myb mutation occurs as a result of a large expansion of all stages of megakaryocytopoiesis. This process occurs at an increased rate than normal in vivo. o As they mature, megakaryocytes increase their DNA content and become polyploid; thus a measure of maturation is DNA content. In experiments where megakaryocytes in bone marrow were examined for DNA ploidy, megakaryocytes from homozygous Plt3 and Plt4 mutants on a Mpl-/- background displayed a modal ploidy of 8N versus 16N for Mpl-/- mice themselves.
- megakaryocytes from one homozygous Plt4 mutant on a wildtype background showed modal ploidy of 8N versus 16N for wildtype controls.
- Heterozygous Plt3 and Plt4 mutants on a Mpl-/- background shown a less dramatic trend towards lower ploidy of the megakaryocyte population.
- the full data is shown in Table 9. It is well established that megakaryocyte ploidy is usually inversely proportional to platelet count.
- Hematopoietic Stem Cell Compartment Cells capable of forming blast colonies in culture are a measure of progenitor cells with multi-lineage potential. As discussed above, the numbers of these progenitors was markedly elevated in the bone marrow and spleen of Plt3 and Plt4 homozygotes on a Mpl-/- background compared with Mpl-/- mice, and evident in the spleens of Plt4 homozygotes on a wildtype background compared with wildtype controls. This data is shown in Table 7. • More primitive cells are the CFU-s, which are multipotential cells assayed by their capacity, upon intravenous injection, to form colonies in the spleens of myeloablated hosts.
- the numbers of these mature stem cells was markedly elevated in the bone marrow of Plt3 and Plt4 heterozygotes and homozygotes on a Mpl-/- background compared with Mpl-/- mice, but apparently unaltered in Plt4 homozygotes on a wildtype background compared with wildtype controls.
- Figure 12a The definitive assay for hematopoietic stem cells in the competitive repopulation assay in which stem cells from a test bone marrow are required to compete with a standard dose of normal stem cells for the long-term and multi-lineage reconstitution of the blood forming system of a transplanted, myeloablated host.
- Mpl-/- mice are known to have defects in the multipotential stem cell pool relative to wild type mice, manifesting as reduced CRU, CFU-s and blast colony-forming cells as well as committed progenitor cells of multiple lineages and this was confirmed in the studies above.
- the novel finding here is that mutation of Myb ameliorates this deficiency at the levels of CFU-s, blast colony-forming progenitors and committed progenitor cells.
- the numbers of CRU are unaffected suggesting that the action of Myb in this regard may not occur in the most primitive stem cells but may be manifest in more mature multipotential cells.
- Lymphocytes Decreased numbers of lymphocytes were observed in cytospin preparations the spleens, bone marrow and peritoneal cavity of homozygous Plt3 and Plt4 mutants on a Mpl-/- background as well as Plt4 homozygotes on a wildtype background (Table 11). This was confirmed by flow cytometric studies showing reduced B lineage cells (B220+) in bone marrow, spleen, lymph node, peritoneal cavity and peripheral blood of homozygous Plt3 and Plt4 mutants on a Mpl-/- background and Plt4 homozygotes on a wild-type background (an example of this is shown graphically in Figure 3b of the manuscript; all the data is in Table 12).
- mice CD5+ IgM+ mice
- DN1 to DN4 maturation series The unconventional B lineage cells in the peritoneal cavity of mice (CD5+ IgM+) were also affected.
- Early T lymphocyte development can be measured using the cell surface markers c-kit and CD25 on CD4-CD8- thymocytes, with differential expression of these markers denoting progression through the so-called DN1 to DN4 maturation series.
- Table 13 homozygous Plt3 and Plt4 mutants on a Mpl-/- background and
- Plt4 homozygotes on a wildtype background displayed a partial block in progression of thymocytes through this series.
- Erythroid cells Increased numbers of nucleated erythroid cells were a common feature of the spleens of homozygous Plt3 and Plt4 mutants on a
- the proportions of cells at these stages is anomalous in homozygous Plt3 and Plt4 mutants on a Mpl-/- background and Plt4 homozygotes on a wildtype background with accumulation of immature (ie stage I and II) cells at the expense of the more mature (stage III and IV) cells, both in bone marrow and in spleen (An example of this is shown in Figure 12b; all the data are shown in Table 15 in this document).
- CFU-e colony-forming units-erythroid
- mice were injected IP with 250 microlitres of saline ( Figure 13 A) or 100 mg/kg carboplatin in approximately 250 microlitres saline ( Figure 13B).
- +/+ Mpl + + , PU4/+ Mpl + + and Plt4/Plt4 Mpl + + were also injected with the same dose of carboplatin ( Figure 13C). All mice were on an inbred C57BL/6 background and were maintained in conventional clean animal facilities. At the indicated time points following injection mice were bled and the numbers of platelets were analyzed using an Advia Automated Haematological Analyser.
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