WO1996009408A1 - Human galactokinase gene - Google Patents

Human galactokinase gene Download PDF

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Publication number
WO1996009408A1
WO1996009408A1 PCT/US1994/010825 US9410825W WO9609408A1 WO 1996009408 A1 WO1996009408 A1 WO 1996009408A1 US 9410825 W US9410825 W US 9410825W WO 9609408 A1 WO9609408 A1 WO 9609408A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
galactokinase
cells
die
dna
Prior art date
Application number
PCT/US1994/010825
Other languages
English (en)
French (fr)
Inventor
Derk Jon Bergsma
Dwight Edward Stambolian
Original Assignee
Smithkline Beecham Corporation
University Of Pennsylvania
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 Smithkline Beecham Corporation, University Of Pennsylvania filed Critical Smithkline Beecham Corporation
Priority to AU79581/94A priority Critical patent/AU7958194A/en
Priority to MXPA94008258A priority patent/MXPA94008258A/es
Priority to BR9408618A priority patent/BR9408618A/pt
Priority to JP8510844A priority patent/JPH10505757A/ja
Priority to PCT/US1994/010825 priority patent/WO1996009408A1/en
Priority to ZA947865A priority patent/ZA947865B/xx
Priority to BR9509211A priority patent/BR9509211A/pt
Priority to PL95319344A priority patent/PL319344A1/xx
Priority to PCT/US1995/006743 priority patent/WO1996009374A1/en
Priority to JP8510858A priority patent/JPH10506529A/ja
Priority to MX9702205A priority patent/MX9702205A/es
Priority to EP95921460A priority patent/EP0783567A4/en
Priority to AU26536/95A priority patent/AU2653695A/en
Publication of WO1996009408A1 publication Critical patent/WO1996009408A1/en
Priority to NO971363A priority patent/NO971363D0/no
Priority to NO971359A priority patent/NO971359L/no
Priority to US08/998,208 priority patent/US5880105A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to human galactokinase, isolated nucleic acids encoding same, recombinant host cell transformed with DNA encoding such protein and to uses of the expressed protein and nucleic acid sequences in therapeutic and diagnostic applications.
  • Galactokinase deficiency is one of three known forms of galactosemia. The other forms are galactose-1 -phosphate uridyltransferase deficiency and UDP- galactose-4-epimerase deficiency. All three enzymes are involved in galactose metabolism, i.e., the conversion of galactose to glucose in the body. Galactokinase deficiency is inherited as an autosomal recessive trait with a heterozygote frequency estimated to be 0.2% in the general population (see, e.g., Levy et al., J. Pediatr.. 22:871-877 (1978)).
  • This invention provides a human galactokinase gene.
  • the DNAs of this invention such as the specific sequences disclosed herein, are useful in that they encode the genetic information required for expression of this protein. Additionally, the sequences may be used as probes in order to isolate and identify additional members, of the family, type and/or subtype as well mutations which may form the basis of galactokinase deficiency which may be characterized by site-specific mutations or by atypical expression of the galactokinase gene.
  • the galactokinase gene is also useful as a diagnostic agent to identify mutant galactokinase proteins or as a therapeutic agent via gene therapy.
  • This invention provides isolated nucleic acid molecules encoding human galactokinase including mRNAs, DNAs (e.g., cDNAs, genomic DNA, etc.), as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
  • This invention also provides recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of human galctokinase proteins, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a human galactokinase nucleic acid sequence.
  • This invention also provides a process for preparing human galactokinase protein which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human galactokinase nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.
  • Another related aspect of this invention is an isolated human galactokinase protein produced by said method.
  • this invention also provides antibodies that are directed to (i.e., bind) human galactokinase.
  • This invention also provides nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human galactokinase sequences.
  • This invention also provides a method to diagnose human galactokinase deficiency which comprises isolating a nucleic acid sample from an individual and assaying the sequence of said nucleic acid sample with the reference gene of the invention and comparing differences between said sample and the nucleic acid of the instant invention, wherein said differences indicate mutations in the human galactokinase gene isolated from an individual.
