EP0817839A1 - Deoxyribonuclease chez l'homme - Google Patents

Deoxyribonuclease chez l'homme

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Publication number
EP0817839A1
EP0817839A1 EP94916012A EP94916012A EP0817839A1 EP 0817839 A1 EP0817839 A1 EP 0817839A1 EP 94916012 A EP94916012 A EP 94916012A EP 94916012 A EP94916012 A EP 94916012A EP 0817839 A1 EP0817839 A1 EP 0817839A1
Authority
EP
European Patent Office
Prior art keywords
dna
polypeptide
dnase
sequence
dna sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94916012A
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German (de)
English (en)
Other versions
EP0817839A4 (fr
Inventor
Craig A. Rosen
Steven Ruben
Mark D. Adams
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
Original Assignee
Human Genome Sciences Inc
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Filing date
Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP0817839A4 publication Critical patent/EP0817839A4/xx
Publication of EP0817839A1 publication Critical patent/EP0817839A1/fr
Withdrawn legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/16Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22Ribonucleases RNAses, DNAses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotide sequences, polypeptides encoded by such sequences, 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 is a human deoxyribonuclease (DNase) . The invention also relates to inhibiting the action of such polypeptide.
  • DNase human deoxyribonuclease
  • DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. It acts to extensively and non- specifically degrade DNA and in this regard it is distinguished from the relatively limited and sequence- specific restriction endonucleases. The degradation activity as stated above is non-specific. However, it also degrades double stranded DNA to yield 5' -oligonucleotides.
  • DNase I has a pH optimum near neutrality, an obligatory requirement for divalent cations, and produces 5' -phosphate nucleotides on hydrolysis of DNA.
  • DNase II exhibits an acid pH optimum, can be activated by divalent cations and produces 3' -phosphate nucleotides on hydrolysis of DNA.
  • Multiple molecular forms of DNase I and II also are known.
  • the DNase I is mainly a digestive enzyme, however, activities similar to DNase I had been found in other tissues suggesting that it may have other functions (Laskowski, ref. 1 9 71 ) .
  • DNase may play a role in the polymerization of actin. (Suck, et al., ref. 1981).
  • DNase's in prokaryotic cells participate in a variety of metabolic functions, including genetic recombination, repair of DNA damage, restriction of foreign DNA and transport of DNA into cells.
  • DNase from various species have been purified to a varying degree.
  • Bovine DNase A, B, C, and D was purified and completely sequenced as early as 1973 (Liao et al., J. Biol. Chem. 248:1489 [1973]) .
  • Porcine and bovine DNase have been purified and fully sequenced (Paudel et al., J. Biol. Chem. 261:16006 [1986]) .
  • Human urinary DNase was reported to have been purified to an electrophoretically homogenous state and the N-terminal amino acid observed to be leucine; no other sequence was reported (Ito et al., J. Biol. Chem. 95:1399 [1984] ) .
  • the Shields et al. reference described the expression cloning of part of the gene for bovine DNase I and expression of a fusion product in E. coli which was biologically and immunologically active (Biochem. Soc. Trans. 16:195 [1988]).
  • the DNase product of Shields et al. was toxic to the host cells and could only be obtained by the use of an inducible promoter.
  • great difficulty was encountered in attempts to isolate plasmid DNA from either clone, an obstacle attributed to constitutive levels of expression of DNase from the clones, so that these authors were unable to determine the sequence for the DNase-encoding nucleic acid.
  • DNase finds a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscosity of pulmonary secretions in such diseases as pneumonia, cystic fibrosis, thereby aiding in the clearing of respiratory airways. Obstruction of airways by secretions can cause respiratory distress, and in some cases, can lead to respiratory failure and death.
  • a novel polypeptide which is a DNase as well as analogs and derivatives thereof.
  • the DNase of the present invention is of human origin.
  • RNA a polynucleotide which encodes such polypeptide.
  • FIG. 1 depicts the a ino acid and DNA sequence of the human DNase of the present invention.
  • the amino acids are represented by their standard one letter abbreviations.
  • the protein shown is the preprocessed form of the protein.
  • FIG. 2 illustrates the amount of DNase released from the pelletted fraction of a bacterial expression system as opposed to the soluble fraction at different time intervals.
  • FIG. 3 shows the rate of success of purification of DNase in different mediums and at different pHs.
  • FIG. 4 demonstrates the tissue distribution of DNase through the use of a Northern blot analysis, indicating that DNase is prevalent in the lungs and heart.
  • FIG. 5 depicts the ability of DNase to digest double- stranded DNA.
  • FIG. 6 demonstrates DNase activity. DNA was labelled with 3 P-dCTP. DNase is then incubated with a fixed amount of labeled DNA and samples were removed and counted by liquid scintillation.
  • FIG. 7 is a schematic representation of the pQE-9 vector..
  • RNA sequence as set forth in Figure 1 of the drawings and/or DNA (RNA) sequences encoding the same polypeptide as the sequence of Figure 1 of the drawings, as well as fragment portions, derivatives, analogs and all allelic variants of such sequences.
  • the polypeptide of Figure 1 is the preprocessed form of the protein and has a leader sequence. The processed or mature form begins at amino acid 19.
  • a polynucleotide which encodes the same polypeptide as the polynucleotide of the cDNA clone deposited as ATCC deposit number 75515, deposited on August 4, 1993, and/or fragments, analogs, derivatives or allelic variants of such polynucleotide.
  • the polynucleotide (DNA or RNA, preferably DNA) includes at least the portion coding for the polypeptide, which coding portion may be the same as that in the deposited clone or may be different than that in the deposited clone provided that it encodes for the same polypeptide or an allelic variant thereof.
