WO1996039508A1 - Fibroblast growth factor 13 - Google Patents
Fibroblast growth factor 13 Download PDFInfo
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- WO1996039508A1 WO1996039508A1 PCT/US1995/007108 US9507108W WO9639508A1 WO 1996039508 A1 WO1996039508 A1 WO 1996039508A1 US 9507108 W US9507108 W US 9507108W WO 9639508 A1 WO9639508 A1 WO 9639508A1
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Classifications
-
- 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
- C07K14/50—Fibroblast growth factor [FGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention have been putatively identified as fibroblast growth factor/heparin binding growth factor, hereinafter referred to as "FGF-13". The invention also relates to inhibiting the action of such polypeptides.
- FGF-13 fibroblast growth factor/heparin binding growth factor
- Fibroblast growth factors are a family of proteins characteristic of binding to heparin and are, therefore, also called heparin binding growth factors (HBGF) . Expression of different members of these proteins are found in various tissues and are under particular temporal and spatial control. These proteins are potent mitogens for a variety of cells of mesodermal, ectodermal, and endodermal origin, including fibroblasts, corneal and vascular endothelial cells, granulocytes, adrenal cortical cells, chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells-.
- HBGF heparin binding growth factors
- FGF-1 and 2 are chemotactic for endothelial cells and FGF-2 has been shown to enable endothelial cells to penetrate the basement membrane. Consistent with these properties, both FGF-1 and 2 have the capacity to stimulate angiogenesis. Another important feature of these growth factors is their ability to promote wound healing. Many other members of the FGF family share similar activities with FGF-1 and 2 such as promoting angiogenesis and wound healing. Several members of the FGF family have been shown to induce mesoderm formation and to modulate differentiation of neuronal cells, adipocytes and skeletal muscle cells.
- FGF proteins have been implicated in promoting tumorigenesis in carcinomas and sarcomas by promoting tumor vascularization and as transforming proteins when their expression is deregulated.
- the FGF family presently consists of eight structurally-related polypeptides: basic FGF, acidic FGF, int 2, hst 1/k- FGF, FGF-5, FGF-6, keratinocyte growth factor, AIGF (FGF-8) and recently a glia-activating factor has been shown to be a novel heparin-binding growth factor which was purified from the culture supernatant of a human glioma cell line (Miyamoto, M. et al., Mol. and Cell. Biol., 13 (7) :4251-4259 (1993) . The genes for each have been cloned and sequenced.
- FGF-1 and FGF-2 Two of the members, FGF-1 and FGF-2, have been characterized under many names, but most often as acidic and basic fibroblast growth factor, respectively.
- the normal gene products influence the general proliferation capacity of the majority of mesoderm and neuroectoderm-derived cells. They are capable of inducing angiogenesis in vivo and may play important roles in early development (Burgess, W.H. and Maciag, T. , Annu. Rev. Biochem., 58:575-606, (1989)). Many of the above-identified members of the FGF family also bind to the same receptors and elicit a second message through binding to these receptors.
- a eukaryotic expression vector encoding a secreted form of FGF-1 has been introduced by gene transfer into porcine arteries. This model defines gene function in the arterial wall in vivo. FGF-1 expression induced intimal thickening in porcine arteries 21 days after gene transfer (Nabel, E.G., et al., Nature, 362:844-6 (1993)). It has further been demonstrated that basic fibroblast growth factor may regulate glioma growth and progression independent of its role in tumor angiogenesis and that basic fibroblast growth factor release or secretion may be required for these actions (Morrison, R.S., et al., J. Neurosci. Res., 34:502-9 (1993)).
- Fibroblast growth factors such as basic FGF
- basic FGF have further been implicated in the growth of Kaposi's sarcoma cells in vi tro (Huang, Y.Q., et al., J. Clin. Invest., 91:1191-7 (1993)).
- the cDNA sequence encoding human basic fibroblast growth factor has been cloned downstream of a transcription promoter recognized by the bacteriophage T7 RNA polymerase.
- Basic fibroblast growth factors so obtained have been shown to have biological activity indistinguishable from human placental fibroblast growth factor in mitogenicity, synthesis of plasminogen activator and angiogenesis assays (Squires, C.H., et al., J. Biol. Chem., 263:16297-302 (1988)).