  • the sample can be assayed by direct sequence comparison (i.e., DNA sequencing), wherein the sample nucleic acid can be compared to the reference galactokinase gene, by hybridization (e.g., mobility shift assays such as heteroduplex gel electrophoresis, SSCP or other techniques such as Northern or Southern blotting which are based upon the length of the nucleic acid sequence) or other known gel electrophoresis methods such as RLFP.
  • the diagnostic method comprises isolating cells from an individual containing genomic DNA and assaying said sample (e.g., cellular RNA) by in situ hybridization using the DNA sequence of the invention as a probe.
  • This invention also provides an antisense oligonucleotide having a sequence capable of binding with mRNAs encoding human galactokinase so as to identify mutant galactokinase genes.
  • This invention also provides transgenic non-human animals comprising a nucleic acid molecule encoding human galactokinase. Also provided are methods for use of said transgenic animals as models for disease states, mutation and S AR. This invention also provides a method for treating conditions which are related to insufficient human galactokinase activity which comprises administering to a patient in need thereof a pharmaceutical composition containing the galactokinase protein of the invention which is effective to supplement a patient's endogenous galactokinase and thereby alleviating said condition.
  • This invention also provides a method for treating conditions which are related to insufficient human galactokinase activity via gene therapy.
  • An additional, reference gene comprising the galactokinase gene of the instant invention is inserted into a patient's cells either in vivo or ex vivo.
  • the reference gene is expressed in transfected cells and as a result, the protein encoded by the reference gene corrects the defect (i.e., galactokinase deficiency) thus permitting the transfected cells to function normally and alleviating disease conditions (or symptoms).
  • This invention relates to human galactokinase and its use as a diagnostic and therapeutic.
  • the particular sequence of human galactokinase is identified as SEQ ED
  • an "antigen” refers to a molecule containing one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular
  • epitope refers to the site on an antigen or hapten to which a specific antibody molecule binds.
  • the term is also used herein interchangeably with "antigenic determinant” or “antigenic determinant site.”
  • a coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences.
  • the coding sequences need not be contiguous to one another so long as the expressed sequence is ultimately processed to produce the desire protein.
  • Recombinant polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
  • synthetic polypeptides are those prepared by chemical synthesis.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo: i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment
  • a “replication-deficient virus” is a virus in which the excision and/or replication functions have been altered such that after transfection into a host cell, the virus is not able to reproduce and/or infect addition cells.
  • a “reference” gene refers to the galactokinase sequence of the invention and is understood to include the various sequence polymorphisms that exist, wherein nucleotide substitutions in the gene sequence exist, but do not affect the essential function of the gene product.
  • a “mutant” gene refers to galactokinase sequences different from the reference gene wherein nucleotide substitutions and or deletions and/or insertions result in impairment of the essential function of the gene product such that the levels of galactose in an individual (or patient) are atypically elevated.
  • a DNA “coding sequence of or a "nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 1 direction) coding sequence.
  • the promoter sequence is bound at the 3' terminus by a translation start codon (e.g., ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with nuclease S 1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • control sequences refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.
  • a control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence.
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.
  • Transfection or “transfected” refers to a process by which cells take up foreign DNA and integrate that foreign DNA into their chromosome. Transfection can be accomplished, for example, by various techniques in which cells take up DNA (e.g., calcium phosphate precipitation, electroporation, assimilation of liposomes, etc.), or by infection, in which viruses are used to transfer DNA into cells.
  • a “target cell” is a cell(s) that is selectively transfected over other cell types (or cell lines).
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a “heterologous” region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a gene, the gene will usually be flanked by DNA that does not flank die gene in the genome of the source animal.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • Constants which are related to insufficient human galactokinase activity or a "deficiency in galactokinase activity” means mutations of the galactokinase protein which affects galactokinase activity or may affect expression of galactokinase or both such that the levels of galactose in a patient are atypically elevated.
  • this definition is intended to cover atypically low levels of galactokinase expression in a patient due to defective control sequences for the reference galactokinase protein.
