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WO1993007270A1 - Production d'un facteur neurotrophique dote d'une activite de promotion de la croissance (gpa) - Google Patents

Production d'un facteur neurotrophique dote d'une activite de promotion de la croissance (gpa) Download PDF

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Publication number
WO1993007270A1
WO1993007270A1 PCT/US1992/008258 US9208258W WO9307270A1 WO 1993007270 A1 WO1993007270 A1 WO 1993007270A1 US 9208258 W US9208258 W US 9208258W WO 9307270 A1 WO9307270 A1 WO 9307270A1
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Prior art keywords
gpa
dna
nucleic acid
leu
cells
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PCT/US1992/008258
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English (en)
Inventor
George Cachianes
Felix P. Eckenstein
David Wai-Hung Leung
Rae Nishi
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Genentech, Inc.
State Of Oregon Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University
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Priority to EP92921199A priority Critical patent/EP0607247A1/fr
Publication of WO1993007270A1 publication Critical patent/WO1993007270A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This application relates to the production of polypeptides involved in neuronal survival and/or growth, in particular the production of purified forms thereof by means of recombinant DNA technology.
  • a number of protein neurotrophic factors have been identified which influence growth and development of the vertebrate nervous system. It is believed that these factors may play an important role in sustaining the survival of neurons in both the mature and immature nervous system.
  • NGF nerve growth factor
  • NGF neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • CNTF ciliary neurotrophic factor
  • Ciliary neurotrophic factors are proteins capable of supporting the survival and growth of chick embryo ciliary ganglion neurons jn vitro. CNTFs have been purified from various tissue sources, including chicken eye and rat sciatic nerve. Manthorpe, et al. J. Neurosci. Res. 38:233-239 (1982); Manthorpe, et al., Brain Res. 3_57:282-286 (1986). The nucleotide sequences encoding rabbit, rat, and human CNTF, as well as the expression of rabbit and rat CNTF in recombinant host cells, has been reported.
  • O ⁇ e of the co-inventors of the present invention has previously reported that an extract of chick eyes contains two independently acting neurotrophic factors, referred to as growth- promoting activity (GPA) and a chol ⁇ ne acetyltransferase-stimulating activity (CSA).
  • GPA growth- promoting activity
  • CSA chol ⁇ ne acetyltransferase-stimulating activity
  • GPA was found to support the survival of ciliary ganglion, dorsal root ganglion, and sympathetic neurons in vitro, jd.
  • Comparison of a partial amino acid sequence obtained for such purified GPA with the known amino acid sequences of rabbit and rat CNTF showed approximately 57% identity between GPA and those CNTFs within the portion of the GPA amino acid sequence that was analyzed. _d.
  • the difficulty of obtaining substantial amounts of GPA from chicken sciatic nerves has interfered with efforts to further characterize GPA, and of course has precluded any possible clinical use of GPA.
  • nucleic acid encoding GPA protein and to use this nucleic acid to produce GPA protein in recombinant host cells for diagnostic use or for therapeutic use with neurological disorders.
  • the invention enables the production of GPA by means of recombinant DNA technology, thereby making available for the first time sufficient quantities of substantially pure GPA protein for diagnostic and therapeutic uses with a variety of neurological disorders.
  • the invention provides GPA that is free of contaminating polypeptides of the animal species in which GPA naturally occurs, and compositions comprising GPA that are free of such contaminating polypeptides.
  • Modified and variant forms of GPA are produced in vitro by means of chemical or enzymatic treatment or in vivo bv means of recombinant DNA technology.
  • Such polypeptides differ from native GPA, for example, by virtue of one or more amino acid substitutions, deletions or insertions, or in the extent or pattern of glycos ⁇ lation, but substantially retain a biological activity of native GPA.
  • Antibodies to GPA are produced by immunizing an animal with GPA or a fragment thereof, optionally in conjunction with an immunogenic polypeptide, and thereafter recovering antibodies from the serum of the immunized animals. Alternatively, monoclonal antibodies are prepared from cells of the immunized animal in conventional fashion. Antibodies obtained by routine screening will bind to GPA but will not substantially bind to (i.e., cross react with) NGF, BDNF, NT-3, CNTF, or other neurotrophic factors. Immobilized anti-GPA antibodies are particularly useful in the detection of GPA in clinical samples for diagnostic purposes, and in the purification of GPA.
  • GPA GPA, its derivatives, or its antibodies are formulated with physiologically acceptable carriers, especially for therapeutic use. Such carriers are used, for example, to provide sustained-release formulations of GPA.
  • the invention provides a method for determining the presence of a nucleic acid molecule encoding GPA in test samples prepared from cells, tissues, or biological fluids, comprising contacting the test sample with isolated DNA comprising the coding sequence for GPA and determining whether the isolated DNA hybridizes to a nucleic acid molecule in the test sample.
  • DNA comprising the coding sequence for GPA is also used in hybridization assays to identify and to isolate nucleic acids sharing substantial sequence identity to the coding sequence for GPA.
  • Figure 1 shows the nucleotide sequence of the cDNA insert in the ⁇ CE15 #19 clone
  • Figure 2 shows the homologies among the amino acid sequences of rabbit, rat, and human CNTF [SEQ ID NOS:3,4,5, respectively] and GPA.
  • Figure 3 shows the effect of conditioned media and cell extracts prepared from cultures of recombinant host cells expressing either CNTF or GPA on ciliary ganglion neuron growth.
  • Figure 4 shows the nucleotide sequence of pHEB030 [SEQ ID NO:6). The location of certain restriction endonuclease cleavage sites and control elements is indicated in brackets. In the sequence, "N” is used to designate the nucleotides that comprise the arbitrary 350 base pair cDNA insert in pHEBO30.
  • GPA or “GPA protein” refers to a polypeptide or protein encoded by the GPA nucleotide sequence set forth in Figure 1 ; a polypeptide that is the translated amino acid sequence set forth in Figure 1 ; fragments thereof having greater than about 5 amino acid residues and comprising an immune epitope or other biologically active site of GPA; amino acid sequence variants of the amino acid sequence set forth in Figure 1 wherein one or more amino acid residues are added at the N- or C-terminus of, or within, said Figure 1 sequence or its fragments as defined above; amino acid sequence variants of said Figure 1 sequence or its fragments as defined above wherein one or more amino acid residues of said Figure 1 sequence or fragment thereof are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above proteins, polypeptides, or fragments thereof, wherein an amino acid residue has been covalently modified so that the resulting product is a non-naturally occurring amino acid.
  • GPA amino acid sequence variants may be made synthetically, for example, by site-directed or PCR mutagenesis, or may exist naturally, as in the case of allelic forms and other naturally occurring variants of the translated amino acid sequence set forth in Figure 1 that may occur in human and other animal species.
  • such fragments, variants, and derivatives exclude any polypeptide heretofore identified, including any known neurotrophic factor, such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), as well as statutorily obvious variants thereof.
  • NGF nerve growth factor
  • BDNF brain derived neurotrophic factor
  • NT-3 neurotrophin-3
  • CNTF ciliary neurotrophic factor
  • a GPA amino acid sequence variant is included within the scope of the invention provided that it is functionally active.
  • functionally active and “functional activity” in reference to GPA means that the GPA is able to promote the growth, survival, and/or differentiation of neurons, especially ciliary ganglion neurons, dorsal root ganglion neurons, or sympathetic neurons, in vivo or in vitro, and/or that the GPA is immunologically cross-reactive with an antibody directed against an epitope of naturally occurring GPA.
  • GPA amino acid sequence variants generally will share at least about 75% (preferably greater than 80% and more preferably greater than 90%) sequence identity with the translated amino acid sequence set forth in Figure 1 , after aligning the sequences to provide for maximum homology, as determined, for example, by the Fitch, et al., Proc. Nat.
  • Amino acid sequence variants of GPA are prepared by introducing appropriate nucleotide changes into GPA DNA and thereafter expressing the resulting modified DNA in a host cell, or by jn vitro synthesis.
  • Such variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the GPA amino acid sequence set forth in Figure 1 . Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of GPA, provided that such variant possesses the desired characteristics described herein.