- U.S. Patent No. 5,155,214 discloses substantially pure mammalian basic fibroblast growth factors and their production.
- the amino acid sequences of bovine and human basic fibroblast growth factor are disclosed, as well as the DNA sequence encoding the polypeptide of the bovine species.
- FGF-9 has around 30% sequence similarity to other members of the FGF family. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic and basic FGF. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol., 13(7) :4251-4259 (1993).
- FGF-9 was found to stimulate the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3, and PC-12 cells but not that of human umbilical vein endothelial cells (Naruo, K., et al., J. Biol. Chem., 268:2857-2864 (1993).
- Basic FGF and acidic FGF are potent modulators of cell proliferation, cell motility, differentiation, and survival and act on cell types from ectoderm, mesoderm and endoderm. These two FGFs, along with KGF and AIGF, were identified by protein purification. However, the other four members were isolated as oncogenes. , expression of which was restricted to embryogenesis and certian types of cancers. FGF-9 was demonstrated to be a mitogen against glial cells. Members of the FGF family are reported to have oncogenic potency. FGF-9 has shown transforming potency when transformed into BALB/c3T3 cells (Miyamoto, M., et al., Mol. Cell. Biol., 13 (7) :4251-4259 (1993) .
- AIGF Androgen induced growth factor
- SC-3 mouse mammary carcinoma cells
- AIGF is a distinctive FGF-like growth factor, having a putative signal peptide and sharing 30-40% homology with known members of the FGF family.
- Mammalian cells transformed with AIGF shows a remarkable stimulatory effect on the growth of SC-3 cells in the absence of androgen. Therefore, AIGF mediates androgen-induced growth of SC-3 cells, and perhaps other cells, since it is secreted by the tumor cells themselves.
- polypeptide of the present invention has been putatively identified as a member of the FGF family as a result of amino acid sequence homology with other members of the FGF family.
- novel mature polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
- the polypeptides of the present invention are of human origin.
- nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
- processes for producing such polypeptides by recombinant techniques through the use of recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides of the present invention, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.
- recombinant vectors such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides of the present invention, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.
- antibodies against such polypeptides there are provided antibodies against such polypeptides.
- antagonists against such polypeptides and processes for their use to inhibit the action of such polypeptides for example, in the treatment of cellular transformation, for example, tumors, to reduce scarring and treat hyper-vascular diseases.
- nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a polynucleotide encoding a polypeptide of the present invention
- diagnostic assays for detecting diseases or susceptibility to diseases related to mutations in a nucleic acid sequence of the present invention and for detecting over-expression of the polypeptides encoded by such sequences.
- Figure 1 depicts the cDNA sequence and corresponding deduced amino acid sequence of FGF-13.
- the initial 21 amino acid residues represent a putative leader sequence.
- isolated nucleic acids molecules polynucleotides which encode for the mature polypeptide having the deduced amino acid sequence of Figure l (SEQ ID NOS:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 97147 on May 12, 1995.
- the polynucleotide encoding FGF-13 of this invention was discovered in a cDNA library derived from human ovarian cancer tissue.
- the FGF-13 polypeptide is structurally related to all members of the fibroblast growth factor family and contains an open reading frame encoding a polypeptide of 212 amino acids of which the first 21 amino acids represent a putative leader sequence such that the mature polypeptide comprises 191 amino acids.
- the top matches are: 1) 69 % identity and 81 % similarity to mouse AIGF over a stretch of 185 amino acids; 2) 30 % identity and 56 % similarity with FGF-4 from chicken in a region of 82 amino acids,- 3) 41 % identity and 64 % similarity with human KGF over a stretch of 78 amino acids.
- the FGF/HBGF family signature, GXLX(S,T,A,G)X6(D,E)CXFXE is conserved in the polypeptide of the present invention, (X means any amino acid residue,- (D,E) means either D or E residue; X6 means any 6 amino acid residues) .
- the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
- the DNA may be double- stranded or single-stranded.
- the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID N0S:1) or that of the deposited clone or may be a different coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1, (SEQ ID N0S:1) or the deposited cDNA.
- the polynucleotides which encodes for the mature polypeptide of Figure 1 (SEQ ID NOS:2) or for the mature polypeptides encoded by the deposited cDNA(s) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
- polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
- the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NOS:2) or the polypeptides encoded by the cDNA(s) of the deposited clone(s).