  • This invention provides an isolated nucleic acid molecule encoding a human galactokinase protein and substantially similar sequences.
  • Isolated nucleic acid sequences are substantially similar if: (i) they are capable of hybridizing under moderately stringent conditions to SEQ ID NO:4; ( ⁇ ) or they encode DNA sequences which are degenerate to SEQ ID NO:4. Degenerate DNA sequences encode the same amino acid sequence as SEQ ID NO:4, but have variation(s) in the nucleotide coding sequences. Hybridization under moderately stringent conditions is outlined below.
  • substantially similar sequences are substantially the same when about 66% (preferably about 75%, and most preferably about 90%) of the nucleotides or amino acids match over a defined length of the molecule.
  • substantially similar refers to the sequences having similar identity to the sequences of the instant invention.
  • nucleotide sequences that are substantially the same can be identified by hybridization or by sequence comparison.
  • Protein sequences that are substantially the same can be identified by one or more of the following: proteolytic digestion, gel electrophoresis and/or microsequencing. Hybridization under moderately stringent conditions can be performed as follows.
  • Nitrocellulose filters are prehybridized at 65°C in a solution containing 6X SSPE, 5X Denhaidt's solution (lOg Ficoll, lOg BSA and lOg Polyvinylpyrrolidone per liter solution), 0.05% SDS and 100 micrograms tRNA.
  • Hybridization probes are labeled, preferably radiolabelled (e.g., using the Bios TAG-IT® kit). Hybridization is then carried out for approximately 18 hours at 65°C.
  • the filters are then washed in a solution of 2X SSC and 0.5% SDS at room temperature for 15 minutes (repeated once). Subsequently, the filters are washed at 58°C, air-dried and exposed to X-ray film overnight at -70°C with an intensifying screen.
  • One means for isolating a nucleic acid molecule encoding for a human galactokinase is to probe a human genomic or cDNA library with a natural or artificially designed probe using art recognized procedures (See for example: "Current Protocols in Molecular Biology", Ausubel, F.M., et al. (eds.) Greene Publishing Assoc. and John Wiley Interscience, New York, 1989,1992). It is appreciated to one skilled in the art that SEQ ID NO:4, or fragments thereof (comprising at least 15 contiguous nucleotides), is a particularly useful probe.
  • probes for this purpose are SEQ ID NOs: 1 , 2 and 3, or hybridizable fragments thereof (i.e., comprising at least 15 contiguous nucleotides). It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe.
  • Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product.
  • the probes are thus useful to isolate complementary copies of genomic DNA, cDNA or RNA from human, mammalian or other animal sources or to screen such sources for related sequences (e.g., additional members of the family, type and/or subtype) and including transcriptional regulatory and control elements defined above as well as other stability, processing, translation and tissue spe ⁇ ficity-dete ⁇ nining regions from 5' and/or 3' regions relative to the coding sequences disclosed herein.
  • related sequences e.g., additional members of the family, type and/or subtype
  • transcriptional regulatory and control elements defined above as well as other stability, processing, translation and tissue spe ⁇ ficity-dete ⁇ nining regions from 5' and/or 3' regions relative to the coding sequences disclosed herein.
  • Gene therapy means gene supplementation. That is, an additional (i.e., reference) copy of the gene of interest is inserted into a patients' cells. As a result, the protein encoded by die reference gene corrects the defect (i.e., galactokinase deficiency) and permits the cells to function normally thus alleviating disease symptoms.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene, and introduction of the genetically altered cells back into the patient.
  • a replication-deficient virus such as a modified retrovirus can be used to introduce the therapeutic gene (galactokinase) into such cells.
  • MMLV mouse Moloney leukemia virus
  • MMLV mouse Moloney leukemia virus
  • the therapeutic gene is typically "packaged” for administration to a patient such as in liposomes or in a replication-deficient virus such as adenovirus (see, e.g., Berkner, K.L., Curr. Top. Microbiol. Immunol..151:39-66 (1992)) or adeno-associated virus (AAV) vectors (see, e.g., Muzyczka, N., Curr. Top. Micmhiol. Immunol..15&97-129 (1992) and U.S. Patent 5,252,479 "Safe Vector for Gene Therapy”).