  • Changes that are made in the amino acid sequence set forth in Figure 1 to arrive at an amino acid sequence variant of GPA also may result in further modifications of GPA upon its expression in host cells, for example, by virtue of such changes introducing or moving sites of glycosyiation, or introducing membrane anchor sequences as described, for example, in PCT Pat. Pub. No. WO 89/01041 (published February 9, 1989).
  • amino acid sequence variants of GPA There are two principal variables in the construction of amino acid sequence variants of GPA: the location of the mutation site and the nature of the mutation. These are variants from the amino acid sequence set forth in Figure 1 , and may represent naturally occurring allelic forms of GPA, or predetermined mutant forms of GPA made by mutating GPA DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the GPA characteristic to be modified. For example, due to the degeneracy of nucleotide coding sequences, mutations can be made in the GPA nucleotide sequence set forth in Figure 1 without affecting the amino acid sequence of the GPA encoded thereby.
  • GPA GPA that has an amino acid sequence different from that set forth in Figure 1 , but which is functionally active.
  • Such functionally active amino acid sequence variants of GPA are selected, for example, by substituting one or more amino acid residues in the amino acid sequence set forth in Figure 1 with other amino acid residues of a similar or different polarity or charge.
  • alanine scanning mutagenesis a an amino acid residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and, by means of recombinant DNA technology, replaced by a neutral or negatively charged amino acid (most preferably alanine or polyaianine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • a neutral or negatively charged amino acid most preferably alanine or polyaianine
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues, and typically are contiguous. Deletions from regions of substantial homology with CNTF, for example, are more likely to affect the functional activity of GPA. Generally, the number of consecutive deletions will be selected so as to preserve the tertiary structure of GPA in the affected domain, e.g., beta-pleated sheet or alpha helix.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one amino acid residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • I ⁇ trasequence insertions i.e., insertions made within the amino acid sequence set forth in Figure 1
  • terminal insertions examples include GPA with an N-terminal methionyl residue (such as may result from the direct expression of GPA in recombinant cell culture), and GPA with a heteroiogous N-termi ⁇ al signal sequence to improve the secretion of GPA from recombinant host cells.
  • signal sequences generally will be homologous to the host cell used for expression of GPA, and include STII or ipp for E. coli. alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.
  • insertions include the fusion to the N- or C- terminus of GPA of immunogenic polypeptides, e.g., bacterial polypeptides such as beta- lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin constant regions, albumin, or ferritin, as described in PCT Pat. Pub. No. WO 89/02922 (published April 6, 1989).
  • the third group of variants are those in which at least one amino acid residue in the amino acid sequence set forth in Figure 1 , and preferably only one, has been removed and a different residue inserted in its place.
  • Insertional, deletional, and substitutional changes in the amino acid sequence set forth in Figure 1 may be made to improve the stability of GPA.
  • trypsin or other protease cleavage sites are identified by inspection of the encoded amino acid sequence for an arginyl or lysinyl residue. These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue.
  • any cysteine residues not involved in maintaining the proper conformation of GPA for functional activity may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • Covalent modifications of GPA molecules also are included within the scope of this invention.
  • covalent modifications are introduced into GPA by reacting targeted amino acid residues of the GPA with an organic derivatizing agent that is capable of reacting with selected amino acid side chains or the N- or C-terminal residues.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo-£-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-riitrobenzo-2-oxa-1 ,3- d ⁇ azole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizi ⁇ g ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; p ⁇ ridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenyigl ⁇ oxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK, of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Tyrosyl residues are iodinated using 1 6 l or 131 l to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
  • aspartyl and giutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Derivatization with bifunctional agents is useful for crosslinking GPA to a water- insoluble support matrix or surface for use in the method for purifying anti-GPA antibodies, or for therapeutic use.
  • Commonly used crosslinking agents include, e.g., 1 ,1-bis(diazoacetyD-
  • 2-phenyIethane, glutaraldehyde, N-hydroxysuccinimide esters for example, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidyipropionate), and bifunctional maleimides such as bis-N-maleimido-1 ,8- octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691 ,01 6; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding giutamyl and aspartyl residues, respectively.
  • these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • GPA also is covalently linked to nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4, 179,337; 4,301 , 144; 4,496,689; 4,640,835; 4,670,417; or 4,791 ,192.
  • Cell "host cell,” “cell line,” and “cell culture” are used interchangeably and all such terms should be understood to include progeny.
  • transformationants and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of times the cultures have been passaged. It should also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations.
  • “Plasmids” are DNA molecules that are capable of replicating within a host cell, either extrachromosomali ⁇ or as part of the host cell chromosome(s), and are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids as disclosed herein and/or in accordance with published procedures. In certain instances, as will be apparent to the ordinarily skilled artisan, other plasmids known in the art may be used interchangeably with plasmids described herein.
  • Control sequences refers to DNA sequences necessary for the expression of an operably linked nucleotide coding sequence in a particular host cell.
  • the control sequences that are suitable for expression in prokar ⁇ otes include origins of replication, promoters, ribosome binding sites, and transcription termination sites.
  • the control sequences that are suitable for expression in eukaryotes include origins of replication, promoters, ribosome binding sites, polyadenylation signals, and enhancers.
  • An "exogenous" element is one that is foreign to the host cell, or homologous to the host cell but in a position within the host cell in which the element is ordinarily not found.
  • “Digestion” of DNA refers to the catalytic cleavage of DNA with an enzyme that acts only at certain Iocations in the DNA. Such enzymes are called restriction enzymes or restriction endonucleases, and the sites within DNA where such enzymes cleave are called restriction sites.
  • restriction enzymes or restriction endonucleases
  • Restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters representing the microorganism from which each restriction enzyme originally was obtained and then a number designating the particular enzyme. In general, about 1 ⁇ g of DNA is digested with about 1 -2 units of enzyme in about 20 ⁇ of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer, and/or are well known in the art.
  • Recovery or “isolation” of a given fragment of DNA from a restriction digest typically is accomplished by separating the digestion products, which are referred to as “restriction fragments," on a polyacrylamide or agarose gel by electrophoresis, identifying the fragment of interest on the basis of its mobility relative to that of marker DNA fragments of known molecular weight, excising the portion of the gel that contains the desired fragment, and separating the DNA from the gel, for example by electroelution.
  • “Ligation” refers to the process of forming phosphodiester bonds between two double- stranded DNA fragments. Unless otherwise specified, ligation is accomplished using . known buffers and conditions with 10 units of T4 DNA l ⁇ gase per 0.5 / g of approximately equimolar amounts of the DNA fragments to be ligated.
  • Oligonucleotides are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (involving, for example, triester, phosphoramidite, or phosphonate chemistry), such as described by Engels, et al., Agnew.
  • PCR Polymerase chain reaction
  • PCR PCR
  • the PCR method involves repeated cycles of primer extension synthesis, using two oligonucleotide primers capable of hybridizing preferentially to a template nucleic acid.
  • the primers used in the PCR method will be complementary to nucleotide sequences within the template at both ends of or flanking the nucleotide sequence to be amplified, although primers complementary to the nucleotide sequence to be amplified also may be used.
  • PCR cloning refers to the use of the PCR method to amplify a specific desired nucleotide sequence that is present amongst the nucleic acids from a suitable cell or tissue source, including total genomic DNA and cDNA transcribed from total cellular RNA.
  • GPA nucleic acid is RNA or DNA that encodes GPA.
  • GPA DNA is DNA that encodes GPA.
  • GPA DNA is obtained from cDNA or genomic DNA libraries, or by jn vitro synthesis. Identification of GPA DNA within a cDNA or a genomic DNA library, or in some other mixture of various DNAs, is conveniently accomplished by the use of an oligonucleotide hybridization probe that is labeled with a detectable moiety, such as a radioisotope. Keller, et al.. DNA Probes, PP.149-213 (Stockton Press, 1989).
  • the nucleotide sequence of the hybridization probe preferably is selected so that the hybridization probe is capable of hybridizing preferentially to DNA encoding the GPA amino acid sequence set forth in Figure 1 , or a variant or derivative thereof as described herein, under the hybridization conditions chosen.