- the variants of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
- the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NOS:2) or the same mature polypeptides encoded by the cDNA(s) of the deposited clone(s) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure l (SEQ ID NOS:2) or the polypeptides encoded by the cDNA(s) of the deposited clone(s).
- Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
- the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure l (SEQ ID NOS.-l) or of the coding sequence of the deposited clone(s).
- an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides.
- the present invention also includes polynu ⁇ _.eotides, wherein the coding sequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
- the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
- the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues.
- a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
- the polynucleotides of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence) .
- the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
- the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
- the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)) .
- gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
- Fragments of the full length FGF-13 gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity.
- Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases.
- the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete FGF-13 gene including regulatory and promotor regions, exons, and introns.
- An example of a screen comprises isolating the coding region of the FGF-13 gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
- the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences.
- the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
- stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
- polypeptides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure l (SEQ ID NO:l) or the deposited cDNA(s) .
- the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.
- such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:l, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
- the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
- the deposit(s) referred to herein will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. These deposits are provided merely as a convenience and are not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
- the sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description of sequences herein.
- a license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
- the present invention further relates to an FGF polypeptide which has the deduced amino acid sequence of Figure l (SEQ ID NOS:2) or which has the amino acid sequence encoded by the deposited cDNA(s) , as well as fragments, analogs and derivatives of such polypeptides.
- fragment when referring to the polypeptide of Figure 1 (SEQ ID NOS:2) or those encoded by the deposited cDNA(s) , means polypeptides which retains essentially the same biological function or activity as such polypeptides.
- an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
- polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
- the fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NOS:2) or that encoded by the deposited cDNA(s) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
- Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from
- polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
- isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
- a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
- Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
- the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
- Host cells may be genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
- the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
- the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the FGF genes.
- 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 polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques.
- the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide.
- vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids,- phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
- any other vector or plasmid may be used as long as they are replicable and viable in the host.
- the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
- the DNA sequence is inserted into an appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
- the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
- promoter for example, LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
- the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression.
- the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
- the vector containing the appropriate DNA sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
- appropriate hosts there may be mentioned: bacterial cells, such as E. coli. Salmonella tvphimurium, Streptomyces; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma,- adenoviruses; plant cells, etc.
- bacterial cells such as E. coli. Salmonella tvphimurium, Streptomyces
- fungal cells such as yeast
- insect cells such as Drosophila S2 and Spodoptera Sf9
- animal cells such as CHO, COS or Bowes melanoma,- adenoviruses
- plant cells etc.
- the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings here
- the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
- the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
- the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
- a promoter operably linked to the sequence.
- Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, ⁇ NH16a, pNHl ⁇ a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) .
- Eukaryotic pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
- any other plasmid or vector may be used as long as they are replicable and viable in the host.
- Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
- Two appropriate vectors are pKK232-8 and pCM7.
- Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
- Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
- the present invention relates to host cells containing the above-described construct.
- the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
- Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, 1986)) .
- the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
- the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
- Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), the disclosure of which is hereby incorporated by reference.
- Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
- promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ex factor, acid phosphatase, or heat shock proteins, among others.
- the heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
- the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
- Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation, initiation and termination signals in operable reading phase with a functional promoter.
- the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
- Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
- useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
- cloning vector pBR322 ATCC 37017
- Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) .
- pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
- the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
- appropriate means e.g., temperature shift or chemical induction
- Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
- Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
- mammalian cell culture systems can also be employed to express recombinant protein.
- mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
- Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
- DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
- the polypeptide of -he present invention may be recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- the polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
- a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
- the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
- Polypeptides of the invention may also include an initial methionine amino acid residue.
- polypeptide of the present invention may be employed in treatment for stimulating re- vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration.
- the polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
- the polypeptide of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage associated with stroke and which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS- related complex.
- FGF-13 has the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
- the polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
- the FGF-13 polypeptide may also be employed for preventing hair loss, since FGF family members activate hair- forming cells and promotes melanocyte growth.
- the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
- the FGF-13 polypeptide may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.
- polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
- This invention provides a method for identification of the receptors for the polypeptides of the present invention.
- the genes encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)).
- expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides.
