[go: up one dir, main page]

WO2005095610A1 - Acide nucleique exprimes dans l'hypothalamus ou les tissus musculaires et leur utilisation a des fins de diagnostic - Google Patents

Acide nucleique exprimes dans l'hypothalamus ou les tissus musculaires et leur utilisation a des fins de diagnostic Download PDF

Info

Publication number
WO2005095610A1
WO2005095610A1 PCT/AU2005/000468 AU2005000468W WO2005095610A1 WO 2005095610 A1 WO2005095610 A1 WO 2005095610A1 AU 2005000468 W AU2005000468 W AU 2005000468W WO 2005095610 A1 WO2005095610 A1 WO 2005095610A1
Authority
WO
WIPO (PCT)
Prior art keywords
agt
syndrome
seq
nucleotide sequence
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2005/000468
Other languages
English (en)
Inventor
Gregory Royce Collier
Kenneth Russell Walder
Lyndal Jane Bayles
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AGT BIOSCIENCES Ltd
Original Assignee
AGT BIOSCIENCES Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AGT BIOSCIENCES Ltd filed Critical AGT BIOSCIENCES Ltd
Publication of WO2005095610A1 publication Critical patent/WO2005095610A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to nucleic acid molecules identified by a pattern of their expression in at least the hypothalamus or skeletal muscle.
  • the nucleic acid molecules or their expression products or fragments of their expression products are associated with or act as markers for conditions of wter alia a healthy or unhealthy state, including the presence or absence of a disorder associated with mitochondrial dysfunction, a myopathy, a genetic disorder and/or a cancer and/or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the subject nucleic acid molecules and their expression products and/or their derivatives, fragments, homologs, analogs and mimetics are proposed to be useful, therefore, as therapeutic and diagnostic agents for wter alia, a disorder associated with mitochondrial dysfunction, a myopathy, a genetic disorder and/or a cancer and/or in modulating apoptosis, signal transduction and/or nuclear targeting or as targets for the design and/or identification of modulators of their activity and/or function.
  • Mitochondrial dysfunction refers to any illness resulting from a deficiency of any mitochondrial-located protein which is involved in energy metabolism. Therefore, deficiencies of the respiratory (electron transport) chain, either resulting from a deficiency in one or more of the mitochondrial or nuclear-encoded proteins, are mitochondrial disorders.
  • disorders of the fatty acid (beta) oxidation, Krebs cycle and pyruvate dehydrogenase complex deficiency are mitochondrial disorders. Although these disorders may be genetically dissimilar, mitochondrial dysfunction results in an energy deficient state.
  • Mitochondrial diseases should be considered in the differential diagnosis when there are unexplained features, especially when these occur in combination. Mitochondrial disease and disorders can affect multiple organs, resulting in a vast array of symptoms. Symptoms which may affect the brain include, developmental delays, mental retardation, dementia, seizures, neuro-psychiatric disturbances, atypical cerebral palsy, migraines, strokes.
  • Cancer is also one of the most debilitating disease conditions affecting predominantly humans but also a range of animals.
  • the health cost to the world- wide community runs into the billions of dollars, let alone the personal cost to families.
  • Mitochondrial disease and cancer are significant conditions requiring expenditure of time and financial resources to develop new methods of treatment, prevention and diagnosis.
  • Group A lean animals
  • Group B obese, non-diabetic animals
  • Group C obese, diabetic animals.
  • mice were maintained under two study conditions: (1) they were either fed ad libitum ("fed”) or fasted for 24 hours (“fasted”) prior to analysis; or (2) maintained by being fed ad libitum (“control”) or placed on an energy restricted diet (“restricted”), and genetic sequences analyzed by differential display analysis. Using these techniques, differentially expressed sequences were identified from hypothalamus cells designated in AGT-106, AGT-113, AGT-201 and AGT-202. Another sequence, designated AGT-203, was differentially expressed in skeletal muscle.
  • the differentially expressed genetic sequences AGT-106, AGT-113, AGT-201, AGT202 and AGT-203 are useful markers and/or drug targets for mitochondrial dysfunction and other conditions such as myopathies, genetic disorders and cancers and/or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • 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, ⁇ 400>2, etc.
  • a sequence listing is provided after the claims.
  • Differentially expressed nucleotide sequences are identified as being useful markers associated with mitochondrial dysfunction, myopathies, genetic disorders and/or cancers and/or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • differential expression means an elevation in levels of expression of a genetic sequence under one set of conditions compared to another.
  • the differentially expressed nucleotide sequences are identified by "AGT” numbers.
  • a summary of the AGT sequences disclosed in WO 02/062994 is provided in Table 1.
  • the identification of these variably expressed sequences permits the rationale design and/or selection of molecules capable of antagonizing or agonizing the expression products and/or permits the development of screening assays.
  • the screening assays include assessing the physiological status of a particular subject. Furthermore, the screening assays are useful in drug target as well as drug evaluation.
  • one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides and/or expression product thereof or a fragment of the expression product which is associated with one or more of mitochondrial dysfunction, myopathies, genetic disorders or cancers and/or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the present invention provides peptide, polypeptide or protein or portion thereof associated with one or more of mitochondrial dysfunction, myopathies, genetic disorders or cancers or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • nucleic acid molecules comprising a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or a human homo log thereof, wherein said nucleic acid molecules or their expression products or a portion of their expression product is associated with one or more of mitochondrial dysfunction, myopathies, genetic disorders or cancers or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • polypeptide or protein compositions comprising an amino acid sequence substantially as set forth in SEQ ID NO:21 or SEQ ID NO:34 or SEQ ID NO:25 or a fragment thereof.
  • the present invention contemplates a method for assessing the presence or absence of a mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targetingby determining the level of expression of a nucleic acid molecule which comprises a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 SEQ ID NO:22 or SEQ ID NO:23 or a nucleotide sequence having at least about 30% similarity to all or part of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 SEQ ID NO:22 or SEQ ID NO:23 and/or is capable of hybridizing to one or more of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 SEQ ID NO:22 or SEQ ID NO:
  • the expression product molecule is generally a peptide, polypeptide and/or a fragment such as a cleavage product, but may also be inter alia an mRNA, intron or exon.
  • the preferred genetic sequences of the present invention are referred to herein as AGT- 106, AGT-113, AGT-201, AGT-202 and AGT-203 (see Table 1).
  • the expression products encoded by AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 are referred to herein as AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203, respectively.
  • the expression products may be an RNA (e.g. mRNA) or a protein. Where the expression produce is an RNA, the present invention extends to RNA-related molecules associated thereto such as RNAi.
  • a further aspect of the present invention relates to a composition
  • a composition comprising AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or its derivatives, homologs, analogs or fragments or mimetics or agonists or antagonists of AGT-106, AGT- 113, AGT-201, AGT- 202 and AGT-203 together with one or more pharmaceutically acceptable carriers and/or diluents for use in treating conditions associated with mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • nucleic acid molecules comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or 22 or SEQ ID NO:23, wherein their expression product or fragment thereof is associated with one or more of mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the present invention contemplates a method for treating a subject having mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting comprising administering to the subject, a treatment effective amount of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or a derivative, homolog, analog or mimetic thereof or a genetic sequence encoding same or an agonist or antagonist of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or AGT-106, AGT-113.
  • AGT-201, AGT-202 and AGT-203 gene expression for a time and under conditions sufficient to effect treatment.
  • Treatment may be by the administration of a pharmaceutical composition or genetic sequences via gene therapy. Treatment is contemplated for human subjects as well as animals such as animals important to livestock industry.
  • Still another aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to a mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, said diagnostic agent selected from an antibody or other ligand to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or its derivatives, homologs, analogs or mimetics and a genetic sequence useful in PCR, hybridization, RFLP, amongst other techniques.
  • a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to a mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, said diagnostic agent selected from an antibody or other ligand to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or its derivatives, homologs, analogs or mimetics and a genetic sequence useful in PCR, hybrid
  • the present invention is predicated in part on the identification of genes associated wter alia with a mitochondrial dysfunction, a myopathy, a genetic disorder or a cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the genes were identified by a number of procedures including differential screening or macroarray analysis of hypothalamus or skeletal muscle mRNA between lean and obese animals normoglycemic (Shafrir and Gutman, J Basic Clin Physiol Pharm 4: 83-99, 1993) and/or between fed animals and fasted animals as disclosed in WO 02/062994.
  • one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, or fragment or homolog, analog or mimetic or portion thereof wherein said nucleic acid molecule is associated with one or more of mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the term "differential" array is used in its broadest sense to include the expression of nucleic acid sequences in one type of tissue relative to another type of tissue in the same or different animals.