  • Another method for obtaining GPA nucleic acid is to chemically synthesize it using one of the methods described, for example, by Engels, et al., Agnew. Chem. Int. Ed. Engl. 28:716-734 (1989).
  • nucleotide coding sequence for GPA is not obtained in a single cDNA, genomic DNA, or other DNA, as determined, for example, by DNA sequencing or restriction endonuclease analysis, then appropriate DNA fragments (e.g., restriction fragments) may be recovered from several DNAs and covalently joined to one another to construct the entire coding sequence.
  • the preferred means of covalently joining DNA fragments is by ligation using a DNA ligase enzyme, such as T4 DNA ligase.
  • isolated GPA nucleic acid is GPA nucleic acid that is identified and separated from (or otherwise free from), contaminant nucleic acid encoding other polypeptides.
  • the isolated GPA nucleic acid may be labeled for diagnostic and probe purposes, using a label as described and defined further below in the discussion of diagnostic assays and nucleic acid hybridization methods.
  • isolated GPA DNA is used as a hybridization probe to detect, diagnose, or monitor disorders or diseases that involve changes in GPA expression, such as may result from sensory neuron damage.
  • total RNA in a tissue sample from a patient can be assayed for the presence of GPA messenger RNA, wherein the decrease in the amount of GPA messenger RNA is indicative of neuronal degeneration.
  • isolated GPA nucleic acid also is used to produce GPA by recombinant DNA and recombinant cell culture methods.
  • host cells are transformed or transfected with recombinant DNA molecules comprising an isolated GPA DNA, to obtain expression of the GPA DNA and thus the production of GPA in large quantities.
  • DNA encoding amino acid sequence variants of GPA is prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants of GPA) or preparation by site-directed (or oligonucieotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding a variant or a non-variant form of GPA.
  • Site-directed mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of GPA DNA. This technique is well known in the art, Zoller, et al., Meth. Enz. 1Q0.4668-500 (1983); Zoller, et al., Meth. Enz. 15.4:329-350 (1987); Carter,
  • the GPA DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such
  • GPA DNA After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of GPA
  • the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.
  • Oligonucleotides for use as hybridization probes or primers may be prepared by any suitable method, such as by purification of a naturally occurring DNA or by jn vitro synthesis.
  • oligonucleotides are readily synthesized using various techniques in organic chemistry, such as described by Narang, et al., Meth. E ⁇ zymol. 6_8:90-98 (1979); Brown, et al., Meth. Enzymol. g£J:109-151 (1979); Caruther, et al., Meth. Enzymol. 1j>4:287-313 (1985).
  • the general approach to selecting a suitable hybridization probe or primer is well known.
  • the hybridization probe or primer will contain 10-25 or more nucleotides, and will include at least 5 nucleotides on either side of the sequence encoding the desired mutation so as to ensure that the oligonucleotide will hybridize preferentially to the single-stranded DNA template molecule.
  • Multiple mutations are introduced into GPA DNA to produce amino acid sequence variants of GPA comprising several or a combination of insertions, deletions, or substitutions of amino acid residues as compared to the amino acid sequence set forth in Figure 1. If the sites to be mutated are located close together, the mutations may be introduced simultaneously using a single oligonucleotide that encodes all of the desired mutations. If, however, the sites to be mutated are located some distance from each other (separated by more than about ten nucleotides), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
  • a separate oligonucleotide is generated for each desired mutation.
  • the oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
  • the alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
  • the first round is as described for introducing a single mutation: a single strand of a previously prepared GPA DNA is used as a template, an oligonucleotide encoding the first desired mutation is annealed to this template, and a heteroduplex DNA molecule is then generated.
  • the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.
  • this template already contains one or more mutations.
  • the oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
  • This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
  • PCR mutagenesis is also suitable for making amino acid sequence variants of GPA. Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990); Vallette, et al., Nuc. Acids Res. 17:723-733 (1989). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
  • one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a nucleotide sequence within the opposite strand of the plasmid DNA, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced.
  • PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.
  • the ratio of template to product amplified DNA is extremely low, the majority of product DNA fragments incorporate the desired mutation(s).
  • This product DNA is used to replace the corresponding region in the plasmid that served as PCR template using standard recombinant DNA methods. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer, or performing a second PCR with different mutant primers and ligat ⁇ ng the two resulting PCR fragments simultaneously to the plasmid fragment in a three (or more)-part ligation.
  • the starting material is the plasmid (or other vector) comprising the GPA DNA to be mutated.
  • the codon(s) in the GPA is the plasmid (or other vector) comprising the GPA DNA to be mutated.
  • DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate Iocations in the GPA DNA. The plasmid DNA is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated GPA DNA sequence.
  • GPA DNA whether cDNA or genomic DNA or a product of in vitro synthesis, is ligated into a replicable vector for further cloning or for expression.
  • Vectors are plasmids and other DNAs that are capable of replicating autonomously within a host cell, and as such, are useful for performing two functions in conjunction with compatible host cells (a vector-host system) .
  • One function is to facilitate the cloning of the nucleic acid that encodes the GPA, i.e., to produce usable quantities of the nucleic acid.
  • the other function is to direct the expression of GPA.
  • One or both of these functions are performed by the vector-host system.
  • the vectors will contain different components depending upon the function they are to perform as well as the host cell with which they are to be used for cloning or expression.
  • an expression vector will contain nucleic acid that encodes GPA as described above.
  • the GPAs of this invention are expressed directly in recombinant cell culture, or as a fusion with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the junction between the heterologous polypeptide and the GPA.
  • mammalian cells are transfected with an expression vector comprising GPA DNA and the GPA encoded thereby is recovered from the culture medium in which the recombinant host cells are grown.
  • the expression vectors and methods disclosed herein are suitable for use over a wide range of prokaryotic and eukaryotic organisms.
  • Prokaryotes may be used for the initial cloning of DNAs and the construction of the vectors useful in the invention. However, prokaryotes may also be used for expression of DNA encoding GPA. Polypeptides that are produced in prokaryotic host cells typically will be non-glycosylated.
  • Plasmid or viral vectors containing replication origins and other control sequences that are derived from species compatible with the host cell are used in connection with prokaryotic host cells, for cloning or expression of an isolated DNA.
  • E. £ ⁇ Ji typically is transformed using pBR322, a plasmid derived from an E. £oji species.
  • PBR322 contains genes for ampicillin and tetracycline resistance so that cells transformed by the plasmid can easily be identified or selected.
  • the pBR322 plasmid, or other plasmid or viral vector must also contain, or be modified to contain, a promoter that functions in the host cell to provide messenger RNA (mRNA) transcripts of a DNA inserted downstream of the promoter.
  • mRNA messenger RNA
  • eukaryotic microbes such as yeast
  • yeast may also be used as hosts for the cloning or expression of DNAs useful in the invention.
  • Saccharomvces cerevisiae. or common baker's yeast is the most commonly used eukaryotic microorganism.
  • Plasmids useful for cloning or expression in yeast cells of a desired DNA are well known, as are various promoters that function in yeast cells to produce mRNA transcripts.
  • cells derived from multiceliular organisms also may be used as hosts for the cloning or expression of DNAs useful in the invention.
  • Mammalian cells are most commonly used, and the procedures for maintaining or propagating such cells jn vitro, which procedures are commonly referred to as tissue culture, are well known. Kruse & Patterson, eds., Tissue Culture (Academic Press, 1977).
  • useful mammalian cells aretiuman cell lines such as 293, HeLa, and WI-38, monkey cell lines such as COS-7 and VERO, and hamster cell lines such as BHK-21 and CHO, all of which are publicly available from the American Type Culture Collection (ATCC), Rockville, Maryland 20852 USA.
  • Expression vectors unlike cloning vectors, should contain a promoter that is recognized by the host organism and is operably linked to the GPA nucleic acid. Promoters are untranslated sequences that are located upstream from the start codon of a gene and that control transcription of the gene (that is, the synthesis of mRNA). Promoters typically fall into two classes, inducibie and constitutive. Inducible promoters are promoters that initiate high level transcription of the DNA under their control in response to some change in culture conditions, for example, the presence or absence of a nutrient or a change in temperature.