- Transfected cells which are grown on glass slides are exposed to the the polypeptide of the present invention, after they have been labelled.
- the polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site- specific protein kinase.
- the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
- This invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention.
- An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3 [H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate.
- a control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3 [H] thymidine in each case.
- the amount of fibroblast cell proliferation is measured by liquid scintillation chromatography whichmeasures the incorporation of 3 [H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
- a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound.
- the ability of the compound to enhance or block this interaction could then be measured.
- the response of a known second messenger system following interaction of a compound to be screened and the FGF-13 receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist.
- second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
- antagonist compounds include antibodies, or in some cases, oligonucleotides, which bind to the receptor for the polypeptide of the present invention but elicit no second messenger response or bind to the FGF-13 polypeptide itself.
- a potential antagonist may be a mutant form of the polypeptide which binds to the receptors, however, no second messenger response is elicited and, therefore, the action of the polypeptide is effectively blocked.
- Another antagonist compound to the FGF-13 gene and gene product is an antisense construct prepared using antisense technology.
- Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
- the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
- a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl.
- the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide (Antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
- the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the polypeptide.
- Potential antagonist compounds also include small molecules which bind to and occupy the binding site of the receptors thereby making the receptor inaccessible to its polypeptide such that normal biological activity is prevented.
- small molecules include, but are not limited to, small peptides or peptide-like molecules.
- Antagonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
- the antagonists may also be employed to prevent hyper- vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.
- the antagonists may also be employed to prevent the growth of scar tissue during wound healing.
- the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
- polypeptides, agonists and antagonists of the present invention may be employed in combination with a suitable pharmaceutical carrier to comprise a pharmaceutical composition for parenteral administration.
- a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
- the formulation should suit the mode of administration.
- the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
- the polypeptides, agonists and antagonists of the present invention may be employed in conjunction with other therapeutic compounds.
- the pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
- the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
- dosages are preferably administered from about 0.1 g to 9 mg per cm 2 .
- polypeptide of the invention and agonist and antagonist compounds which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptide in vivo, which is often referred to as "gene therapy.”
- cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide.
- a polynucleotide DNA or RNA
- Such methods are well-known in the art.
- cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding for the polypeptide of the present invention.
- cells may be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art.
- a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
- the expression vehicle for engineering cells may be other than a retroviral particle, for example, an adenovirus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
- Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
- the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
- the vector includes one or more promoters.
- Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechni ⁇ ues. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 3-actin promoters) .
- CMV human cytomegalovirus
- viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
- Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter,- or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter,- heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described) ; the jS-actin promoter; and human growth hormone promoters.
- the promoter also may be the native promoter which controls the
- the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
- packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CKE , ⁇ CRIP , GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
- the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
- the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
- the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence (s) encoding the polypeptides.
- retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
- the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
- Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
- This invention is also related to the use of the genes of the present invention as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences encoding the polypeptide of the present invention.
- Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
- the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analysis.
- RNA or cDNA may also be used for the same purpose.
- PCR primers complementary to the nucleic acid encoding a polypeptide of the present invention can be used to identify and analyze mutations.
- deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
- Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
- DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985)).
- Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).
- nuclease protection assays such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).
- the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
- restriction enzymes e.g., Restriction Fragment Length Polymorphisms (RFLP)
- mutations can also be detected by in si tu analysis.
- the present invention also relates to a diagnostic assay for detecting altered levels of FGF-13 proteins in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of abnormal cellular proliferation, for example, a tumor.
- Assays used to detect levels of protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich” assay.
- An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to an antigen to the polypeptides of the present invention, preferably a monoclonal antibody.
- a reporter antibody is prepared against the monoclonal antibody.
- a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme.
- a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like bovine serum albumen.
- the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any polypeptides of the present invention attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
- the reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the protein of interest.
- Unattached reporter antibody is then washed out.
- Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of a polypeptide of the present invention present in a given volume of patient sample when compared against a standard curve.
- a competition assay may be employed wherein antibodies specific to a polypeptide of the present invention are attached to a solid support and labeled FGF-13 and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of a polypeptide of the present invention in the sample.
- a “sandwich” assay is similar to an ELISA assay.
- a polypeptide of the present invention is passed over a solid support and binds to antibody attached to a solid support.