  • adenovirus see, e.g., Berkner, K.L., Curr. Top. Microbiol. Immunol..151:39-66 (1992)
  • AAV adeno-associated virus
  • Another approach is administration of so-called “naked DNA” in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • Cell types useful for gene therapy of the present invention include hepatocytes, fibroblasts, lymphocytes, any cell of the eye (e.g., retina), epithelial and endothelial cells.
  • the cells are hepatocytes, any cell of the eye or respiratory (or pulmonary) epithelial cells.
  • Transfection of (pulmonary) epithelial cells can occur via inhalation of a neubulized preparation of DNA vectors in liposomes, DNA-protein complexes or replication-deficient adenoviruses (see, e.g., U.S. Patent 5,240,846 "Gene Therapy Vector for Cystic Fibrosis".
  • This invention also provides for a process to prepare human galactokinase protein.
  • This protein is defined with reference to the amino acid sequence listed in SEQ ID NO:4 and includes variants with a substantially similar amino acid sequence that have the same galactokinase activity.
  • the proteins of this invention are preferably made by recombinant genetic engineering techniques.
  • the isolated nucleic acids particularly the DNAs can be introduced into expression vectors by operatively linking the DNA to the necessary expression control regions (e.g., regulatory regions) required for gene expression.
  • the vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (Ausubel et al., sucia).
  • the coding sequences for the desired proteins having been prepared or isolated can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include, but is not limited to, the bacteri ⁇ phage ⁇ (£. cjoJi), pBR322 (E.
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in die host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • the subunit antigens of the present invention can be expressed using, for example, the £. coji tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g.. U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.
  • regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in die vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the sequences encoding die particular antigen of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • die coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by die deletion of a portion of d e sequence encoding die protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art Sfifi. e * , T. Maniatis et al., supra; DNA Cloning. Vols. I and ⁇ , supra; Nucleic Acid HyhriHirati n supra. A number of prokaryotic expression vectors are known in the art
  • pSV2neo (as described in J. Mol. Appl. Gene 1:327-341) which uses the SV40 late promoter to drive expression in mammalian cells or pCDNAlneo, a vector derived from pCDNAl (Mol. Cell Biol. 7:4125-29) which uses the CMV promoter to drive expression. Both these latter two vectors can be employed for transient or stable (using G418 resistance) expression in mammalian cells. Insect cell expression systems, e.g., Drosophila. are also useful, see for example, PCT applications WO 90 06358 and WO 92 06212 as well as EP 290,261-B1.
  • die proteins of die present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed.
  • Preferred mammalian cells include human embryonic kidney cells, monkey kidney (HEK-293cclls), fibroblast (COS) cells, Chinese hamster ovary (CHO) cells, Drosophila or murine L-cells. If the expression system secretes the protein into growth media, die protein can be purified direcdy from the media. If the protein is not secreted, it is isolated from cell ly sates or recovered from the cell membrane fraction. The selection of the appropriate growtii conditions and recovery methods are within the skill of die art
  • An alternative method to identify proteins of the present invention is by constructing gene libraries, using the resulting clones to transform I £ ⁇ li and pooling and screening individual colonies using polyclonal serum or monoclonal antibodies to galactokinase.
  • the proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from die DNA sequence of die genes of interest Such methods are known to those skilled in d e art. Chemical synthesis of peptides is not particularly preferred.
  • the proteins of the present invention or their fragments comprising at least one epitope can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit goat horse, etc.) is immunized with a receptor of de present invention, or its fragment or a mutated receptor. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, die polyclonal antibodies can be purified by immunoaffinity chromatography or other known procedures. Monoclonal antibodies to die proteins of the present invention, and to the fragments thereof, can also be readily produced by one skilled in the art.
  • a selected mammal e.g., mouse, rabbit goat horse, etc.