  • promoters A large number of promoters are known, that may be operably linked to GPA DNA to achieve expression of GPA in a host cell. This is not to say that the promoter associated with naturally occurring GPA DNA is not usable. However, heterologous promoters generally will result in greater transcription and higher yields of expressed GPA.
  • Promoters suitable for use with prokaryotic hosts include the Mactamase and lactose promoters, Goeddel, et al., Nature 281 :544-548 (1 979), tryptophan (trp) promoter, Goeddel, et al., Nuc. Acids Res.1:4057-4074 (1980), and hybrid promoters such as the tac promoter, deBoer, et al., Proc. Natl. Acad. Sci. USA 8_Q_:21 -25 (1983).
  • other known bacterial promoters are suitable. Their nucleotide sequences have been published, Siebenlist, et al..
  • Suitable promoters for use with yeast hosts include the promoters for 3- phosphoglycerate kinase, Hitzeman, et al., J. Biol. Chem. 255:12073-12080 (1980); Kingsman, et al., Meth. Enz.
  • glycolytic enzymes such as enofase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarbox ⁇ lase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and giucokinase.
  • enofase glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarbox ⁇ lase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and giucokinase.
  • Expression vectors useful in mammalian cells typically include a promoter derived from a virus.
  • promoters derived from polyoma virus, adenovirus, cytomegalovirus (CMV), and simian virus 40 (SV40) are commonly used.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • Other control sequences that are desirable in an expression vector in addition to a promoter are a ribosome binding site, and in the case of an expression vector used with eukaryotic host cells, an enhancer.
  • Enhancers are cis-ac ⁇ ing elements of DNA, usually about from 10-300 bp, that act on a promoter to increase the level of transcription.
  • Many enhancer sequences are now known from mammalian genes (for example, the genes for globi ⁇ , elastase, albumin, ⁇ -fetoprotein and insulin).
  • the enhancer used will be one from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Kriegler, Meth. Enz. J_85_:512-527 (1990).
  • Expression vectors may also contain sequences necessary for the termination of transcription and for stabilizing the messenger RNA (mRNA). Balbas, et al., Meth. Enz. 185:14-37 (1990); Levinson, Meth. Enz. 185:485-51 1 (1990).
  • transcription termination sequences may be obtained from the untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain polyaden ⁇ lation sites as well as transcription termination sites. Birnsteil, et al.. Cell 41:349-359 (1985).
  • control sequences are DNA sequences necessary for the expression of an operably liked coding sequence in a particular host cell.
  • “Expression” refers to transcription and/or translation.
  • “Operably linked” refers to the covalent joining of two or more DNA sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.
  • Expression and cloning vectors also will contain a sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosome(s), and includes origins of replication or autonomously replicating sequences.
  • Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (for example, from SV40, polyoma, or adenovirus) are useful for cloning vectors in mammalian cells.
  • Most expression vectors are "shuttle" vectors, i.e.
  • a vector may be cloned in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome.
  • the expression vector may also include an ampiifiabie gene, such as that comprising the coding sequence for dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • DHFR protein encoded by the expression vector also may be used as a selectable marker of successful transfection. For example, if the host cell prior to transformation is lacking in DHFR activity, successful transformation by an expression vector comprising DNA sequences encoding GPA and DHFR protein can be determined by cell growth in medium containing methotrexate. Also, mammalian cells transformed by an expression vector comprising DNA sequences encoding GPA, DHFR protein, and aminoglycoside 3' phosphotransferase (APH) can be determined by cell growth in medium containing an aminoglycoside antibiotic such as kanamycin or neomyci ⁇ .
  • APH aminoglycoside 3' phosphotransferase
  • genes encoding APH protein may be used as dominant selectable markers in a wide range of eukaryotic host cells, by which cells transfected by the vector can easily be identified or selected.
  • Jiminez, et al.. Nature, 287:869-871 (1980); Colbere-Garapin, et al., J. Mol. Biol. 150:1-14 (1981 ); Okayama & Berg, Mol. Cell. Biol., 2 280-289 (1983).
  • a suitable selection marker for use in yeast is the trpl gene present in the yeast plasmid YRp7. Stinchcomb, et al., Nature 2 ⁇ 2:39-43 (1979); Kingsman, et al., Gene 2:141-152 (1979); Tschemper, et al.. Gene 1jQ:157-166 (1980).
  • the trp 1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (available from the American Type Culture Collection, Rockville, Maryland 20852 USA).
  • Leu2-deficient yeast strains (ATCC Nos. 20622 or 38626) are complemented by known plasmids bearing the Leu2 gene.
  • transient expression involves the use of an expression vector that is able to efficiently replicate in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector.
  • Transient expression systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties.
  • transient expression systems are particularly useful in the invention for expressing DNAs encoding amino acid sequence variants of GPA, to identify those variants which are functionally active. Since it is often difficult to predict in advance the characteristics of an amino acid sequence variant of GPA, it will be appreciated that some screening of such variants will be needed to identify those that are functionally active. Such screening may be performed vitro, using routine assays for neuronal survival, Eckenstein, et al..
  • GPA expression in mammalian host cells is accomplished by the use of an expression vector comprising the oriP origin of replication from Epstein-Barr virus (EBV) and a host cell that is transformed with and constitutively expresses the EBNA-1 gene of EBV.
  • EBV Epstein-Barr virus
  • Plasmids containing the oriP sequence of EBV are able to replicate in EBV-transformed host cells that express the EBV nuclear antigen (EBNA-1 ).
  • EBNA-1 EBV nuclear antigen
  • the expression of GPA that is described in the Examples below involves the use of a plasmid expression vector (pHEBO30) containing the oriP region from EBV, and a cell line (CEN4) that constitutively expresses EBNA-1 .
  • pHEBO30 comprises the strong CMV promoter, a multiple cloning region for insertion of foreign (exogenous) genes downstream of the CMV promoter, the oriP region of
  • EBV for plasmid replication in host cells expressing EBNA-1 (for example, CEN4), a hygromycin resistance gene for selection in eukaryotes, the origin of replication from pBR322 for replication in prokaryotes, and an ampicillin resistance gene for selection in prokaryotes.
  • EBNA-1 for example, CEN4
  • a hygromycin resistance gene for selection in eukaryotes
  • the origin of replication from pBR322 for replication in prokaryotes
  • an ampicillin resistance gene for selection in prokaryotes.
  • pHEBO30 (including recombinant derivatives thereof) is stably maintained as an episome in the nuclei of host cells expressing EBNA-1 .
  • the efficiency of stable transfection of such host cells with pHEBO30 is several fold greater than obtained with a plasmid containing both the oriP region and the EBNA-1 gene from EBV.
  • the efficiency of stable transfection of CEN4 cells with pHEBO30 is from about 5% to 25% or more.
  • pHEBO30 and CEN4 include: (1 ) The level of transient expression in CEN4 cells of foreign genes cloned in pHEBO30 (for example, the genes encoding human tissue-type plasminogen activator (tPA) and human soluble alkaline phosphatase) is several fold higher than obtained with expression vectors lacking the oriP from EBV; (2) Stable expression in CEN4 cells of foreign genes cloned in pHEBO30 can be maintained for four months or more with appropriate selection (for example, hygromycin selection); and (3) pHEB030 and recombinant derivatives thereof are readily recovered from transfected cells, for analysis or modification.
  • tPA tissue-type plasminogen activator
  • pHEB030 and recombinant derivatives thereof are readily recovered from transfected cells, for analysis or modification.
  • transformation and transfection refer to the process of introducing a desired nucleic acid, such a plasmid or an expression vector, into a host cell.
  • a desired nucleic acid such as a plasmid or an expression vector
  • transformation and transfection are available, depending on the nature of the host cell.
  • E. c_2lj cells the most common methods involve treating the cells with aqueous solutions of calcium chloride and other salts.
  • mammalian cells the most common methods are transfection mediated by either calcium phosphate or DEAE-dextran, or electroporation.