- a second antibody is then bound to the polypeptide of interest.
- a third antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.
- sequences of the present invention are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
- Few chromosome marking reagents based on actual sequence data (repeat polymorphism's) are presently available for marking chromosomal location.
- the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
- sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
- mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
- Fluorescence in si tu hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
- FISH Fluorescence in si tu hybridization
- a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
- polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
- These antibodies can be, for example, polyclonal or monoclonal antibodies.
- the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
- Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
- any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
- Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
- the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
- equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
- “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
- the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
- For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
- For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
- Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .
- Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
- Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
- ligase T4 DNA ligase
- the DNA sequence encoding FGF-13 ATCC # 97147 is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequences of the processed protein (minus the signal peptide sequence) and the vector sequences 3' to the gene. Additional nucleotides corresponding to the gene are added to the 5' and 3' sequences respectively.
- the 5' oligonucleotide primer 5' GCCAGACCATGGAGAATCACCCGTCTCCTAAT 3' contains a Nco restriction enzyme site.
- the 3' sequence 5' GATTTAAGATCTCGTGAGGGGCTGGGGCCG 3' contains complementary sequences to a BglU site and is followed by 18 nucleotides of FGF-13 coding sequence.
- the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, CA 91311) .
- pQE-60 encodes antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and restriction enzyme sites.
- pQE-60 was then digested with Ncol and BglU. The amplified sequences are ligated into pQE-60 and are inserted in frame with the sequence encoding for the histidine tag and the ribosome binding site (RBS) . The ligation mixture is then used to transform E.
- M15/rep 4 coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) .
- M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan r ) .
- Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA is isolated and confirmed by restriction analysis.
- Clones containing the desired constructs are grown overnight (O/N) in liquid culturein LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
- the O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
- the cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
- IPTG Isopropyl-B-D-thiogalacto pyranoside
- IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression.
- Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation.
- the cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
- solubilized FGF-13 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., j. Chromatography 411:177-184 (1984)) .
- the proteins are eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the proteins are dialyzed to 10 mmolar sodium phosphate.
- the DNA sequence encoding the full length FGF-13 protein, ATCC # 97147, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
- the FGF-13 5' primer has the sequence 5' CTAGTGGATCCCGA GAATCACCCGTCTCCT 3' (SEQ ID NO:5) and contains a BamHl restriction enzyme site (in bold) such that cloning at this site will put the baculovirus signal sequence in frame with 18 nucleotides of the FGF-13 gene downstream of the putative FGF-13 signal peptide cleavage site.
- the 3' primer has the sequence 5' CGACTTCTAGAACCT CGGGGATCTGGCTCC 3' (SEQ ID NO:6) and contains the cleavage site for the restriction endonuclease Xbal and 18 nucleotides complementary to the 3' non-translated sequence of the gene.
- the amplified sequences are isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.) .
- the fragment is then digested with the respective endonucleases and purified again on a 1% agarose gel. This fragment is designated F2.
- the vector pA2gp (modification of pVL94l vector, discussed below) is used for the expression of the proteins using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
- This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHl and Xbal.
- the polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation.
- the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
- the polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA.
- Many other baculovirus vectors could be used in place of pA2 such as pRGl, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
- the plasmid is digested with the restriction enzymes and dephosphorylated using calf intestinal phosphatase by procedures known in the art.
- the DNA is then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated V2.
- Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli DH5c. cells are then transformed and bacteria identified that contained the plasmid (pBacFGF-13) using the respective restriction enzymes. The sequence of the cloned fragment are confirmed by DNA sequencing.
- 5 ⁇ g of the plasmid pBacFGF-13 are co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
- BaculoGoldTM virus DNA and 5 ⁇ g of the plasmids, in each case, are mixed in a sterile well of microtiter plates containing 50 ⁇ l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) .
- plaque assays performed similar as described by Summers and Smith (supra) .
- an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques.
- a detailed description of a "plaque assay” can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .
- the virus is added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette.
- the agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
- the agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
- the supernatants of these culture dishes are harvested and then stored at 4°C.
- Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
- the cells are infected with the recombinant baculovirus V-FGF-13 at a multiplicity of infection (MOD of 2.
- MOD multiplicity of infection
- the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 35 S cysteine (Amersham) are added.