  • Serum from the immunized animal is collected and treated according to known procedures.
  • die polyclonal antibodies can be purified by immunoaffinity chromatography or other known procedures.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g.. M. Schreierel L, "Hybridoma Techniques” (1980); Hammerling&al-, "Monoclonal Antibodies and T-cell Hybridomas” (1981); Kennett£tal-» "Monoclonal Antibodies” (1980); acs lS ⁇ U.S. Patent Nos.
  • Panels of monoclonal antibodies produced against the antigen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against Alternatively, genes encoding the monoclonals of interest may be isolated from the hybridomas by PCR techniques known in d e art and cloned and expressed in the appropriate vectors.
  • the antibodies of this invention may be employed reagents in immunoassays, RIA, ELISA, and die like.
  • monoclonal antibody is understood to include antibodies derived from one species (e.g., murine, rabbit goat rat human, etc.) as well as antibodies derived from two (or perhaps more) species (e.g., chimeric and humanized antibodies).
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (sec, £_£. Liu et al., Proc. Natl Acad. Sci. USA. 84:3439 (1987)), may also be used in assays or therapeutically.
  • a therapeutic monoclonal antibody would be "humanized” as described in Jones et al., Nature.
  • this invention also contemplates antibodies, polyclonal or monoclonal (including chimeric and "humanized") directed to epitopes corresponding to amino acid sequences disclosed herein from human galactokinase.
  • Methods for the production of polyclonal and monoclonal antibodies are well known, see for example Chap. 11 of Ausubel et al. (supra).
  • an analytically detectable reagent such as radioactivity, fluorescence, or an enzyme
  • the antibody can be use to detect die presence or absence of human galactokinase and/or its quantitative level.
  • pharmaceutical compositions comprising an effective amount of the galactokinase protein of the invention and a pharmaceutically acceptable carrier.
  • compositions of proteineous drugs of this invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously.
  • the galactokinase protein is surrounded by a membrane bound vesicle, such as a liposome.
  • compositions for parenteral administration will commonly comprise a solution of d e compounds of die invention or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be employed, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and die like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • the concentration of die compound of die invention in such pharmaceutical formulation can very widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and 50 mg of a compound of die invention.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of a compound of the invention.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in die art and are described in more detail in, for example, Remington's
  • die dosage of die present therapeutic agents which will be most suitable and it will vary with the form of administration and die particular compound chosen, and furthermore, it will vary with the particular patient under patient undo * treatment He will generally wish to initiate treatment with small dosages substantially less than the optimum dose of die compound and increase the dosage by small increments until the optimum effect under the circumstances is reached. It will generally be found that when the composition is administered orally, larger quantities of die active agent will be required to produce die same effect as a smaller quantity given parenterally.
  • the therapeutic dosage will generally be from 1 to 10 milligrams per day and higher although it may be administered in several different dosage units.
  • the pharmaceutical composition of die invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest die disease and its complications.
  • compositions containing the present compounds or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Single or multiple administrations of the pharmaceutical compositions can be carried out with dose levels and pattern being selected by die treating physician.
  • the pharmaceutical composition of die invention should provide a quantity of the compounds of the invention sufficient to effectively treat the patient
  • This invention also contemplates use of the galactokinase gene as a diagnostic. For example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequence of the defective gene with that of a normal one. Subsequendy, one can verify that a "mutant" gene is associated with galactokinase deficiency by measurement of galactose.
  • mutant gene would be associated witii (atypically) elevated levels of galactose in a patient
  • mutant galactokinase genes into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, e.g, in a galactokinase deficient strain of yeast or E. col ⁇ ) as yet another means to verify or identify galactokinase mutations.
  • RNA from an individual can be transcribed with reverse transcriptase to cDNA which can then be amplified by polymerase chain reaction (PCR), cloned into an E.