  • Reference to "different” animals include the same animals but in different gastronomical states such as in a fed or non-fed state.
  • Macroarray (i.e. membrane- based microarray) analysis preferably includes sets of arrays of nucleic and expression products (e.g. mRNA or PCR products) which display differential hybridization characteristics.
  • An "animal” in this context includes a human or non-human animal.
  • the expression product may be a peptide, polypeptide or protein or mRNA or may be an exon or intron spliced, for example, from an RNA construct.
  • the expression product may also be a hairpin structure which induces or is associated with RNAi.
  • a fragment includes a part, portion, region, domain, N-terminal fragment, a C-terminal fragment, an internal fragments and/or an enzymatically cleaved protein, such as by a membrane cleaving protease.
  • N-terminal fragments include amino acid residues 1- 5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1- 37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44, 1-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1- 68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82,
  • examples of C-terminal fragments include amino acid residues 376-371, 376-370, 376-369, 376-368, 376-367, 376-366, 376-365, 376-364, 376-363, 376- 362, 376-361, 376-360, 376-359, 376-358, 376-357, 376-356, 376-355, 376-354, 376-353, 376-352, 376-351, 376-350, 376-349, 376-348, 376-347, 376-346, 376-345, 376-344, 376- 343, 376-342, 376-341, 376-340, 376-339, 376-338, 376-337, 376-336, 376-335, 376-334, 376-333, 376-332, 376-331, 376-330, 376-329, 376-328, 376-327, 376-326, 376-325, 376- 324, 376-323, 376-322, 376-321, 376-320, 376-319, 376-318, 376-317, 376-316, 376-315, 376-3
  • fragments are those which are nuclear targeted peptides, such as those which regulate auto dynamics, signal transduction and apoptosis.
  • the fragment corresponds to AGT-203 amino acid residues 53-77, FRKAPRKVEPRRSDPGTSGEAYKRS (SEQ ID NO:21).
  • the altered expression levels may be in healthy animals or in lean, obese, diabetic or non- diabetic animals as disclosed in WO 02/062994.
  • lean and “obese” are used in their most general sense but should be considered relative to the standard criteria for determining obesity.
  • BMI>30 (Risk Factor Prevalence Study Management Committee. Risk Factor Prevalence Study: Survey No. 3 1989. Sydney: National Heart Foundation of Australia and Australian Institute of Health, 1990; Waters and Bennett, Risk Factors for cardiovascular disease: A summary of Australian data. Canberra: Australian Institute of Health and Welfare, 1995).
  • WO 02/062994 exemplified differentially expressed genes using the Psammomys obesus (the Israeli sand rat) animal model of dietary-induced obesity and NIDDM. In its natural desert habitat, an active lifestyle and saltbush diet ensure that they remain lean and normoglycemic (Shafrir and Gutman, J Basic Clin Physiol Pharm 4: 83- 99, 1993).
  • Psammomys obesus exhibit a range of bodyweight and blood glucose and insulin levels which forms a continuous curve that closely resembles the patterns found in human populations, including the inverted U- shaped relationship between blood glucose and insulin levels known as "Starling's curve of the pancreas" (Barnett et al, [1994a; supra]). It is the heterogeneity of the phenotypic response of Psammomys obesus which make it an ideal model to study the etiology and pathophysiology of obesity and NIDDM.
  • Psammomys obesus animals are conveniently divided into three groups viz Group A animals which are lean, normoglycemic and normoinsulinemic, Group B animals which are obese, normoglycemic and hyperinuslinemic and Group C animals which are obese, hyperglycemic and hyperinsulinemic.
  • the present invention provides, in one aspect, nucleic acid molecule compositions. Therefore, the present invention provides compositions which comprise some or all of a nucleic acid molecule set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO: or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23.
  • Another aspect of the present invention provides a method for assessing the presence or absence of a mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting by determining the level of expression of a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative, homolog or mimetic thereof wherein said nucleotide sequence is as substantially set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 SEQ ID NO:22 or SEQ ID NO:23 or a nucleotide sequence having at least about 30% similarity to all or part of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 SEQ ID NO:22 or SEQ ID NO:23 and/or is capable of hybridizing to one or more of SEQ ID NO:l or S
  • Reference herein to similarity is generally at a level of comparison of at least 15 consecutive or substantially consecutive nucleotides or at least 5, 6, 7, 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, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1
  • Preferred percentage similarities have at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% and at least about 90% or above.
  • Examples include 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, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • 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 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, examples include 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25. 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 computerized 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).
  • a 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, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 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, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% and from at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 and 0.9 M for hybridization, and at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 and 0.9 M 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, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.
  • T m 69.3 + 0.41 (G+C)% (Marmur and Doty, J. Mol. Biol 5: 109, 1962).
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Ewr. J. Biochem. 46: 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.
  • nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in S ⁇ Q ID NO:l or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of S ⁇ Q ID NO: 1.
  • nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in S ⁇ Q ID NO:2 or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of S ⁇ Q ID NO:2.
  • Still yet another aspect of the present invention provides a nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO: 3 or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of SEQ ID NO:3.
  • nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:4 or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of SEQ ID NO:4.
  • nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO: 5 or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of SEQ ID NO:5.
  • nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:22 or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of SEQ ID NO:22.
  • nucleic acid molecule or derivative, homolog or analog thereof associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:23 or a derivative, homolog or mimetic thereof or having at least about 40% identity to all or part of SEQ ID NO:23.
  • the expression pattern of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 has been determined, ter alia, to indicate an involvement in the regulation of one or more of a associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting said nucleic acid molecule.
  • the subject nucleic acid molecules are preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence.
  • a genomic sequence may also comprise exons and introns.
  • a genomic sequence may also include a promoter region or other regulatory regions.
  • the present invention extends, however, to expression products such as mRNA, introns and exons which may also be involved in genetic networking, whether or not they are translated into proteins.
  • the expression products may include complexes comprising RNA or may comprising RNAi or RNAi-type molecules.
  • a homolog is considered to be a AGT-106, AGT-113, AGT-201, AGT-202 or AGT-203 gene from another animal species.
  • the AGT-106, AGT-113, AGT-201, AGT-202 or AGT- 203 genes are exemplified in WO 02/062994 as being derived from Psammomys obesus hypothalamus.
  • the present invention extends, however, to the homologous gene, as determined by nucleotide sequence and/or amino acid sequences and/or function, from primates, including humans, marmosets, orangutans and gorillas, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g.
  • mice mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos).
  • the present invention also contemplates deimmunized forms of the expression products from one species relative to another species.
  • the deimmunized form of the expression product is a malianized form relative to a particular target animal.
  • the target mammal is a human
  • the present invention contemplates use of a humanized form of a non- human expression product.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 and its derivatives and homologs may be in isolated or purified form and/or may be ligated to a vector such as an expression vector.
  • Expression may be in a eukaryotic cell line (e.g. mammalian, insect or yeast cells) or in microbial cells (e.g. E. coli) or both.
  • isolated is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means.
  • the nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector.
  • the nucleotide sequence corresponding to AGT-106 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:l or a derivative, homolog or analog thereof including a nucleotide sequence having similarity to SEQ ID NO: 1.
  • the nucleotide sequence corresponding to AGT-113 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:2 or a derivative, homolog or analog thereof including a nucleotide sequence having similarity to SEQ ID NO:2.
  • the nucleotide sequence corresponding to AGT-201 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO: 3 or a derivative, homolog or analog thereof including a nucleotide sequence having similarity to SEQ ID NO:3.
  • the nucleotide sequence corresponding to AGT-202 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:4 or a derivative, homolog or analog thereof including a nucleotide sequence having similarity to SEQ ID NO:4.
  • the nucleotide sequence corresponding to AGT-203 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or a derivative, homolog or analog thereof including a nucleotide sequence having similarity to SEQ ID NO:5or SEQ ID NO:22 or SEQ ID NO:23.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E.col ⁇ ) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector. The latter embodiment facilitates production of recombinant forms of sphingosine kinase which forms are encompassed by the present invention.
  • the derivatives of the nucleic acid molecule of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co- suppression (e.g. RNAi) and fusion nucleic acid molecules.