  • Sambrook, et al., eds., Molecular Cloning, pp. 1 .74-1 .84 and 16.30-16.55 Cold Spring Harbor Laboratory Press, 1989.
  • the desired nucleic acid may integrate into the host cell genome, or may exist as an extrachromosomal element.
  • Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype.
  • the culture conditions such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent those skilled in the art.
  • Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, vertebrate, and mammalian host cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli. Bacillus species such as j
  • eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for GPA-encoding vectors. Saccharomvces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Suitable host cells for the expression of GPA also are derived from ⁇ ulticellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture. It will be appreciated, however, that because of the species-, tissue-, and cell-specificity of glycosylation, Rademacher, et al., Ann. Rev. Biochem. 57:785-838 (1988), the extent or pattern of glycosylation of GPA in a foreign host cell typically will differ from that of GPA obtained from a cell in which it is naturally expressed.
  • invertebrate cells include insect and plant cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera fruoiperda (caterpillar), Aedes aeovpti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombvx mori host cells have been identified. Luckow, et al., Bio Technology 5:47-55 (1988); Miller, et al., in Genetic Engineering, vol. 8, pp.277- 279 (Plenum Publishing, 1986); Maeda, et al.. Nature 315:592-594 (1985).
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts.
  • plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens. which has been previously altered to contain
  • GPA DNA During incubation of the plant cells with A. tumefaciens. the DNA encoding the
  • GPA is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the GPA DNA.
  • regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences, and the ribulose biphosphate carboxylase promoter. Depicker, et al., J.
  • DNA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required.
  • the vectors are analyzed by restriction digestion (to confirm the presence in the vector of predicted restriction endonuclease) and/or by sequencing by the dideoxy chain termination method of Sanger, et al., Proc. Nat. Acad. Sci. USA 72:3918-3921 (1979).
  • the mammalian host cells used to produce the GPA of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal
  • WO 90/03430 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromoiar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the host cells referred to in this disclosure encompass cells in culture jn vitro as well as cells that are within a host animal, for example, as a result of transplantation or implantation.
  • GPA of this invention may be produced by homologous recombination, for example, as described in PCT Pat. Pub. No. WO 91 /06667 (published May
  • this method involves transforming cells containing an endogenous gene encoding GPA with a homologous DNA, which homologous DNA comprises (1 ) an amplifiable gene, such as DHFR, and (2) at least one flanking sequence, having a length of at least about
  • the transformation is carried out under conditions such that the homologous DNA integrates into the cell genome by recombination.
  • Cells having integrated the homologous DNA then are subjected to conditions which select for amplification of the amplifiable gene, whereby the GPA gene amplified concomitantly.
  • Flanking sequences that are in proximity to a gene encoding GPA are readily identified, for example, by the method of genomic walking, using as a starting point the GPA nucleotide sequence set forth in Figure 1. Spoerel, et al., Meth. Enz. 152:598-603 (1987).
  • Gene amplification and/or gene expression may be measured in a sample directly, for example, by conventional Southern blotting to quantitate DNA, or Northern blotting to quantitate mRNA, using an appropriately labeled oligonucleotide hybridization probe, based on the sequences provided herein.
  • Various labels may be employed, most commonly radioisotopes, particularly 32 P.
  • other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a pol ⁇ nucleotide.
  • the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radioisotopes, fluorophores, chromophores, or the like.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of the gene product, GPA.
  • immunohistochemical staining techniques a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like.
  • a particularly sensitive staining technique suitable for use in the present invention is described by Hsu, et al., Am. J. Clin. Path., 75:734-738 (1980).
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or poiyclonal. Conveniently, the antibodies may be prepared against a synthetic peptide based on the DNA sequences provided herein.
  • GPA preferably is recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates.
  • GPA thereafter is purified from contaminant soluble proteins and polypeptides, for example, by ammonium sulfate or ethanol precipitation, gel filtration (molecular exclusion chromatography), ion- exchange chromatography, immunoaffinity chromatography, reverse phase HPLC, and/or gel electrophoresis.
  • Amino acid sequence variants and derivatives of GPA are recovered in the same fashion, taking account of any distinguishing features or physical properties of the particular GPA.
  • a significant degree of purification may be obtained by using an immunoaffinity column containing antibody to the antigen.
  • purification methods suitable for naturally occurring GPA may require modification to account for changes in the character of GPA or its variants or derivatives produced in recombinant host cells.
  • the purity of GPA produced according to the present invention is determined according to methods well known in the art, such as by analytical sodium dodecyl sulfate (SDS) gel electrophoresis, immunoassa ⁇ , or amino acid composition or sequence analysis electrophoresis.
  • the GPA is purified to such an extent that it is substantially free of other proteins.
  • the purified GPA will be greater than 99% GPA and, accordingly, non-GPA proteins will comprise less than 1 % of the total protein in the purified GPA composition.
  • GPA may be used as an immunogen to generate a ⁇ ti-GPA antibodies.
  • Such antibodies which specifically bind to GPA, are useful as standards in assays for GPA, such as by labeling purified GPA for use as a standard in a radioimmunoassay, enzyme-linked immunoassay, or competitive-type receptor binding assays radioreceptor assay, as well as in affinity purification techniques.
  • GPA-carrier protein conjugates combining 1 mg or 1 ⁇ g of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1 /5th to 1 /10th the original amount of conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • 7 to 14 days later animals are bled and the serum is assayed for anti-GPA antibody titer. Animals are boosted until the antibody titer plateaus.
  • the animal is boosted by injection with a conjugate of the same GPA with a different carrier protein and/or through a different cross-linking agent.
  • Conjugates of GPA and a suitable carrier protein also can be made in recombinant cell -culture as fusion proteins.
  • aggregating agents such as alum are used to enhance the immune response.
  • Monoclonal antibodies directed toward GPA are produced using any method which provides for the production of antibody molecules by continuous cell lines in culture. Examples of such methods include the original hybridoma method of Kohler, et al.. Nature 256:495-497 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133:3001 (1984); Brodeur, et al.. Monoclonal Antibodv Production Technioues and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987).
  • anti-GPA antibodies typically will be labeled with a detectable moiety.
  • the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a rad ⁇ oisotope, such as 3 H, 14 C, 32 P, 3B S, or 125 l, a fluorescent or chemiluminescent compound, such as fluorescein isothioc ⁇ anate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., t26 l, 32 P, W C, or 3 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by David, et al., Biochemistry 12:1014-1021 (1974); Pain, et al., J. Immunol. Meth. 4Q:219-231 (1981 ); and Bayer, et al., Meth. Enz. 1_8_4:138-163 (1990).
  • the anti-GPA antibodies may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Technioues. pp.147-158 (CRC Press, Inc., 1987).
  • GPA labeled standard
  • GPA analyte
  • the amount of GPA in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunogiobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the anti-GPA antibodies of the invention also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety is administered to a host, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed.
  • This imaging technique is useful in the staging and treatment of various neurological disorders.
  • the antibody may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • GPA is believed to be useful in promoting the development, maintenance, or regeneration of neurons jn vivo, including ciliary, sensory, and sympathetic neurons. Accordingly, GPA may be utilized in methods for the diagnosis and/or treatment of a variety of neurologic diseases and disorders.
  • purified GPA can be administered to patients in whom the nervous system has been damaged by trauma, surgery, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, or toxic agents, to promote the survival or growth of neurons.
  • GPA can be used to promote the survival or growth of motorneurons that are damaged by trauma or surgery.
  • GPA can be used to treat motorneuron disorders, such as amyotrophic lateral sclerosis (Lou Gehrig's disease), Bell's palsy, and various conditions involving spinal muscular atrophy, or paralysis.
  • GPA can be used to treat human neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, and Down's Syndrome.
  • antibodies directed toward GPA can be administered to patients suffering from neurologic diseases and disorders characterized by excessive production of GPA.
  • Anti-GPA antibodies can be used in the prevention of aberrant regeneration of sensory neurons such as may occur post-operatively, or in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.
  • Therapeutic formulations of GPA and anti-GPA antibodies for treating neurologic diseases and disorders are prepared by mixing GPA or anti-GPA antibody, having the desired degree of purity, with optional physiologically acceptable carriers, excipients, or stabilizers which are well known.