  • PCR polymerase chain reaction
  • galactokinase-deficient cells When grown on MacConkey indicator plates, galactokinase-deficient cells will produce colonies that are white in color, whereas cells that have been transformed/complemented widi a functional galactokinase gene will be red (see, e.g., Examples section). If most to all of the colonies from an individual are red, then the indivdual is considered to be normal with respect to galactokinase activity. If approximately 50% of the colonies are red (die other 50% white), then that individual is likely to be a carrier for galactokinase deficiency.
  • Nucleic acids used for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy (e.g., chorionic villi sampling or removal of amniotic fluid cells), and autopsy material.
  • the genomic DNA may be used direcdy for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplifcation (SDA), etc.
  • RNA or cDNA my also be used for the same purpose.
  • PCR primers complementary to the nucleic acid of die instant invention can be used to identify and analyze galactokinase mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal galactokinase genotype.
  • Point mutations can be identified by hybridizing amplified DNA to rabiolabeled galactokinase RNA (of the invention) or alternatively, radiolabelled galactokinase antisense DNA sequences (of die invention). Perfectiy matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures (Tm). Such a diagnostic would be particularly useful for prenatal and even neonatal testing.
  • point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by yet otiier well-known techniques, e.g., direct DNA sequencing, single-strand conformational polymorphim (SSCP; Qrita et al., Geno ics.5:874-879 (1989)).
  • SSCP single-strand conformational polymorphim
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures witii radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is gready enhanced when combined with PCR.
  • the presence of nucleotide repeats may correlate to a change in galactokinase activity (causative change) or serve as marker for various polymorphims.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in die gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science.220:1242 (1985)).
  • sequence alterations in particular small deletions, may be detected as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel electrophoresis (i.e., heteroduplex electrophoresis) (see, e.g., Nagamine et al., Am. J. Hum. Genet. 45:337-339 (1989)).
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or d e chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA. 85:4397-4401 (1985)).
  • nuclease protection assays such as RNase and SI protection or d e chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA. 85:4397-4401 (1985)).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization (e.g., heteroduplex electroporation, see, White et al., Genomics.12:301-306 (1992), RNAse protection (e.g., Myers et al., Science, 220:1242 (1985)) chemical cleavage (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA.
  • hybridization e.g., heteroduplex electroporation, see, White et al., Genomics.12:301-306 (1992
  • RNAse protection e.g., Myers et al., Science, 220:1242 (1985)
  • chemical cleavage e.g., Cotton et al., Proc. Natl. Acad. Sci. USA.
  • restriction enzymes e.g., restriction fragment length polymorphisms (RFLP) in which variations in die number and size of restriction fragments can indicate insertions, deletions, presence of nucleotide repeats and any other mutation which creates or destroys an endonuclease restriction sequence.
  • RFLP restriction fragment length polymorphisms
  • Southen blotting of genomic DNA may also be used to identify large (i.e., greater than 100 base pair) deletions and insertions.
  • mutations e.g., microdeletions, aneuploidies, translocations, inversions
  • in situ analysis See, e.g., Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)). That is, DNA (or RNA) sequences in cells can be analyzed for mutations without isolation and/or immobilization onto a membrane.
  • Fluorescence in situ hybridization FISH is presendy the most commonly applied mediod and numerous reviews of FISH have appeared.
  • some diseases are a result of, or are characterized by, changes in gene expression which can be detected by changes in the mRNA.
  • the galactokinase gene can be used as a reference to identify individuals expressing a decreased level of galactokinase, e.g., by Northern blotting or in situ hybridization. Defining appropriate hybridization conditions is within the skill of the art. See, e.g.. "Current Protocols in Mol. Biol.” Vol. I & II, Wiley Interscience. Ausbel £1 al- (ed.) (1992).
  • probes can vary widely but it is preferred tiiat the probe be at least 15 nucleotides in length. It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of die probe.
  • useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. As a general rule the more stringent the hybridization conditions die more closely related genes will be that are recovered.
  • antisense oligonucleotides predicated upon die sequences disclosed herein for human galactokinase are antisense oligonucleotides predicated upon die sequences disclosed herein for human galactokinase.