  • Ribozymes and DNA enzymes are also contemplated by the present invention directed to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or its mRNA.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 are conveniently encompassed by those nucleotide sequences capable of hybridizing to SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or their complementary form under low stringency conditions.
  • the present invention extends to expression products of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • the preferred expression products are proteins or mutants, derivatives, homologs or analogs thereof as well as a range of RNA molecules.
  • An expression product includes an RNA molecule such as a mRNA transcript as well as a protein.
  • Some genes are non-protein encoding genes and produce mRNA or other RNA type molecules and are involved in regulation by RNA:DNA, RNA:RNA or RNA:protein interaction.
  • the RNA e.g. mRNA
  • the RNA may act directly or via the induction of other molecules such as RNAi or via products mediated from splicing events (e.g. exons or introns).
  • Other genes encode mRNA transcripts which are then translated into proteins.
  • a protein includes a polypeptide.
  • the differentially expressed nucleic acid molecules therefore, may encode mRNAs only or, in addition, proteins. Both mRNAs and proteins are forms of "expression products".
  • Derivatives include fragments, parts, portions, mutants, variants and mimetics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of AGT-106, AGT-113, AGT-202 or AGT-203. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • An example of substitutional amino acid variants are conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
  • AGT-106, AGT-113, AGT-202 or AGT-203 should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
  • the derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • Another aspect of the present invention provides an isolated protein or other expression product or a derivative, homolog, analog or mimetic thereof which is associated with one or more of mitochondrial disease, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • an isolated protein or derivative, homolog, analog, fragment or mimetic thereof wherein said protein or polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:21 or SEQ ID NO:24 or SEQ ID NO:25 or an amino acid sequence having at least 30% similarity to all or part thereof and wherein said protein or expression product is associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • an isolated protein or a derivative, homolog, analog or mimetic thereof or other expression product wherein said protein or expression product comprises an amino acid or nucleotide sequence substantially encoded by a nucleotide sequence as set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or an amino acid sequence having at least 30% similarity to all or part thereof and wherein said protein or expression product is associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a further aspect of the present invention is directed to an isolated protein or a derivative, homolog, analog or mimetic thereof or other expression product wherein said protein or expression product is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or a nucleotide sequence having at least 30% similarity to all or part of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 and/or is capable of hybridizing to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or their complementary forms under low stringency conditions at a specified temperature, wherein said protein or expression product is associated with a mitochondrial dysfunction,
  • references herein to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 includes reference to isolated or purified naturally occurring AGT-106, AGT-113, AGT-201, AGT- 202 and AGT-203 protein or expression product molecules as well as any derivatives, homologs, analogs and mimetics thereof.
  • Derivatives include parts, fragments and portions of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 as well as single and multiple amino acid substitutions, deletions and/or additions to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • a derivative of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 is conveniently encompassed by molecules encoded by a nucleotide sequence capable of hybridizing to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 under low stringency conditions at a specified temperature.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 include chemical analogs.
  • Analogs of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confirmational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 -phosphate followed by reduction with NaBH 4 .
  • 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 3. TABLE 3 Codes for non-conventional amino acids
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and Cp atoms of amino acids and the formation of cyclic peptides or analogs 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 expression product may be an RNA or protein.
  • the term "protein” should be understood to encompass peptides, polypeptides and proteins.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:l or a derivative, homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:l.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:2 or a derivative, homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:2.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO: 3 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:3.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:4 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:4.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO: 5 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:5.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:22 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:22.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NO:23 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 40% identity to SEQ ID NO:23.
  • Another aspect of the present invention is directed to an isolated expression product associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, said expression product selected from the list consisting of:-
  • (x) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:23 or a derivative, homolog or analog thereof or a sequence encoding an amino acid sequence having at least about 40% similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of said protein.
  • the present invention extends to the expression product of the nucleic acid molecules as hereinbefore defined.
  • the expression product is preferably a protein but extends to mRNA,
  • RNA introns and exons.
  • the expression products are AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 encoded by SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23, respectively or are derivatives, analogs, homologs, chemical equivalents or mimetics thereof.
  • Another aspect of the present invention is directed to an isolated protein associated with one or more of mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, said protein selected from the list consisting of:-
  • a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:l or a derivative, homolog or analog thereof under low stringency conditions (viii) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:l or a derivative, homolog or analog thereof under low stringency conditions; (ix) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:2 or a derivative, homolog or analog thereof under low stringency conditions;
  • (x) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO: 3 or a derivative, homolog or analog thereof under low stringency conditions;
  • xi a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:4 or a derivative, homolog or analog thereof under low stringency conditions;
  • xii a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:5 or SEQ ID NO:22 or SEQ ID NO:23 or a derivative, homolog or analog thereof under low stringency conditions;
  • the protein of the present invention is preferably in isolated form.
  • isolated is meant a protein having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10%) subject protein, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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, 80, 81, 82, 83,
  • sequence similarity and “sequence identity” as used herein refers 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 nucleotide sequence or amino acid sequence of the present invention may correspond to exactly the same sequence of the naturally occurring gene (or corresponding cDNA) or protein or may carry one or more nucleotide or amino acid substitutions, additions and/or deletions.
  • the nucleotide sequences set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 SEQ ID NO:22 or SEQ ID NO:23 correspond to the genes referred to herein as AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203, respectively.
  • the corresponding expression products are AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203, respectively.
  • references herein to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 includes, where appropriate, reference to the genomic gene or cDNA as well as any naturally occurring or induced derivatives. Apart from the substitutions, deletions and/or additions to the nucleotide sequence, the present invention further encompasses mutants, fragments, parts and portions of the nucleotide sequence corresponding to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 permits the generation of a range of therapeutic molecules capable of modulating expression of AGT- 106, AGT-113, AGT-201, AGT-202 and AGT-203 or modulating the activity of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • Modulators contemplated by the present invention includes agonists and antagonists of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 expression.
  • Antagonists of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 expression include antisense molecules, ribozymes and co-suppression molecules.
  • Antagonists of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 include antibodies and inhibitor peptide fragments. All such molecules may first need to be modified to enable such molecules to penetrate cell membranes. Alternatively, viral agents may be employed to introduce genetic elements to modulate expression of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • the present invention contemplates, therefore, a method for modulating expression of one or more of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 in a mammal, said method comprising contacting the AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 gene with an effective amount of a modulator of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of AGT-106, AGT-113, AGT-201, AGT- 202 and AGT-203.
  • a nucleic acid molecule encoding AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or a derivative or homolog thereof may be introduced into a cell to enhance the ability of that cell to produce AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203, conversely, AGT-106, AGT-113, AGT-201, AGT-202 and AGT- 203 antisense sequences such as oligonucleotides may be introduced to decrease the availability of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 molecules.