  • Acceptable carriers, excipients or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylp ⁇ rrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such
  • adsorb GPA onto a membrane such as a silastic membrane, which can be implanted in proximity to damaged neural tissue, or to complex GPA with liposomes.
  • a membrane such as a silastic membrane
  • GPA optionally is combined with or administered in concert with other neurotrophic factors to achieve a desired therapeutic effect.
  • GPA may be used together with NGF or BDNF or another neurotrophic factor to achieve a synergistic stimulatory effect on the growth of sensory neurons, wherein the term "synergistic" means that the effect of the combination of GPA with a second neurotrophic factor is greater than that achieved with either substance used individually.
  • GPA and anti-GPA antibodies to be used for in vivo administration must be sterile. This is readily accomplished by filtration of a solution of GPA or anti-GPA antibody through sterile filtration membranes. Thereafter, the filtered solution may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The filtered solution also may be lyophiiized to produce sterile GPA or anti-GPA antibody in a powder form.
  • Methods for administering GPA and anti-GPA antibodies _n vivo include injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, and by means of sustained-release formulations.
  • Sustained-release formulations generally consist of GPA or anti-GPA antibodies and a matrix from which the GPA or anti-GPA antibodies are released over some period of time.
  • Suitable matrices include semipermeable polymer matrices in the form of shaped articles, for example, membranes, fibers, or microcapsules.
  • Sustained release matrices may comprise polyesters, hydrogeis, polylactides, U.S. Pat. No.
  • the sustained release formulation comprises GPA or anti-GPA antibodies entrapped within or complexed with liposomes.
  • the sustained release formulation comprises cells actively producing GPA or anti-GPA antibodies. Such cells may be directly introduced into the tissue of a patient, or may be encapsulated within porous membranes which are then implanted in a patient, in either case providing for the delivery of GPA or anti-GPA antibody into areas within the body of the patient in need of increased or decreased concentrations of GPA.
  • An effective amount of GPA or anti-GPA antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient.
  • a typical daily dosage might range from about 1 g/kg to up to 100 mg/kg or more, depending on the factors mentioned above.
  • a pharmaceutical composition effective in promoting the survival or growth of neurons will provide a local GPA concentration in vivo of between about 0.1 and 10 ng/ml.
  • the present invention enables for the first time the production of GPA by recombinant DNA methods, thus providing a reliable source of sufficient quantities of GPA for use in various diagnostic and therapeutic applications.
  • purified recombinant GPA will be especially useful in a variety of circumstances where it is necessary or desirable to assure neuronal growth and survival, but where other neurotrophic factors either cannot be used or are ineffective.
  • the following examples are offered by way of illustration only and are not intended to limit the invention in any manner.
  • pRK.CXRHN contains an Sfii restriction endonuclease site downstream of its multiple cloning region. An additional Sfil site was introduced into the multiple cloning region by inserting between the Clal and NotI restriction endonuclease sites a synthetic DNA having the sequence:
  • the resulting plasmid then was cleaved with Spel and HinPI restriction endonucleases, and the 1 1 10 base pair restriction fragment was isolated. That restriction fragment, which comprises the entire expression unit from pRK.CXRHN, including the CMV promoter, two Sfil sites for efficient cDNA cloning, and the SV40 pol ⁇ -adenylation sequence for transcription termination, was inserted at the unique Xbal restriction endonuclease site of the plasmid
  • Plasmid p220.2 was obtained from Dr. William Sugden, McCardle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin USA.
  • p220.2 is a derivative of the plasmid p201 , Yates, et al., Nature 313:812-815 (1985), constructed by inserting the multiple cloning region from pUC12 (containing BamHi, Xbal, Sail, and Hindlll restriction endonuclease sites) at the unique Narl site within the herpes simplex virus (HSV) thymidine kinase (tk) terminator sequence of p201.
  • HSV herpes simplex virus
  • tk thymidine kinase terminator sequence of p201.
  • p220.2 should contain a Narl restriction endonuclease site, it was determined both by restriction analysis and DNA sequence analysis that the particular p220.2 cloned used in this instance did not. Therefore, the above 1 1 10 base pair Spel-H ⁇ nPI restriction fragment was joined to Xbal digested p220.2 by way of legitimate ligation of Spel end of the restriction fragment to one Xbal end of the linearized p220.2 plasmid, and forced ligation of the HinPI end of the restriction fragment to the other Xbal end of that plasmid.
  • pHEB02 contains the CMV promoter, the EB A1 gene and oriP region of Epstein-Barr virus, and a hygromycin resistance gene, and the pML sequence for replication and selection in E. coli.
  • the ⁇ -HEBO vector was constructed by inserting BamHI linearized pHEB02 DNA at the BamHI site of the ⁇ -DASHII vector (Stratagene, Inc., La Jolla, California USA).
  • eyes from embryonic day 15-17 chickens contain relatively greater amounts of GPA than other embryonic chicken tissues
  • eyes from embryonic day 15 chicken were used as a source for preparing a cDNA library.
  • RNA messenger RNA
  • Poly(A) + RNA messenger RNA
  • cDNA was selected by affinity chromatography of total RNA on a column of oligo(dT)-cellulose essentially as described, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., pp.7.26-7.29 (Cold Spring Harbor Laboratory Press, 1989), except that poly(A) * RNA that was eluted from the column was passed over the column a second time, for a total of two cycles of selection. Approximately 5% of the total RNA was recovered as poly(A) + RNA.
  • cDNA was prepared by reverse transcription of the poly(A) + RNA using an oiigo-dT primer and a reverse transcriptase that lacks RNase H activity (Bethesda Research Laboratories, Gaithersburg, Maryland). The reaction conditions for the first and second strand synthesis were as specified by the manufacturer.
  • the resulting double-stranded cDNAs were ligated with a molar excess of 5' phosphorylated Sfil restriction endonuclease adaptors having the sequence:
  • the cDNAs with cohesive Sfil ends were fractionated by polyacrylamide gel electrophoresis, and those cDNAs greater than 600 base pairs in length ends were eluted from the gel and ligated to Sfil digested ⁇ -HEBO vector.
  • oligonucleotide probes were designed on the basis of partial amino acid sequences of GPA obtained by microsequencing of three different peptide fragments of purified GPA protein.
  • o-GPA-1 Three oiigonucleotides, referred to as o-GPA-1 , o-GPA-2, and o-GPA-3, having the following sequences:
  • Denhardt's solution (1X Denhardt's solution is 0.02% Ficoll, 0.02% bovine serum albumin, and 0.02% polyvinyl-pyrrolidone), 0.1 % SDS (sodium dodecyl sulfate), and 50 //g/ml salmon sperm DNA.
  • Each of the radiolabeled probes, o-GPA-1 , o-GPA-2, and o-GPA-3 then was separately hybridized to the filters. Hybridization was carried out in the same buffer as used for prehybridization. After hybridization, filters were washed in 1 X SSC at 42° C, and autoradiographed.
  • Nucleotide sequencing of the cDNA insert in the ⁇ CE15 #19 clone was accomplished by the dideoxy chain termination method of Sanger, et al., Proc. Nat. Acad. Sci. USA 72:3918-3921 (1979), but with several modifications. Specifically, various anomalies in the electrophoretic mobility of the DNA sequencing fragments that were initially encountered were overcome by the use of the single-stranded binding protein gp32, Schwarz, et al., Nuc.
  • ⁇ CE15 #19 DNA was digested with the restriction endonuclease NotI, thereby generating a restriction fragment comprising the complete nucleotide coding sequence for
  • GPA flanked by nucleotide sequences of the pHEB02 plasmid. That fragment was circularized by ligation with T4 DNA ligase to generate the plasmid pHEB02-GPA. 6. Cloning and Expression of GPA DNA in E. coli
  • the complete nucleotide coding sequence for GPA was excised from pHEB02-GPA by complete digestion of that plasmid DNA with restriction endonuclease Ncol and partial digestion with restriction endonuclease Espl.