  • Synthetic oligonucleotides or related antisense chemical structural analogs are designed to recognize and specifically bind to a target nucleic acid encoding galactokinase and galactokinase mutations.
  • the general field of antisense technology is illustrated by the following disclosures which are incorporated herein by reference for purposes of background (Cohen, J.S., Trends in Pharm. Sci.. 10:435(1989) and Weintraub, H.M. Scientific American, Jan.(1990) at page 40).
  • Transgenic, non-human, animals may be obtained by transfecting appropriate fertilized eggs or embryos of a host with nucleic acids encoding human galactokinase disclosed herein, see for example U.S. Patents 4,736,866; 5,175,385; 5,175,384 and 5,175,386.
  • the resultant transgenic animal may be used as a model for die study of galactokinase.
  • useful transgenic animals are those which display a detectable phenotype associated with the expression of the receptor. Drugs may then be screened for dieir ability to reverse or exacerbate the relevant phenotype.
  • This invention also contemplates operatively linking the receptor coding gene to regulatory elements which are differentially responsive to various temperature Or metabolic conditions, thereby effectively turning on or off the phenotypic expression in response to those conditions.
  • Purified protein was obtained following separation by SDS polyacrylamide electrophoresis and then Western blotted using standard techniques (see, Laemmli, ⁇ aill ⁇ e, 221:680-685 (1970), or LeGendre et al., Biotechniques. £: 154 (1988)). Minute amounts of galactokinase were isolated (micrograms) from multiple rounds of protein purification. After a trypsin peptide digest, 7 peptide sequences were eventually isolated and identified.
  • the fragments were compared widi peptide sequences encoded by cDNAs, in which the cDNAs were partially sequenced.
  • the cDNAs also known as expressed sequence tags or ESTs
  • SEQ ID NO:4 A full-length cDN A from the human osteoclastoma stromal cell library was identified and sequenced (SEQ ID NO:4).
  • SEQ ID NO: 4 The corresponding amino acid sequence (SEQ ID NO:4) was compared against die peptide fragments identified above.
  • SEQ ID NO: 1 corresponds to amino acids 38-68 of the full-length human galactokinase protein.
  • SEQ ID NOs: 2 and 3 correspond to amino acids 367-388 and 167-195, respectively, of human galactokinase.
  • the GK2 gene maps to human chromosome 15 which is in contrast to the gene of the present invention which maps to human chromosome 17, position q24 as determined by fluorescence in situ hybridization (FISH) analysis.
  • FISH fluorescence in situ hybridization
  • SEQ ID NO:4 was hybridized against a Northern blot containing human messenger RNA from placenta, brain, skeletal muscle, kidney, intestine, heart lung and liver according to standard procedures (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, 1989). Hybridization was strongest with human liver and lung tissue.
  • SEQ ID NO:4 was subcloned into an E. coli vector, plasmid pBluescript [Stratagene]. When transformed into C600K-, a galactokinase-deficient strain, the transformed E. coli grew on MacConkey agar plates, and produced brick red colonies, indicating sugar fermentation.
  • SEQ ID NO:4 was also subcloned into COS-1 cells [ATCC CRL 1650]. The cells were transfected, grown, and cell lysates were prepared. The lysates were assayed by a , C galactokinase assay as described by Stambolian et al. ( Exp. Eve Res.. 2£:231-237 (1984)) which is hereby incorporated by reference in its entirety. When expressed in transiendy transfected COS cells, galactokinase activity was tenfold higher than control levels (6600 vs. 640 counts per minute - repeated three times). These results definitively confirm that SEQ ID NO:4 encodes a full-length, biologically active, human galactokinase gene.
  • the nucleic acid molecule of the invention can also be subcloned into an expression vector to produce high levels of human galactokinase (either fused to anodier protein, e.g., operatively linked at die 5' end widi another coding sequence, or unfiised) in transfected cells.
  • the expression vector would optionally encode a neomycin resistance gene to select for transfectants on the basis of ability to grow in G418 and a dihydrofolate reductase gene which permits amplification of the transfected gene in DHFR ⁇ cells.