  • Another aspect of the present invention contemplates a method of modulating activity of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 in a mammal, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease AGT-106, AGT-113, AGT- 201, AGT-202 and AGT-203 activity.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or its ligand.
  • Modulating levels of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 expression is important in the treatment of a range of conditions such as mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the present invention has application in the treatment of humans as well as in the veterinary and animal husbandry industries. Accordingly, subjects contemplated for treatment in accordance with the present invention includes, but is not limited to humans, primates, livestock animals (e.g. pigs, sheep, cows, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. dogs, cats) and captured wild animals (e.g. foxes, kangaroos, deer).
  • a particularly preferred host is a human, primate or livestock animal.
  • the present invention contemplates therapeutic and prophylactic uses of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 amino acid and nucleic acid molecules in addition to AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 agonistic and antagonistic agents.
  • the present invention contemplates, therefore, a method of modulating expression of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 in a mammal, said method comprising contacting the AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 genes with an effective amount of an agent for a time and under conditions sufficient to up- regulate, down-regulate or otherwise module expression of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203.
  • antisense sequences such as oligonucleotides may be utilized.
  • nucleic acid molecules encoding AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or derivatives thereof may be introduced to up-regulate one or more specific functional activities.
  • Another aspect of the present invention contemplates a method of modulating activity of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 in a subject, said method comprising administering to said subject a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease AGT-106, AGT-113, AGT- 201, AGT-202 and/or AGT-203 activity.
  • Modulation of activity by the administration of an agent to a mammal can be achieved by one of several techniques, including but in no way limited to introducing into said mammal a proteinaceous or non-proteinaceous molecule which:
  • the proteinaceous molecule may be derived from natural or recombinant sources including fusion proteins or following, for example, natural product screening or the screening of chemical libraries.
  • the non-proteinaceous molecule may be, for example, a nucleic acid molecule or may be derived from natural sources, such as for example natural product screening or may be chemically synthesized.
  • the present invention contemplates chemical analogs of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or small molecules capable of acting as agonists or antagonists.
  • Chemical agonists may not necessarily be derived from AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 from carrying out their normal biological functions. Antagonists include monoclonal antibodies, antisense and sense nucleic acids which prevent transcription or translation of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 genes or mRNA in mammalian cells. Modulation of expression may also be achieved utilizing antigens, RNA, RNAi, ribosomes, DNAzymes, RNA aptamers or antibodies.
  • the proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the expression of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or the activity of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203. Said molecule acts directly if it associates With AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 to modulate expression or activity.
  • Said molecule acts indirectly if it associates with a molecule other than AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or AGT-106, AGT-113, AGT-201, AGT- 202 and/or AGT-203 which other molecule either directly or indirectly modulates the expression or activity of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203.
  • the method of the present invention encompasses the regulation of AGT-106, AGT-113, AGT-201, AGT- 202 and/or AGT-203 or AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 expression or activity via the induction of a cascade of regulatory steps.
  • the molecules which may be administered to a mammal in accordance with the present invention may also be linked to a targeting means such as a monoclonal or polyclonal antibody, which provides specific delivery of these molecules to the target cells.
  • a further aspect of the present invention relates to the use of the invention in relation to mammalian disease conditions.
  • the present invention is particularly useful but in no way limited to use in a therapeutic or prophylactic treatment of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • another aspect of the present invention relates to a method of treating a mammal suffering from a condition characterized by one or more symptoms of a mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of AGT-106, AGT-113, AGT-201, AGT- 202 and/or AGT-203 or sufficient to modulate the activity of AGT-106, AGT-113, AGT- 201, AGT-202 and/or AGT-203.
  • the present invention relates to a method of treating a mammal suffering from a disease condition characterized by one or more symptoms of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, said method comprising administering to said mammal an effective amount of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT- 203 or AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203.
  • myopathy refers to any abnormal conditions or disease of the muscle tissues, which include the muscles over our bones (skeletal muscle) and the heart (cardiac muscle). Mitochondrial dysfunction relates to abnormalities in mitochondria. Mitochondria are part of the cell (organelle) that is responsible for energy production.
  • the organelle consists of two sets of membranes, a smooth continuous outer coat and an inner membrane arranged in tubules or in folds that form plate-like double membranes (cristae).
  • Mitochondria are the principal energy source of the cell, containing the cytochrome enzymes of terminal electron transport and the enzymes of the citric acid cycle, fatty acid oxidation, and oxidative phosphorylation.
  • Mitochondria are complex organelles located in virtually all cells of the body. A large degree of their complexity is due to the fact that over 1000 proteins are located in the mitochondria. Thirteen of these proteins are encoded by the mitochondrial DNA (mfDNA), while the remainder are nuclear-encoded, and imported into the mitochondria.
  • mfDNA mitochondrial DNA
  • Symptoms of mitochondrial dysfunction include weakness (which may be intermittent), neuropathic pain, absent reflexes, gastrointestinal problem (gastroesophogeal reflux, delayed gastric emptying, constipation, pseudo-obstruction), fainting, absent or excessive sweating resulting in temperature regulation problems, hypotonia, cramping and muscle pain, proximal renal tubular wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes, cardiac conduction defects (heart blocks) and cardiomyopathy, hypoglycemia (low blood sugar) and liver failure, visual loss and blindness, hearing loss and deafness, and diabetes and exocrine pancreatic failure (inability to make digestive enzymes).
  • mitochondrial dysfunction There may also be systemic problems associated with mitochondrial dysfunction, including failure to gain weight, short stature, fatigue, respiratory problems.
  • Mitochondrial defects have been linked to Alzheimer's, Parkinson's, diabetes, autism, and the aging process.
  • Other disease associated with mitochondrial dysfunction include, LIC (Lethal Infantile Cardiomyopathy), Beta-oxidation Defects, COX Deficiency, Mitochondrial Cytopathy, Alpers Disease, Barth syndrome, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency, Co-Enzyme Q10 Deficiency, Complex I Deficiency, Complex II Deficiency, Complex III Deficiency, Complex IN Deficiency, Complex V Deficiency, CPEO, CPT I Deficiency, Glutaric Aciduria Type II, KSS, lactic acidosis, LCAD, LCHAD, Leigh Disease, LHO ⁇ , Luft Disease, MAD, MCA, MELAS, MERRF, mitochondrial D ⁇ A depletion, Mitochondrial Encephalopathy, M ⁇ GIE, ⁇ ARP, Pearson Syndrome, Pyruvate Car
  • Alpers Disease or Progressive Infantile Poliodystrophy, includes symptoms such as seizures, dementia, spasticity, blindness, liver dysfunction, and cerebral degeneration. (Luft; Proceedings of the National Academy of Sciences of the United States of America; P7(79j:8731-8, 1994).
  • Barth syndrome or LIC Lethal Infantile Cardiomyopathy
  • LIC Lethal Infantile Cardiomyopathy
  • Carnitine-Acyl-Carnitine Deficiency is an autosomal recessive disorder, the symptoms of which are seizures, apnea, bradycardia, vomiting, lethargy, coma, enlarged liver, limb weakness, myoglobin in the urine, Reye-like symptoms triggered by fasting.
  • Carnitine Deficiency is an autosomal recessive disease, the symptoms of which include Cardiomyopathy, failure to thrive, and altered consciousness or coma, sometimes hypotonia.
  • Co-Enzyme Q10 Deficiency is most likely an autosomal recessive disease, the symptoms of which include Encephalomyopathy, mental retardation, exercise intolerance, ragged-red fibers, and recurrent myoglobin in the urine.
  • ⁇ ADH dehydrogenase ⁇ ADH-CoQ reductase
  • ⁇ ADH-CoQ reductase ⁇ ADH-CoQ reductase
  • ⁇ ADH-CoQ reductase ⁇ ADH-CoQ reductase
  • MELAS mitochondrial encephalomyopathy
  • encephalomyopathy which is typically normal for the first 6 to 12 months of life and then show developmental regression, ataxia, lactic acidosis, optic atrophy, ophthalmoplegia, nystagmus, dystonia, pyramidal signs, respiratory problems and frequent seizures; and
  • myopathy Two main variants: (a) Fatal infantile myopathy: may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged-red fibers, respiratory failure, and kidney problems: and (b) Benign infantile myopathy: may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged- red fibers, respiratory problems, but (if the child survives) followed by spontaneous improvement.
  • Complex V Deficiency or ATP synthase deficiency includes symptoms such as slow, progressive myopathy.
  • CPEO or Chronic Progressive External Ophthalmoplegia Syndrome includes symptoms such as visual myopathy, retinitis pigmentosa, dysfunction of the central nervous system. It is caused by single mitochondrial DNA deletions, with Mitochondrial DNA point mutation, A3243G being the most common (Luft; 1994 [supra]).
  • CPT I Deficiency is an autosomal recessive disease and includes symptoms such as enlarged liver and recurrent Reye-like episodes triggered by fasting or illnesses.
  • CPT II Deficiency is an autosomal recessive disease, the symptoms of which include exercise intolerance, fasting intolerance, muscle pain, muscle stiffness, and myoglobin in the urine and in infants, Reye-like syndrome, enlarged liver, hypoglycemia, enlarged heart and cardiac arrhythmia.
  • KSS KSS or Kearns-Sayre Syndrome
  • Symptoms associated with KSS include progressive external ophthalmoplegia, pigmentary retinopathy, heart block, and high cerebrospinal protein.