  • the Ncol-Espl DNA fragment comprising the complete coding sequence for GPA was modified by iigating to the Ncol cohesive-end of the fragment a Xbal-Ncol adaptor having the sequence:
  • Xbal-Ncol adaptor includes a ribosome binding site (Shine-Dalgarno sequence), and by Iigating to the Espl cohesive-end of the fragment a Espl-Bglll adaptor having the sequence:
  • plasmid pHGH207-1 (described in U.S. Patent No. 4,551 ,433), which contains the human growth hormone gene under the control of a trp promoter-operator, was digested with restriction endonucleases Xbal and Bglll to release the human growth hormone gene.
  • the Xbal site in pHGH207-1 is immediately downstream of the trp promoter-operator.
  • the above Xbal-Bglll DNA fragment encoding GPA then was joined to the remaining restriction DNA fragment of pHGH207-1 by ligation, thereby effectively substituting the GPA nucleotide coding sequence for the human growth hormone gene.
  • the resulting plasmid thus contains the complete GPA nucleotide coding sequence downstream of and in the correct orientation for expression under the control of the trp promoter of pHGH207-1 and the ribosome binding site provided by the above Xbal-Ncol adaptor.
  • E. coli DH1 OB cells (BRL/Gibco, Gaithersburg, Maryland 20877 USA) were transformed with ptrp-GPA according to the method of Chung, et al., Proc. Nat. Acad. Sci. 86:2172-2175 (1989). Expression of the cloned GPA DNA in the transformed E. coli cells was confirmed by polyacrylamide gel electrophoresis of total cellular protein.
  • Transformed E. coli cells, but not untransformed E. coli cells contained a protein of the size expected for GPA, approximately 21 , 500 Daltons .
  • the plasmid pHEB02 contains the EBNA1 gene and the oriP region of Epstein-Barr virus. A plasmid containing these two viral elements can replicate autosomally in the nucleus of a host cell. Because the function of the EBNA1 gene can be provided by a cell that expresses EBNA1 constitutively, it was possible to reduce the size of the plasmid pHEB02 by removing an 1840 base pair EcoNI-SgrAI restriction fragment from pHEB02 that spans most of the EBNA1 gene.
  • pHEB02 DNA was digested with the restriction endonucleases EcoNI and SgrAI and the cohesive ends of the larger resulting restriction fragment were filled-in with Klenow DNA polymerase I in the presence of all four deoxyribonucleotides. The resulting blunt-end fragment then was circularized by ligation with
  • pHEBO30 350 base pair cDNA insert at the Sfil site of pHEBO20, is referred to as pHEBO30.
  • pCEN1 The starting plasmids for the construction of pCEN1 were pRK.CXRHN, pHEB02, pKAN2, and pSW2.RXR. pRK.CXRHN and pHEB02 are described above.
  • pKAN2 is described in Yates, et al., Proc. Nat. Acad. Sci 81 :3806-3810 (1984), and was obtained from Dr.
  • pSW2.RXR was derived from pRK.CXRHN by deleting three small regions within the pUCI 18-derived sequence of pRK.CXRHN that are not essential for replication and selection of the plasmid in E. £2il - n d by introducing an EcoRI site upstream of the M13 intergenic region of pRK.CXRHN, using PCR mutagenesis. Higuchi, in PCR Protocols, pp.177-
  • pRK.CXRHN DNA was digested with the restriction endonuclease Xhol, and the resulting Xhol cohesive ends were filled-in with Klenow DNA polymerase in the presence of all four deoxyribonucleotides.
  • the blunt end DNA then was digested with the restriction endonuclease Spel, and a 900 base pair Spel-Xhol' restriction fragment, comprising the CMV promoter of pRK.CXRHN, was isolated.
  • pHEB02 DNA was digested with the restriction endonuclease Hinfl, and the resulting
  • Hinfl cohesive ends were filled-in with Klenow DNA polymerase in the presence of all four deoxyribonucleotides.
  • the blunt end DNA then was digested with the restriction endonuclease Mrol, and a 215 base pair Hinfl'-Mrol restriction fragment, comprising the N- terminal coding sequence of the EBNA1 gene, was isolated.
  • EBNA1 gene i.e., that portion of the EBNA1 gene not present in the above 215 base pair
  • Hinfl'-Mrol restriction fragment and oriP, (2) the 1600 base pair BamHI-EcoRI restriction fragment from pKan2 which comprises the G418 resistance gene, and (3) the 2900 base pair EcoRI-Spel restriction fragment from pSW.RXR which comprises the elements necessary for replication and selection in E. coli. and those four DNA fragments were joined together by ligation with T4 DNA ligase to generate the plasmid pC.EBNA.
  • pC.EBNA was digested with the restriction endonucleases Accl and
  • the cell line CEN4 is a derivative of the human embryonic kidney cell line 293, which was prepared by stably integrating into the genome of 293 cells the plasmid pCEN1 .
  • pCEN1 DNA was linearized by digestion with the restriction endonuclease Seal (the unique Seal site in pCEN1 occurs within the ampicillin resistance gene).
  • the linearized plasmid DNA then was transfected into 293 cells by electroporation. Potter, et al., Proc. Nat. Acad. Sci. 51:7161 -7165 (1984). Linearizing the pCEN1 DNA increased the frequency of stable integration of the plasmid DNA into the cell chromosomal DNA.
  • transfected cells which had incorporated the pCEN1 DNA
  • the cells were grown in the presence of neomycin, and six neomycin resistant clones were selected for further study. Specifically, pHEB0.20 was transfected into ceils of each of the six clones by electroporation, and in each case the efficiency of transfection was determined by counting the number of hygromycin resistant colonies obtained. Because pHEBO20 lacks the EBNA1 gene, but requires the EBNA1 gene product for replication from oriP, efficient transfection depends upon the host cell being able to express the EBNA1 gene that is present within the cell by virtue of the integrated pCEN1 DNA. One of the six clones was found to be most efficiently transfected with pHEBO20 DNA. The cells of that clone are referred to as CEN4 cells.
  • GPA DNA Cloning and Expression of GPA DNA in Mammalian Cells
  • the complete nucleotide coding sequence for GPA was excised from pHEB02-GPA by digestion of that plasmid DNA with the restriction endonuclease Sstl.
  • the GPA DNA then was ligated to Sstl linearized plasmid pUC219, to form plasmid pUC219-GPA.
  • pUC219-GPA then was digested with restriction endonucleases Eagl and EcoNI.
  • the cohesive ends of the resulting DNA fragment having the complete nucleotide coding sequence for GPA were filled in with Klenow DNA polymerase and all four deoxynucloside triphosphates, and adaptors having Sfil cohesive ends then were joined to the DNA fragment by ligation. That fragment with Sfil cohesive ends then was ligated to Sfil linearized pHEBO30.
  • the resulting plasmid, referred to as pHEBO30-GPA thus contains the complete GPA nucleotide coding sequence downstream of, and in the correct orientation for transcription from the CMV promoter of the plasmid.
  • CEN4 cells were transfected with pHEBO30-GPA by electroporation. Potter, et al., Proc. Nat. Acad. Sci.
  • Transfected cells were cultured in high glucose DMEM, 10% fetal bovine serum, 200 //g/ml hygromycin, 800 ⁇ g/ml neomycin.
  • the transfected cells were transferred to serum free medium, and the serum free conditioned medium then was assayed for growth promoting activity.
  • Ciliary ganglion neurons were cultured for a total of nine days, and were fed with medium containing freshly thawed samples of conditioned media or cell extract every third day.
  • the amount of neuronal cytoplasm present after nine days was quantitated by measuring the amount of lactate deh ⁇ drogenase (LDH) released upon extracting the neurons in each well of the tissue culture plate with detergent.
  • LDH lactate deh ⁇ drogenase
  • the neuron cultures were washed once with balanced salt solution and then extracted with 100 ⁇ homogenate buffer (0.05M. Tris, pH 7.2, 1 mM EDTA, 0.5% Triton X-100, and 2mg/ml bovine serum albumin).