  • the plasmid can then be introduced into host cell lines e.g., CHO ACC98, a nonadherem, DHFR" cell line adapted to grow in serum free medium, and human embryonic kidney 293 cells (ATCC CRL 1573), and transfected cell lines can be selected by G418 resistance.
  • host cell lines e.g., CHO ACC98, a nonadherem, DHFR" cell line adapted to grow in serum free medium, and human embryonic kidney 293 cells (ATCC CRL 1573), and transfected cell lines can be selected by G418 resistance.
  • GCT GCC AGG GAC CTG GTG AGC AAA GAG GGC TTC CGG CGG GCC CGG CAC 868 Ala Ala Arg Asp Leu Val Ser Lys Glu Gly Phe Arg Arg Ala Arg His 265 270 275 280

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PCT/US1994/010825 1994-09-23 1994-09-23 Human galactokinase gene WO1996009408A1 (en)

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AU79581/94A AU7958194A (en) 1994-09-23 1994-09-23 Human galactokinase gene
MXPA94008258A MXPA94008258A (es) 1994-09-23 1994-09-23 Gen de la galactocinasa humana.
BR9408618A BR9408618A (pt) 1994-09-23 1994-09-23 Gene de galactocinase humana
JP8510844A JPH10505757A (ja) 1994-09-23 1994-09-23 ヒト・ガラクトキナーゼ遺伝子
PCT/US1994/010825 WO1996009408A1 (en) 1994-09-23 1994-09-23 Human galactokinase gene
ZA947865A ZA947865B (en) 1994-09-23 1994-10-07 Human glactokinase
PL95319344A PL319344A1 (en) 1994-09-23 1995-05-26 Gene of human galactokinase
BR9509211A BR9509211A (pt) 1994-09-23 1995-05-26 Gene de galactocinase humana
PCT/US1995/006743 WO1996009374A1 (en) 1994-09-23 1995-05-26 Human galactokinase gene
JP8510858A JPH10506529A (ja) 1994-09-23 1995-05-26 ヒト・ガラクトキナーゼ遺伝子
MX9702205A MX9702205A (es) 1994-09-23 1995-05-26 Gen de la galactocinasa humana.
EP95921460A EP0783567A4 (en) 1994-09-23 1995-05-26 HUMAN GALACTOKINASE GEN
AU26536/95A AU2653695A (en) 1994-09-23 1995-05-26 Human galactokinase gene
NO971363A NO971363D0 (no) 1994-09-23 1997-03-21 Human galaktokinasegen
NO971359A NO971359L (no) 1994-09-23 1997-03-21 Humant galaktokinase-gen
US08/998,208 US5880105A (en) 1994-09-23 1997-12-24 Human galactokinase gene

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Title
BIOCHIM. BIOPHYS. ACTA, Volume 831, issued 1985, D. STAMBOLIAN et al., "Purification of Human Galactokinase and Evidence for its Existence as a Monomer Form", pages 306-312. *
NUCLEIC ACIDS RESEARCH, Volume 13, No. 6, issued 1985, C. DEBOUCK et al., "Structure of the Galactokinase Gene of Escherichia Coli, the Last (?) Gene of the Gal Operon", pages 1841-1853. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 78, No. 11, issued November 1981, S.V. SUGGS et al., "Use of Synthetic Oligonucleotides as Hybridization Probes: Isolation of Cloned cDNA Sequences for Human beta2-Microglobulin", pages 6613-6617. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, issued November 1992, R.T. LEE et al., "Cloning of A Human Galactokinase Gene (GK2) On Chromosome 15 by Complementation in Yeast", pages 10887-10891. *
SCIENCE, Volume 222, issued 18 November 1983, R.A. YOUNG et al., "Yeast RNA Polymerase II Genes: Isolation with Antibody Probes", pages 778-782. *
SCIENCE, Volume 239, issued 11 March 1988, C.C. LEE et al., "Generation of cDNA Probes Directed by Amino Acid Sequence: Cloning of Urate Oxidase", pages 1288-1291. *

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