  • Lactic Acidosis is associated with the accumulation of lactic acid due to its production exceeding its use. Chronic lactic acidosis is a common symptom of mitochondrial disease.
  • LCAD or Long-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, which causes a fatal syndrome, in infants, typified by failure to thrive, enlarged liver, enlarged heart, metabolic encephalopathy and hypotonia.
  • LCHAD is an autosomal recessive disorder, characterized by symptoms such as encephalopathy, liver dysfunction, cardiomyopathy, and myopathy. Also pigmentary retinopathy and peripheral neuropathy.
  • Leigh Disease or Syndrome or Subacute Necrotizing Encephalomyelopathy is characterized by symptoms such as Seizures, hypotonia, fatigue, nystagmus, poor reflexes, eating and swallowing difficulties, breathing problems and poor motor function.
  • LHON or Leber Hereditary Optic Neuropathy is caused by mitochondrial DNA point mutations, including G14459A, among others. Symptoms associated with LHON include primarily blindness in young men. Less common symptoms include mild dementia, ataxia, spasticity, peripheral neuropathy and heart conduction defects.
  • MAD or Glutaric Aciduria Type II or multiple Acyl-CoA Dehydrogenase Deficiency is caused by defects of the flavoproteins responsible for transferring electrons (ETF or ETF- dehydrogenase) therefore affecting the function of all six ETF-funneling acyl-CoA dehydrogenases
  • MCAD or Medium-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, which afflicts infants or young children with episodes of encephalopathy, enlarged and fatty degeneration of the liver, and low carnitine in the blood.
  • MELAS or Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes is caused by mitochondrial DNA point mutations, the most common of which is A3243G. It is characterized by symptoms: Short stature, seizures, stroke-like episodes with focused neurological deficits, recurrent headaches, cognitive regression, disease progression ragged-red fibers (Koo, et. al; Annals of Neurology; 34(l):25-32, 1993).
  • MERRF or Myoclonic Epilepsy and Ragged-Red Fiber Disease is caused by the mitochondrial DNA point mutations A8344G and T8356C. Its symptoms include myoclonus, epilepsy, progressive ataxia, muscle weakness and degeneration, deafness and dementia (Luft; 1994 [supra]).
  • mitochondrial DNA Depletion There are three forms of mitochondrial DNA Depletion. These include: (1) congenital myopathy: Neonatal weakness, hypotonia requiring assisted ventilation, possible renal dysfunction. Severe lactic acidosis. Prominent ragged-red fibers. Death due to respiratory failure usually occurs prior to one year of age; (2) infantile myopathy: Following normal early development until one year old, weakness appears and worsens rapidly, causing respiratory failure and death typically within a few years; and (3) hepatopathy, enlarged liver and intractable liver failure, myopathy. Severe lactic acidosis. Death is typical within the first year.
  • Mitochondrial Encephalopathy also includes Encephalomyopathy and Encephalomyelopathy.
  • MNGIE Myoneurogastrointestinal Disorder and Encephalopathy
  • symptoms such as progressive external ophthalmoplegia, limb weakness, peripheral neuropathy, digestive tract disorders, leukodystrophy, lactic acidosis and ragged red fibers.
  • NARP or Neuropathy, Ataxia, and Retinitis Pigmentosa is caused by mitochondrial DNA point mutations in genes associated with Complex V, including T8993G, (also T8993C by some researchers). Leigh Syndrome may result if the percentage of mutation is high enough. Pearson Syndrome is characterized by symptoms associated with bone marrow and pancreas dysfunction. It is caused by single mitochondrial DNA deletions. Inheritance is usually sporadic. Those who survive infancy usually develop Kearns-Sayre Syndrome.
  • Pyruvate Carboxylase Deficiency is an autosomal recessive disorder, the symptoms of which include lactic acidosis, hypoglycemia, severe retardation, failure to thrive, in addition to seizures and spasticity.
  • Pyruvate Dehydrogenase Deficiency is characterized by symptoms such as lactic acidosis, ataxia, pyruvic acidosis, spinal and cerebellar degeneration. Less common symptoms include agenesis of the corpus callosum and lesions in the basal ganglia, cerebellum, and brain stem, growth delay, hypotonia, seizures and polyneuropathy.
  • SCAD Short-Chain Acyl-CoA Dehydrogenase Deficiency
  • SCAD Short-Chain Acyl-CoA Dehydrogenase Deficiency
  • SCHAD is an autosomal recessive disorder, characterized by encephalopathy and possibly liver disease or cardiomyopathy.
  • VLCAD or Very Long-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, characterized by various manifestations, ranging from fatal infantile encephalopathy to recurrent myoglobin in the urine, similar to the myopathic form of CPT II deficiency.
  • Hyperphenylalaninemia Auditory Canal Atresia, Auriculotemporal Syndrome, Autism, Autism Asperger's Type, Autism Dementia Ataxia and Loss of Purposeful Hand Use, Autism Infantile Autism, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune-Polyendocrinopathy-Candidias, Autoimmune Polyglandular Disease Type I, Autosomal Dominant Albinism, Autosomal Dominant Compelling Helioophthalmic Outburst Syndrome, Autosomal Dominant Desmin Distal Myopathy with Late Onset, Autosomal Dominant EDS, Autosomal Dominant Emery-Dreifuss Muscular Dystrophy, Autosomal Dominant Keratoconus, Autosomal Dominant Pelizaeus-Merzbacher Brain Sclerosis, Autosomal Dominant Polycystic Kidney Disease, Autosomal Dominant Spinocerebell
  • Hypervalinemia Hypocalcified (Hypomineralized) Type
  • Hypochondrogenesis Hypochrondroplasia
  • Hypogammaglobulinemia Hypogammaglobulinemia Transient of Infancy
  • Hypogenital Dystrophy with Diabetic Tendency Hypoglossia-Hypodactylia Syndrome
  • Hypoglycemia Hypoglycemia
  • Exogenous Hypoglycemia Hypoglycemia with Macroglossia
  • Hypoglycosylation Syndrome Type la Hypogonadism with Anosmia
  • Hypogonadotropic Hypogonadism and Anosmia Hypohidrotic Ectodermal Dysplasia
  • Hypohidrotic Ectodermal Dysplasia Hypohidrotic Ectodermal Dysplasia Autosomal Dominant type, Hypohidrotic Ectodermal Dysplasias autorecessive, Hypokalemia, Hypokalemic Alkalosis with Hypercalciuria, Hypokalemic Syndrome, Hypolactasia, Hypo
  • Oculomandibulofacial Syndrome Oculomotor with Congenital Contractures and Muscle Atrophy
  • Oculosympathetic Palsy ODD Syndrome, ODOD, Odontogenic Tumor, Odontotrichomelic Syndrome, OFD, OFD Syndrome, Ohio Type Amyloidosis (Type VII), OI, OI Congenita, OI Tarda, Oldfield Syndrome, Oligohydramnios Sequence, Oligophrenia Microphthalmos, Oligophrenic Polydystrophy, Olivopontocerebellar Atrophy, Olivopontocerebellar Atrophy with Dementia and Extrapyramidal Signs, Olivopontocerebellar Atrophy with Retinal Degeneration, Olivopontocerebellar Atrophy I, Olivopontocerebellar Atrophy II, Olivopontocerebellar Atrophy III, Olivopontocerebellar Atrophy IV, Olivopontocerebellar Atrophy V, Oilier Disease, Oilier Osteochondr
  • Pericardial Constriction with Growth Failure Pericollagen Amyloidosis, Perinatal Polycystic Kidney Diseases, Perineal Anus, Periodic Amyloid Syndrome, Periodic Peritonitis Syndrome, Periodic Somnolence and Morbid Hunger, Periodic Syndrome, Peripheral Cystoid Degeneration of the Retina, Peripheral Dysostosis-Nasal Hypoplasia-Mental Retardation, Peripheral Neuritis, Peripheral Neuropathy, Peritoneopericardial Diaphragmatic Hemia, Pernicious Anemia, Peromelia with Micrognathia, Peroneal Muscular Atrophy, Peroneal Nerve Palsy, Peroutka Sneeze, Peroxisomal Acyl-CoA Oxidase, Peroxisomal Beta-Oxidation Disorders, Peroxisomal Bifunctional Enzyme, Peroxisomal Thiolase, Peroxisomal Thiolase Deficiency, Persistent Truncus Ar
  • Pseudohermaphroditism-Nephron Disorder-Wilm's Tumor Pseudohypertrophic Muscular Dystrophy, Pseudohypoparathyroidism, Pseudohypophosphatasia, Pseudopolydystrophy, Pseudothalidomide Syndrome, Pseudoxanthoma Elasticum, Psoriasis, Psorospermosis Follicularis, PSP, PSS, Psychomotor Convulsion, Psychomotor Epilepsy, Psychomotor Equivalent Epilepsy, PTC Deficiency, Pterygium, Pterygium Colli Syndrome, Pterygium Universale, Pterygolymphangiectasia, Pulmonary Atresia, Pulmonary
  • Lymphangiomyomatosis Pulmonary Stenosis, Pulmonic Stenosis-Ventricular Septal Defect, Pulp Stones, Pulpal Dysplasia, Pulseless Disease, Pure Alymphocytosis, Pure Cutaneous Histiocytosis, Purine Nucleoside Phosphorylase Deficiency, Pu ⁇ ura Hemorrhagica, Purtilo Syndrome, PXE, PXE Dominant Type, PXE Recessive Type, Pycnodysostosis, Pyknoepilepsy, Pyroglutamic Aciduria, Pyroglutamicaciduria, Pyrroline Carboxylate Dehydrogenase Deficiency, Pymvate Carboxylase Deficiency, Pymvate Carboxylase Deficiency Group A, Pymvate Carboxylase Deficiency Group B, Pymvate Dehydrogenase Deficiency, Pymvate Kinase Deficiency, q25-q
  • Type I Urinary Tract Defects, Urofacial Syndrome, Uropo ⁇ hyrinogen III cosynthase, Urticaria pigmentosa, Usher Syndrome, Usher Type I, Usher Type II, Usher Type III, Usher Type IV, Uterine Synechiae, Uopo ⁇ hyrinogen I- synthase, Uveitis, Uveomeningitis Syndrome, V-CJD, VACTEL Association, VACTERL Association, VACTERL Syndrome, Valgus Calcaneus, Valine Transaminase Deficiency, Valinemia, Valproic Acid, Valproate acid exposure, Valproic acid exposure, Valproic acid, Van Buren's Disease, Van der Hoeve-Habertsma-Waardenburg-Gauldi Syndrome, Variable Onset Immunoglobulin Deficiency Dysgammaglobulinemia, Variant Creutzfeldt- Jakob Disease (V-CJD), Varicella
  • cancers contemplated include ABL1 protooncogene, AIDS Related Cancers, Acoustic Neuroma, Acute Lymphocytic Leukaemia, Acute Myeloid Leukaemia, Adenocystic carcinoma, Adrenocortical Cancer, Agnogenic myeloid metaplasia, Alopecia, Alveolar soft-part sarcoma, Anal cancer, Angiosarcoma, Aplastic Anaemia, Astrocytoma, Ataxia-telangiectasia, Basal Cell Carcinoma (Skin), Bladder Cancer, Bone Cancers, Bowel cancer, Brain Stem Glioma, Brain and CNS Tumours, Breast Cancer, CNS tumours, Carcinoid Tumours, Cervical Cancer, Childhood Brain Tumours, Childhood Cancer, Childhood Leukaemia, Childhood Soft Tissue Sarcoma, Chondrosarcoma, Choriocarcinoma,
  • an “effective amount” means an amount necessary at least partly to attain the desired immune response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT- 203 or AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or agents capable of modulating the expression or activity of said molecules may be co-administered with one or more other compounds or other molecules.
  • co-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.
  • 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 present invention relates to the use of an agent capable of modulating the expression of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or a derivative, homolog or analog thereof in the manufacture of a medicament for the treatment of a condition characterized by mitochondrial dysfunction, myopathy, genetic disorder or cancer.