  • LDH activity in duplicate 20 - 25 ⁇ aliquots of the detergent extracts was determined using a spectrophotometric assay that measures the conversion of lactate to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD) and a tetrazolium dye. Reaction rates were measured on a Molecular Devices kinetic microplate reader.
  • one unit of activity is the amount of sample which gives half-maximal levels of LDH in the neuron cultures. N/D indicates that no activity greater than background levels was detectable. TABLE I
  • LDH activity is indicated in terms of the reaction rate (change in optical density (OD) per minute) in the LDH spectrophotometric assay. N/D indicates that no activity greater than background levels was detectable.
  • the absence of detectable LDH activity in the samples of conditioned medium indicates that the GPA in the conditioned medium results from secretion of the GPA from the recombinant CEN4 cells rather than from cell lysis.
  • Conditioned media from CEN4 cells transfected with pHEBO30-GPA contained significant amounts of neuron growth promoting activity, whereas conditioned media from
  • CEN4 cells transfected with pHEB030-CNTF contained no such activity in excess of that found in the conditioned media of non-transfected CEN4 cells.
  • extracts prepared from CEN4 cells transfected with pHEBO30-CNTF did have significant neuron growth promoting activity, indicating that pHEBO30-CNTF DNA was being expressed in those cells.
  • CAG ACG GGC ACC CAG CGC CTC CTG GAC AAC CTG GCC GCC 331 Gin Thr Gly Thr Gin Arg Leu Leu Asp Asn Leu Ala Ala 70 75 TAC CGG GCC TTC CGC ACG CTG CTG GCG CAG ATG CTG GAG 370
  • GGC GCG GCG CTG GCG GCC ATG CTG CTG CAG GTC TCG GCC 448 Gly Pro Ala Leu Ala Ala Met Leu Leu Gin Val Ser Ala 1 10 1 15
  • GGCCATGCTA CTAACCCAGG ACACTTCTGC TTCCTAATGG GCCCACTTCC 830
  • Ala Ser lie Ser Val Ala Ala Val Asp Gly Val Pro Thr Ala Ala 50 55 60
  • Lys Asn lie Asn Leu Asp Ser Val Asp Gly Val Pro Met Ala Ser 50 55 60 Thr Asp Gin Trp Ser Glu Leu Thr Glu Ala Glu Arg Leu Gin Glu
  • Lys Asn lie Asn Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser 50 55 60 Thr Asp Arg Trp Ser Glu Met Thr Glu Ala Glu Arg Leu Gin Glu
  • CTTAAATTCA CCTAAGAATG GGAGCAACCA GCAGGAAAAG GACAAGCAGC 800
  • TTGTCTTTGT TTATGGGCCC CATTGGCGTG GAGCCCCGTT TAATTTTCGG 1850
  • AACTACAGTC AGAGAACCCC TTTGTGTTTG GTCCCCCCCC GTGTCACATG 2900
  • ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT 3250

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Abstract

L'invention concerne des acides nucléiques codant pour la protéine GPA, et aussi sur la protéine GPA produite par des procédés impliquant de l'ADN recombiné. Cette protéine GPA est utile pour préparer des anticorps et diagnostiquer et traiter divers troubles neuronaux.
PCT/US1992/008258 1991-10-01 1992-09-29 Production d'un facteur neurotrophique dote d'une activite de promotion de la croissance (gpa) WO1993007270A1 (fr)

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DE4237047A1 (en) * 1991-11-04 1993-05-06 I N R O Handels-Gmbh, 8750 Aschaffenburg, De Drug contg. polypeptide homologous to nerve growth factor and slow virus sequence - for treating amyotrophic lateral sclerosis, spinal muscle atrophy or Parkinson's disease
FR2727867A1 (fr) * 1994-12-13 1996-06-14 Rhone Poulenc Rorer Sa Transfert de genes dans les motoneurones medullaires au moyen de vecteurs adenoviraux
US5534615A (en) * 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5571675A (en) * 1994-04-25 1996-11-05 Genentech, Inc. Detection and amplification of candiotrophin-1(cardiac hypertrophy factor)
US6472585B1 (en) 1994-04-25 2002-10-29 Genentech, Inc. Cardiotrophin-1 defective mouse
US7227004B2 (en) 1991-03-29 2007-06-05 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
US9150884B2 (en) 2011-03-08 2015-10-06 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona, Acting For And On Behalf Of Arizona State University Microbial conversion of glucose to styrene and its derivatives
US11613768B2 (en) 2017-07-25 2023-03-28 Arizona Board Of Regents On Behalf Of Arizona State University Microbial production of 2-phenylethanol from renewable substrates

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A. PARENT ET AL. 'Gene expression of a trophic factor during development of embryonic chick ciliary ganglion neurons' 20th annual meeting of the society for neuroscience, St. Louis, Missouri, USA, october 28- november 2, 1990 Abstract no. 467.8 the whole document *
NATURE vol. 342, no. 6252, 21 December 1989, MACMILLAN JOURNALS LTD., LONDON,UK; pages 920 - 923 K.A. ST\CKLI ET AL. 'Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor' cited in the application *
NEURON vol. 4, no. 4, April 1990, CELL PRESS,CAMBRIDGE,NA; pages 623 - 631 F.P. ECKENSTEIN ET AL. 'Purification and characterization of a trophic factor for embryonic peripheral neurons: comparison with fibroblast growth factors' cited in the application *
NEURON vol. 8, no. 6, June 1992, CELL PRESS, CAMBRIDGE, NA; pages 1045 - 1053 D.W. LEUNG ET AL. 'Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons' *
SCIENCE vol. 246, 24 November 1989, AAAS, WASHINGTON,DC,US; pages 1023 - 1025 L.-F. LIN ET AL. 'Purification, cloning, and expression of ciliary neurotrophic factor (CNTF)' cited in the application *
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US7482005B2 (en) 1991-03-29 2009-01-27 Genentech, Inc. Methods of treating diseases with anti-VEGF antibodies
US7227004B2 (en) 1991-03-29 2007-06-05 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
DE4237047A1 (en) * 1991-11-04 1993-05-06 I N R O Handels-Gmbh, 8750 Aschaffenburg, De Drug contg. polypeptide homologous to nerve growth factor and slow virus sequence - for treating amyotrophic lateral sclerosis, spinal muscle atrophy or Parkinson's disease
US5571675A (en) * 1994-04-25 1996-11-05 Genentech, Inc. Detection and amplification of candiotrophin-1(cardiac hypertrophy factor)
US6472585B1 (en) 1994-04-25 2002-10-29 Genentech, Inc. Cardiotrophin-1 defective mouse
US5624806A (en) * 1994-04-25 1997-04-29 Genentech, Inc. Antibodies to cardiac hypertrophy factor and uses thereof
US5627073A (en) * 1994-04-25 1997-05-06 Genentech, Inc. Hybridomas producing antibodies to cardiac hypertrophy factor
US5679545A (en) * 1994-04-25 1997-10-21 Genentech, Inc. Gene encoding cardiac hypertrophy factor
US5723585A (en) * 1994-04-25 1998-03-03 Genentech, Inc. Method of purifying cardiac hypertrophy factor
US6117650A (en) * 1994-04-25 2000-09-12 Genentech, Inc. Assay for cardiac hypertrophy
US5534615A (en) * 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
WO1996018740A1 (fr) * 1994-12-13 1996-06-20 Rhone-Poulenc Rorer S.A. Transfert de genes dans les motoneurones medullaires au moyen de vecteurs adenoviraux
US6632427B1 (en) 1994-12-13 2003-10-14 Aventis Pharma S.A. Adenoviral-vector-mediated gene transfer into medullary motor neurons
FR2727867A1 (fr) * 1994-12-13 1996-06-14 Rhone Poulenc Rorer Sa Transfert de genes dans les motoneurones medullaires au moyen de vecteurs adenoviraux
US9150884B2 (en) 2011-03-08 2015-10-06 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona, Acting For And On Behalf Of Arizona State University Microbial conversion of glucose to styrene and its derivatives
US11613768B2 (en) 2017-07-25 2023-03-28 Arizona Board Of Regents On Behalf Of Arizona State University Microbial production of 2-phenylethanol from renewable substrates

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