- the cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
- plasmids FGF-13-HA derived from a vector pcDNA3/Amp (Invitrogen) containing: l) SV40 origin of re lication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site.
- DNA fragments encoding the entire FGF-13 precursor and an HA tag fused in frame to the 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
- the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
- HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
- the PCR amplified DNA fragments and the vector, pcDNA3/Amp, are digested with the respective restriction enzymes and ligated.
- the ligation mixture is transformed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
- FGF-13 COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)).
- FGF-13-HA protein The expression of the FGF-13-HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 35 S-cysteine two days post transfection. Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with an HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
- RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5
- Fibroblasts are obtained from a subject by skin biopsy.
- the resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask.
- the flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37°C for approximately one week. At this time, fresh media is added and subsequently changed every several days.
- fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
- pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phosphatase.
- the linear vector is fractionated on agarose gel and purified, using glass beads.
- the cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively.
- the 5' primer containing an EcoRI site and the 3' primer having contains a Hindlll site.
- Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and Himdlll fragment are added together, in the presence of T4 DNA ligase.
- the resulting mixture is maintained under conditions appropriate for ligation of the two fragments.
- the ligation mixture is used to transform bacteria HB101, which are then plated onto agar- containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
- the amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin.
- DMEM Dulbecco's Modified Eagles Medium
- CS calf serum
- penicillin and streptomycin The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector.
- the packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells) .
- Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells.
- the spent media containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells.
- Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
- the engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
- the fibroblasts now produce the protein product.
- ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
- MOLECULE TYPE Oligonucleotide
- xi SEQUENCE DESCRIPTION: SEQ ID NO:3:
- MOLECULE TYPE Oligonucleotide
- xi SEQUENCE DESCRIPTION: SEQ ID NO:4:
- MOLECULE TYPE Oligonucleotide
- xi SEQUENCE DESCRIPTION: SEQ ID NO:5:
- MOLECULE TYPE Oligonucleotide
- xi SEQUENCE DESCRIPTION: SEQ ID NO:6:
- MOLECULE TYPE Oligonucleotide
- xi SEQUENCE DESCRIPTION: SEQ ID NO:7: (2) INFORMATION FOR SEQ ID NO:8:
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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JP9500361A JPH11507504A (ja) | 1995-06-05 | 1995-06-05 | 線維芽細胞増殖因子13 |
PCT/US1995/007108 WO1996039508A1 (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
EP95922214A EP0857209A1 (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
AU26979/95A AU702682B2 (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
KR1019970708795A KR19990022315A (ko) | 1995-06-05 | 1995-06-05 | 섬유아세포 성장 인자 13 |
US10/123,481 US7208584B2 (en) | 1995-06-05 | 2002-04-17 | Fibroblast growth factor-13 |
US11/738,563 US20070281890A1 (en) | 1995-06-05 | 2007-04-23 | Fibroblast Growth Factor-13 |
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PCT/US1995/007108 WO1996039508A1 (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
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US08/462,965 A-371-Of-International US5728546A (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
US08/462,965 Continuation-In-Part US5728546A (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
US08/976,315 Continuation-In-Part US6403557B1 (en) | 1995-06-05 | 1997-11-21 | Fibroblast growth factor-13 |
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PCT/US1995/007108 WO1996039508A1 (en) | 1995-06-05 | 1995-06-05 | Fibroblast growth factor 13 |