  • the present invention relates to the use of an agent capable of modulating the activity of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or a derivative, homolog, analog, chemical equivalent or mimetic thereof in the manufacture of a medicament for the treatment of a condition characterized by mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a further aspect of the present invention relates to the use of AGT-106, AGT-113, AGT- 201, AGT-202 and/or AGT-203 or derivative, homolog or analog thereof or AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or derivative, homolog, analog, chemical equivalent or mimetic thereof in the manufacture of a medicament for the treatment of a condition characterized by mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • Still yet another aspect of the present invention relates to agents for use in modulating the expression of AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or a derivative, homolog or analog thereof.
  • a further aspect relates to agents for use in modulating AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 activity or a derivative, homolog, analog, chemical equivalent or mimetic thereof.
  • Still another aspect of the present invention relates to AGT-106, AGT-113, AGT-201, AGT-202 and/or AGT-203 or derivative, homolog or analog thereof or AGT-106, AGT- 113, AGT-201, AGT-202 and/or AGT-203 or derivative, homolog, analog, chemical equivalent or mimetic thereof for use in treating a condition characterized by one or more symptoms of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the mammal undergoing treatment may be a human or an animal in need of therapeutic or prophylactic treatment.
  • treating and “treatment” as used herein refer to a reduction in the severity and/or frequency of symptoms associated with wter alia a mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, elimination of symptoms and/or the underlying cause, prevention of the occurrence of symptoms of disease and/or the underlying cause and improvement or remediation of damage.
  • Treating a subject may involve prevention of the disorder or disease condition or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting a disease or disorder.
  • such conditions involve, weakness (which may be intermittent), neuropathic pain, absent reflexes, gastrointestinal problem (gastroesophogeal reflux, delayed gastric emptying, constipation, pseudo-obstruction), fainting, absent or excessive sweating resulting in temperature regulation problems weakness, hypotonia, cramping, muscle pain, proximal renal tubular wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes, cardiac conduction defects (heart blocks) and cardiomyopathy, hypoglycemia (low blood sugar) and liver failure, visual loss and blindness, hearing loss and deafness, diabetes and exocrine pancreatic failure (inability to make digestive enzymes), mitochondrial dysfunction, including failure to gain weight, short statue, fatigue and respiratory problems.
  • the present invention contemplates in one embodiment a composition comprising a modulator of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 expression or AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 activity and one or more pharmaceutically acceptable carriers and/or diluents.
  • the composition comprises AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or a derivative, homolog, analog or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.
  • active components all such components of such a composition are referred to as "active components”.
  • compositions of active components in a form suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active components in the required amount in the appropriate solvent with optionally other ingredients, as required, followed by sterilization by, for example, filter sterilization, irradiation or other convenient means.
  • sterilization by, for example, filter sterilization, irradiation or other convenient means.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 and AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 including AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 itself are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be inco ⁇ orated directly with the food of the diet.
  • the active compound may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1% by weight of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, com starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as com starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be inco ⁇ orated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active component may be compounded for convenient and effective administration in sufficient amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active component in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 will range from 0.01 ng/kg/body weight to above 10,000 mg/kg/body weight. Alternative amounts range from 0.1 ng/kg/body weight is above 1000 mg/kg/body weight.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 may be administered per minute, hour, day, week, month or year depending on the condition being treated.
  • the route of administration may vary and includes intravenous, intraperitoneal, sub-cutaneous, intramuscular, intranasal, via suppository, via infusion, via drip, orally or via other convenient means.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 expression or AGT- 106, AGT-113, AGT-201, AGT-202 and AGT-203 activity.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies to AGT-106, AGT- 113, AGT-201, AGT-202 and AGT-203 and their derivatives and homologs.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or may be specifically raised to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or derivatives or homologs thereof.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or their derivatives or homologs may first need to be associated with a carrier molecule.
  • the antibodies and/or recombinant AGT-106, AGT-113, AGT- 201, AGT-202 and AGT-203 or their derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 and their derivatives can be used to screen for naturally occurring antibodies to AGT-106, AGT- 113, AGT-201, AGT-202 and AGT-203 which may occur in certain autoimmune diseases or where cell death is occurring. These may occur, for example in some autoimmune diseases.
  • specific antibodies can be used to screen for AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.
  • Antibodies to AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 of the present invention may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or may be specifically raised to the AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or their derivatives. In the case of the latter, the AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 protein may need first to be associated with a carrier molecule. Alternatively, fragments of antibodies may be used such as Fab fragments.
  • the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
  • a "synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • the antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool or as a means for purifying AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • specific antibodies can be used to screen for AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 proteins.
  • the latter would be important, for example, as a means for screening for levels of AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 in a cell extract or other biological fluid or purifying AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed 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 AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein 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 AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203, 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. (See, for example, Douillard and Hoffman, Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein, European Journal of Immunology 6: 511-519, 1976).
  • Another aspect of the present invention contemplates a method for detecting AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or a derivative or homolog thereof in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for AGT-106, AGT-113, AGT-201, AGT-202 and AGT- 203 or their antigenic derivatives or homologs for a time and under conditions sufficient for a complex to form, and then detecting said complex.
  • the presence of the complex is indicative of the presence of AGT-106, AGT-113, AGT- 201, AGT-202 and AGT-203.
  • This assay may be quantitated or semi-quantitated to determine a propensity to develop mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting or to monitor a therapeutic periodine.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 may be accomplished 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. These, of course, includes both single- site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, 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 AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203, 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-AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 -labeled antibody.
  • AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody.
  • the sample is one which might contain AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • 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 overnight if more convenient) and under suitable conditions (e.g.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of AGT- 106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to AGT- 106, AGT-113, AGT-201, AGT-202 and AGT-203.
  • 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 labeling 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.
  • 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 antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • 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, ⁇ -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. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • 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.
  • a "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 fluorecein and rhodamine
  • fluorecein and rhodamine 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.
  • the present invention also contemplates genetic assays such as involving PCR analysis to detect AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or their derivatives.
  • the assays of the present invention may also extend to measuring AGT-106, AGT-113, AGT-201, AGT-202 and AGT-203 or AGT-106, AGT-113, AGT-201, AGT-202 and AGT- 203 in association with another gene or molecule.
  • a Psammomys obesus colony is maintained at Deakin University, Waum Ponds, Geelong, Victoria, Australia with the breeding pairs fed ad libitum a diet of lucerne and standard laboratory chow. Animals are weaned at four weeks of age and sustained on a diet of standard laboratory chow from which 12% of energy was derived from fat, 63% from carbohydrate and 25% from protein (Barastoc, Pakenham, Australia). Animals are housed in a humidity and temperature controlled room (22 ⁇ 1°C) with a 12- 12-hour light-dark cycle.
  • Group A animals are lean, normoglycemic and normoinsulinemic
  • group B animals are obese, normoglycemic and hyperinsulinemic
  • group C animals are obese, hyperglycemic and hyperinsulinemia.
  • Plasma insulin concentrations were determined using a double antibody solid phase radioimmunoassay (Phadeseph, Kabi Pharmacia Diagnostics, Sweden).
  • RNAimage mRNA Differential Display System GenHunter Co ⁇ oration, Nashville, Tennessee.