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EP (1) | EP0857209A1 (ja) |
JP (1) | JPH11507504A (ja) |
KR (1) | KR19990022315A (ja) |
AU (1) | AU702682B2 (ja) |
WO (1) | WO1996039508A1 (ja) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998014467A1 (en) * | 1996-09-30 | 1998-04-09 | Human Genome Sciences, Inc. | Method of purifying protein from inclusion bodies |
WO1998023749A1 (en) * | 1996-11-27 | 1998-06-04 | Human Genome Sciences, Inc. | Fibroblast growth factor-13 |
WO1999014327A2 (en) * | 1997-09-17 | 1999-03-25 | Genentech, Inc. | Genes amplified in tumours, antibodies against the proteins coded thereby, and their use in diagnosis and treatment of cancer |
WO2001064744A1 (fr) * | 2000-03-02 | 2001-09-07 | Biowindow Gene Development Inc. | Nouveau polypeptide, facteur de croissance du fibroblaste 14, et polynucleotide codant pour ce polypeptide |
US6368822B1 (en) | 1995-06-05 | 2002-04-09 | Human Genome Sciences, Inc. | Fibroblast growth factor 13 |
US6486133B1 (en) * | 1997-03-07 | 2002-11-26 | The Wistar Institute Of Anatomy And Biology | Methods for inducing localized vascular development and enhancing the repair of a wound in the mammamian dermis |
US6632425B1 (en) | 1997-03-20 | 2003-10-14 | Human Genome Sciences, Inc. | Chemokine compositions |
US7217570B2 (en) | 2001-08-23 | 2007-05-15 | The Wistar Institute Of Anatomy And Biology | Organotypic intestinal culture and methods of use thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000071567A2 (en) * | 1999-05-06 | 2000-11-30 | Human Genome Sciences, Inc. | Fibroblast growth factor 13 |
-
1995
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- 1995-06-05 JP JP9500361A patent/JPH11507504A/ja active Pending
- 1995-06-05 WO PCT/US1995/007108 patent/WO1996039508A1/en not_active Application Discontinuation
- 1995-06-05 EP EP95922214A patent/EP0857209A1/en not_active Withdrawn
- 1995-06-05 AU AU26979/95A patent/AU702682B2/en not_active Ceased
Non-Patent Citations (3)
Title |
---|
J. BRUGGE et al., "Origins of Human Cancer: A Comprehensive Review", Published 1991, by COLD SPRING HARBOR LABORATORY PRESS, (N.Y.), pages 675-683. * |
PROGRESS IN GROWTH FACTOR RESEARCH, Volume 1, issued 1989, M. KLAGSBRUN, "The Fibroblast Growth Factor Family: Structural and Biological Properties", pages 207-235. * |
See also references of EP0857209A4 * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6368822B1 (en) | 1995-06-05 | 2002-04-09 | Human Genome Sciences, Inc. | Fibroblast growth factor 13 |
US7208584B2 (en) * | 1995-06-05 | 2007-04-24 | Human Genome Sciences, Inc. | Fibroblast growth factor-13 |
WO1998014467A1 (en) * | 1996-09-30 | 1998-04-09 | Human Genome Sciences, Inc. | Method of purifying protein from inclusion bodies |
US5912327A (en) * | 1996-09-30 | 1999-06-15 | Human Genome Sciences, Inc. | Method of purifying chemokines from inclusion bodies |
WO1998023749A1 (en) * | 1996-11-27 | 1998-06-04 | Human Genome Sciences, Inc. | Fibroblast growth factor-13 |
US6403557B1 (en) | 1996-11-27 | 2002-06-11 | Human Genome Sciences, Inc. | Fibroblast growth factor-13 |
US6486133B1 (en) * | 1997-03-07 | 2002-11-26 | The Wistar Institute Of Anatomy And Biology | Methods for inducing localized vascular development and enhancing the repair of a wound in the mammamian dermis |
US6632425B1 (en) | 1997-03-20 | 2003-10-14 | Human Genome Sciences, Inc. | Chemokine compositions |
WO1999014327A2 (en) * | 1997-09-17 | 1999-03-25 | Genentech, Inc. | Genes amplified in tumours, antibodies against the proteins coded thereby, and their use in diagnosis and treatment of cancer |
WO1999014327A3 (en) * | 1997-09-17 | 1999-09-30 | Genentech Inc | Genes amplified in tumours, antibodies against the proteins coded thereby, and their use in diagnosis and treatment of cancer |
WO2001064744A1 (fr) * | 2000-03-02 | 2001-09-07 | Biowindow Gene Development Inc. | Nouveau polypeptide, facteur de croissance du fibroblaste 14, et polynucleotide codant pour ce polypeptide |
US7217570B2 (en) | 2001-08-23 | 2007-05-15 | The Wistar Institute Of Anatomy And Biology | Organotypic intestinal culture and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
KR19990022315A (ko) | 1999-03-25 |
AU702682B2 (en) | 1999-03-04 |
EP0857209A1 (en) | 1998-08-12 |
EP0857209A4 (ja) | 1998-08-12 |
JPH11507504A (ja) | 1999-07-06 |
AU2697995A (en) | 1996-12-24 |
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