  • the PCR products were separated on a 6% w/v polyacrylamide gel, and differentially expressed PCR fragments were visualized by exposing the dried gel to x-ray film.
  • Candidate bands were excised from the gel and reamplified by PCR using the appropriate primer combination.
  • Sequencing reactions were carried out using ABI PRISM Big-Dye terminator cycle sequencing ready reaction kits and analyzed on an ABI 373A DNA sequencer. Gene database searches were performed at the National Centre for Biotechnology Information using the BLAST network service.
  • AGT-106 was identified as being differentially expressed using the technique of differential display PCR in the hypothalamus of the Israeli Sand Rat (ISR) and expression of AGT-106 was higher in fed compared to fasted Sand rats.
  • the partial nucleotide sequence of Psammomys obesus AGT-106 cDNA is as follows :-
  • TROY is a newly identified member of the Tumour Necrosis Factor Receptor Superfamily (Kojima et al, J. Biol Chem. 275(27): 20742-20747, 2000). Nucleotide sequence homology of ISR TROY with mouse TROY mRNA is 85%.
  • hypothalamic AGT-106 expression was decreased with fasting in Psammomys obesus. The main effect was observed in group A fed and A fasted animals with an approximately 50% reduction in AGT-106 expression with fasting. This dramatic reduction in AGT-106 expression with fasting was not evident in group B and C animals, and the fed animals in both of these obese groups were similar to the fasted group A animals.
  • AGT-106 a member of the Tumor Necrosin Factor Receptor Superfamily (TNFRSF) in the regulation of energy utilisation and bodyweight in response to fasting in rodents.
  • AGT-106 is a receptor in the hypothalamus, a key site within the brain for the regulation of bodyweight and energy balance, this regulation may involve downstream transcriptional regulation of genes involved in homeostasis via the NF- ⁇ B pathway, or other as yet unidentified pathways. Alternatively, this regulation may involve the action of circulating messengers/molecules feeding back information to the hypothalamus on the state of energy balance within the body. It is proposed that AGT-106 is associated with one or more of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • AGT-113 was discovered using differential display and it appeared to be expressed at higher levels in the hypothalamus of group C (obese, diabetic) animals than group A (lean healthy) animals.
  • AGT- 201 (Example 10), AGT-203 (Example 11) and cyclophilin, fluorogenic probes which had the reporter dye FAM attached to the 5' end and the quencher dye TAMRA attached to the 3' end were used with Taqman Universal PCR Master Mix (PE Applied Biosystems).
  • AGT-202 AGT-106 (Troy) and AGT-113, no probe was used, and SYBR Green Master Mix (PE Applied Biosystems) was used instead.
  • the level of expression of the "housekeeping gene” cyclophilin was examined in each group and was shown not to be altered in the obese, diabetic or fasted state.
  • Reverse primer 5'-TCCCAAAGTAAATTAAACACATCAGAA-3' [ SEQ I D NO : 17 ]
  • Reverse primer 5'-CCA GTG CTC AGA GCA CGA AA-3' [ SEQ ID NO : 19]
  • the Psammomys obesus AGT-113 partial nucleotide sequence is as follows:
  • This sequence has some homology (84% homology over 65 nucleotides) to human clone RP11-368J13 (GenBank accession number AC008070).
  • AGT-113 plays a role in body weight regulation and energy homeostasis through its actions in the hypothalamus. AGT-113 may also be involved in insulin's action or insulin resistance in the hypothalamus. It is proposed that AGT-113 is associated with one or more of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • AGT-201 was determined to be differentially expressed in Psammomys obesus by membrane-based microarray (macroarray) analysis of the hypothalamus.
  • Reverse primer 5'-TTTCAAGATGGCCTGGCG-3' [ SEQ ID NO : IO ]
  • the partial nucleotide sequence of Psammomys obesus AGT-201 is as follows:
  • AGT-201 is known to be located on chromosome 12 and has been mapped to the interval D12S78-D12S79 (12q21-22). 2 animal QTLs are located in the vicinity of AGT-201, Qsbw and Weightl (Chagnon et al, Obes Res. 8(1): 89-117, 2000).
  • hypothalamic AGT-201 expression was decreased with fasting significantly in group A and B animals however there was no reduction of expression with fasting in group C animals.
  • hypothalamic AGT-201 expression There was significantly lower hypothalamic AGT-201 expression in the fasted animals compared to the fed animals.
  • AGT-201 gene expression in the hypothalamus did not correlate with body weight, blood glucose or insulin concentration in the fed or fasted state.
  • AGT-201 in the central response to fasting and energy homeostasis, possibly by altering the protein expression, retention, retrieval or folding of secretory proteins exiting the endoplasmic reticulum.
  • these results suggest that the role of AGT-201 in this regulation may be altered in the diabetic state thereby identifying this gene as a potential target for the development of diabetic treatments. It is proposed that AGT-201 is associated with one or more of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • AGT-202 was determined to be differentially expressed in the hypothalamus of Psammomys obesus by macroarray analysis.
  • Reverse primer 5'-TCATAGTCTCGCTCGCAGTAGG-3' [ SEQ- ID NO . 15] A portion of the Psammomys obesus AGT-202 sequence was obtained in order to design primers for gene expression studies.
  • AGT-202 is located on human chromosome 5. No QTL's relevant to obesity or diabetes were found.
  • AGT-202 gene expression appears to be widespread.
  • cDNA sources include aorta, blood, brain, breast, colon, germ cell, kidney, larynx, lung, muscle, ovary, pancreas, pooled, prostate, stomach, testis, tonsil, ute s, whole embryo, brain, cervix, colon, eye, neck, kidney, lung, ovary, pancreas, prostate, tumor, skin, thymus, pooled, utems, whole blood
  • AGT-202 is associated with one or more of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • AGT-203 Presenilins interacting rhomboid-like protease
  • a human gene in the form of an expressed sequence tag (EST) with Accession Number AA131464 was found to be differentially expressed in skeletal muscle of lean, non- diabetic versus obese, diabetic animals by membrane-based microarray (macroarray).
  • a human mRNA with a complete coding sequence that matched EST AA131464 was recently added to GenBank (on 1 November, 2000, Accession Number AF 197937).
  • GenBank GenBank
  • the mRNA codes for a 379 amino acid protein they have named Presenilins interacting rhomboid-like protease (AGT-203).
  • Reverse primer 5'-CCTGTGAACCCAACAGTGAAGA-3' [ SEQ ID NO : 7 ]
  • the AGT-203 gene appears to have a widespread tissue expression pattern. ESTs that correspond to the AGT-203 mRNA have been found in adrenal gland, blood, bone, brain, breast, colon, foreskin, germ cell, heart, kidney, lung, lymph, marrow, muscle, ovary, pancreas, parathyroid, placenta, prostate, skin, spleen, stomach, testis, tonsil, utems and whole embryo.
  • the exon/intron stmcture of the human AGT-203 gene was deduced by aligning the mRNA sequence to human high throughput genome sequencing clones, then applying the GT-AG mle (where all introns start with GT and end with AG).
  • the human AGT-203 gene has 10 exons. The first 4 exons were on clone RP11-315J22 (Accession Number AC068644) which is from chromosome 3. The last 6 exons were on clone RP11-637N15 (Accession Number AC020694) which is from chromosome 17. It is considered likely that one of the clones has been localized incorrectly. Studies are currently underway to determine which is the correct chromosome.
  • AGT-203 gene expression was found to be reduced in the skeletal muscle of obese, hyperinsulinemic Psammomys obesus. A negative correlation was seen with body weight and plasma insulin levels. AGT-203 is thought to interact with presenilin proteins and be involved with protein cleavage.
  • the presenilins are involved in the proteolytic processing of transmembrane proteins such as APP and Notch.
  • APP is most highly expressed in the central nervous system, it is ubiquitously expressed and its role in the skeletal muscle is not known.
  • Diabetes is known to be a risk factor for Alzheimer's disease, and AGT-203 may play a role in both diseases.
  • NOTCH is a membrane receptor and, after proteolytic processing within the membrane, has the ability to move to the nucleus and activate gene expression.
  • AGT-203 may act through NOTCH to affect expression of genes involved in metabolism.
  • the role of AGT-203 in obesity or diabetes may be through processing of another, as yet unidentified, transmembrane protein.
  • AGT-203 is associated with one or more of mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a lysate of cells transfected with a tagged AGT-203 is immunoprecipitated with antibodies directed to the N-terminal, C-terminal or internal portions of AGT-203 (anti- AGT-203) and with antibodies against the tag.
  • antibodies directed to the N-terminal, C-terminal or internal portions of AGT-203 anti- AGT-203
  • immunoprecipitated bands are detected.
  • the formation of smaller fragments are also sought.
  • the absence of any smaller is then screened in the presence of mutations at potential cleavage sites. N-terminal, C-terminal and internal AGT-203 fragments are sought.
  • AGT-203 To map the potential cleavage sites, cells are transfected with AGT-203 containing a tag inserted upstream of potential cleavage sites. To identify the cleavage sites within AGT- 203, fragments are isolated and subjected to sequencing by Edman degradation.
  • N-terminal fragments generated could be a nucleus-target molecule.
  • cells are transfected with constmcts expressing different portions of AGT-203 conjugated to a flurochrome, such as GFP, and the location of the peptides identified by fluorescent microscopy analysis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte d'une manière générale sur des molécules d'acides nucléiques identifiées par leur motif d'expression dans au moins l'hypothalamus ou des muscles du squelette. Lesdites molécules ou leurs produits d'expression ou des fragments de ces produits sont associées à des marqueurs ou servent de marqueurs notamment d'états de bonne santé ou morbides dont: la présence ou l'absence d'un trouble associé à une dysfonction mitochondrienne, à une myopathie, à un trouble génétique, et/ou à un cancer, et/ou à moduler l'apoptose et la transduction de signaux, et/ou au ciblage de noyaux. Lesdites molécules et/ou leurs produits d'expression et/ou leurs dérivés, fragments, homologues, analogues et mimétiques, peuvent servir d'agents thérapeutiques et diagnostiques notamment pour des troubles associés à une dysfonction mitochondrienne, à une myopathie, à un trouble génétique et/ou à un cancer, et/ou à moduler l'apoptose et la transduction de signaux, et/ou au ciblage de noyaux ou de cibles, en vue de la conception et/ou de l'identification de modulateurs de leur activité et/ou de leur fonction.
PCT/AU2005/000468 2004-03-31 2005-03-31 Acide nucleique exprimes dans l'hypothalamus ou les tissus musculaires et leur utilisation a des fins de diagnostic Ceased WO2005095610A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US55826104P 2004-03-31 2004-03-31
US60/558,261 2004-03-31

Publications (1)

Publication Number Publication Date
WO2005095610A1 true WO2005095610A1 (fr) 2005-10-13

Family

ID=35063787

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2005/000468 Ceased WO2005095610A1 (fr) 2004-03-31 2005-03-31 Acide nucleique exprimes dans l'hypothalamus ou les tissus musculaires et leur utilisation a des fins de diagnostic

Country Status (1)

Country Link
WO (1) WO2005095610A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1795198A1 (fr) * 2005-12-09 2007-06-13 Hubrecht Laboratorium Traitement de l'esophage de Barret
CN112870234A (zh) * 2021-01-27 2021-06-01 四川九章生物科技有限公司 包含绿原酸的药物组合物在制备治疗病理性黄疸的药物中的用途
EP3668891B1 (fr) * 2017-08-16 2023-07-26 Lgv1 S.R.L. Isoform vtft d'une proteine bpifb4 destinee aux maladies neuronales et aux blessures

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006994A2 (fr) * 2000-07-14 2002-01-24 Sunil Paul Systeme et procede determinant un itineraire de transport optimal
WO2003070771A1 (fr) * 2002-01-23 2003-08-28 Exelixis, Inc. Sequences de rrp, souris knock-out et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006994A2 (fr) * 2000-07-14 2002-01-24 Sunil Paul Systeme et procede determinant un itineraire de transport optimal
US20030165497A1 (en) * 2000-07-19 2003-09-04 Lioubin Mario N. RRP sequences and knockout mice and uses thereof
WO2003070771A1 (fr) * 2002-01-23 2003-08-28 Exelixis, Inc. Sequences de rrp, souris knock-out et leurs utilisations

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK *
DATABASE GENBANK "Has 100% identity with a 1131 bp stretch of SEQ ID NO:23" *
DATABASE GENBANK 2001 *
DATABASE GENBANK 2002 *
DATABASE GENBANK 2002, "Has 94% identity with a 1119 bp stretch of SEQ ID NO:22" *
DATABASE GENBANK 2002, "Has 94% identity with a 815 bp stretch of SEQ ID NO:22" *
J. ALRHEIMERS DIS, vol. 3, no. 2, 2001, pages 181 - 190 *
J. ALTYHEIMERS DIS, vol. 3, no. 2, 2001 *
J. ALZHEIMERS DIS, vol. 3, no. 2, 2001, pages 181 - 190 *
PROC. NATH. ACAD SCI, vol. 99, no. 26, 2002, USA, pages 16899 - 16903 *
PROC. NATL. ACAD SCI USA, vol. 99, no. 26, 2002, USA, pages 16899 - 16903 *
PROC. NATL. ACAD SCI, vol. 99, no. 26, 2002, pages 16899 - 16903 *
PROC. NATL. ACAD. SCI, vol. 99, no. 26, 2002, USA, pages 16899 - 16903 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1795198A1 (fr) * 2005-12-09 2007-06-13 Hubrecht Laboratorium Traitement de l'esophage de Barret
WO2007067048A1 (fr) * 2005-12-09 2007-06-14 Hubrecht Laboratorium Traitement de l’endobrachyœsophage
RU2432163C2 (ru) * 2005-12-09 2011-10-27 Хубрехт Лабораториум Лечение пищевода барретта
EP3668891B1 (fr) * 2017-08-16 2023-07-26 Lgv1 S.R.L. Isoform vtft d'une proteine bpifb4 destinee aux maladies neuronales et aux blessures
CN112870234A (zh) * 2021-01-27 2021-06-01 四川九章生物科技有限公司 包含绿原酸的药物组合物在制备治疗病理性黄疸的药物中的用途

Similar Documents

Publication Publication Date Title
US20080153159A1 (en) Therapeutic and diagnostic agents
US20070135335A1 (en) Therapeutic molecules
US20060228775A1 (en) Differential expression of nucleic acid molecules
US7662929B2 (en) Antibody that specifically binds hyaluronan synthase
US20090232808A1 (en) Molecules and chimeric molecules thereof
WO2005014634A1 (fr) Gene et ses applications
EP1861423A1 (fr) Molécule et molécules chimères de celle-ci
US20070009887A1 (en) Genotyping of deafness by oligonucleotide microarray analysis
US20070021589A1 (en) Obesity-related genes
US20090136444A1 (en) molecule and chimeric molecules thereof
WO2005095610A1 (fr) Acide nucleique exprimes dans l'hypothalamus ou les tissus musculaires et leur utilisation a des fins de diagnostic
WO2005026358A9 (fr) Polymorphismes dans les selenoproteines s
AU2003283054B2 (en) Genotyping of deafness by oligonucleotide microarray analysis
AU2013205487A1 (en) Therapeutic and diagnostic agents
US20070184036A1 (en) Isolated cd4+ t cells defined by cmrf-35
AU2004279475A1 (en) The moduilation of hyaluronan synthesis and degradation in the treatment of disease
AU2004285986A1 (en) Isolated CD4+ T cells defined by CMRF-35 and CD45RO

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
122 Ep: pct application non-entry in european phase