  • the coding portion preferably encodes at least the mature form of the polypeptide of the present invention.
  • the present invention further relates to polynucleotide sequences which hybridize to the hereinabove-described polynucleotide sequences if there is at least 50% and preferably at least 70% identity between the sequences.
  • the present invention relates to polynucleotide sequences which hybridize under stringent conditions to the hereinabove-described polynucleotide sequences.
  • stringent conditions means hybridization will occurs if there is at least 95 % and preferably at least 97 % identity between the segments.
  • the present invention includes DNA (RNA) sequences encoding allelic variant forms of the peptide encoded by the DNA of Figure 1.
  • the present invention provides isolated DNA (RNA) encoding for a naturally occurring human polypeptide which is a DNase, as well as allelic variants thereof.
  • the DNA (RNA) is preferably provided in a purified and isolated form.
  • the present invention further relates to a polypeptide which is a DNase, and which, has the structure shown in Figure 1, as well as allelic variants thereof, and analogs, fragments, derivatives and allelic variants thereof and which have the same function as the naturally occurring polypeptide.
  • the present invention further relates to a polypeptide encoded by the DNA contained in the clone deposited as ATCC number 75515 on August 4, 1993, as well as analogs, fragments, derivatives and allelic variants thereof.
  • polypeptide of the present invention is preferably provided in an isolated form, and preferably is purified.
  • 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.
  • the DNase is a full length mature human DNase or an allelic or glycosylated variant thereof.
  • the polynucleotide may also encode a preprotein which is processed and secreted from mammalian cells as the mature protein.
  • the polynucleotide sequence of the present invention may encode for the mature form of the polypeptide or may encode for a leader sequence.
  • the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in the expression and secretion of the polypeptide, for example, a leader sequence which acts as a secretory sequence for controlling transportation of the polypeptide from the cell of the host.
  • the protein 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 protein.
  • the polynucleotide of the present invention may also be fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention, for example, a hexa-histidine tag.
  • polypeptide(s) of the present invention may be the mature form of the DNase of the present invention; or may be in the form of a preprotein or prepolypeptide wherein the DNase includes a leader or secretory sequence; or may be in the form of a fusion protein wherein additional amino acids which aid in, for example, purification of the polypeptide are fused to the mature or preprotein at either the 3' or 5' end thereof.
  • the marker sequence is a hexa-histidine tag, which is supplied by the plasmid pQE-9.
  • the present invention also includes variants of the polypeptide which is encoded by the DNA of Figure 1 or and variants of the DNA contained in the deposited clone, which retains the qualitative activity of such a polypeptide which is a DNase.
  • the variant may be a substitutional variant, or an insertion variant or a deletional variant.
  • Such variants can be naturally occurring allelic variants such as for example, those with different glycosylation patterns or substitution at the amino acid level or deletion at the amino acid level.
  • Such variants may also be produced by site specific mutagenesis.
  • the substitutional variant may be a substituted conserved amino acid or a substituted non-conserved amino acid, and preferably a conserved amino acid.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained from one or more libraries prepared from one of the following tissues: heart, lung, infant brain and placenta. It contains an open reading frame of 298 amino acids encoding for a preprocessed form which exhibit some homology to both human and non-human pancreatic DNase. Among the top matches are: 34% identity and 53% similarity to the human pancreatic DNase over a stretch of 282 amino acids. The coding sequence also contains regions of up to 60% identity and 75% similarity to DNase I from a pig and an overall similarity of 54%. The predicted molecular weight based on amino acid composition is 33kDa.
  • Host cells are transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying the DNase gene.
  • 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.
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.
  • the method used herein for transformation of the host cells is the method of Graham, F. and dan der Eb, A., Virolocry 52:456-457 (1973).
  • other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used.
  • the preferred method of transfection is calcium treatment using calcium chloride as described by Cohen, F.N. et al., Proc. Natl. Acad. Sci. (USA), 69:2110 (1972).
  • Transfection refers to the introduction of DNA into a host cell whether or not any coding sequences are ultimately
  • SUBST1TUTE SHEET (RULE 26) expressed.
  • Cells do not naturally take up DNA.
  • a variety of technical "tricks" have been utilized to facilitate gene transfer. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 4 and electroporation. Transformation of the host cell is the indicia of successful transfection.
  • 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 vectors or plasmids for expressing a polypeptide.
  • vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA'S; yeast plasmids; vectors derived from combinations of plasmids and phage DNAS, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • 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 site(s) 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 PL 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 hereinabove 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.
  • E. coli e.g., E. coli is shown in Figure 2 and Figure 3, lane 5.
  • appropriate hosts there may be mentioned: bacterial cells, such as E. coli. Salmonella typhimurium; fungal cells, such as yeast; animal cells such as Cos-7 cells, CHO or Bowes melanoma; 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 herein.
  • the DNase of the present invention was purified from an E. coli host through the use of a nickel chelate resin that has a high affinity for the hexa-histidine tag.
  • 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 pQE-9 (Qiagen) , pBs, phagescript, pDIO, PsiXI74, pbluescript SK, pBsKS, PNH8A, PNE16A, PNH18A, PNH46A (Stratagene) ; Ptrc99a_, PKK223-3, PKK233-3, PDR540, PRIT5 (Pharmacia).
  • Eukaryotic pWLneo, PSV2CAT, POG44, PXTI, pSG (Stratagene) PSVK3, PBPV, PMSG, PSVL (Pharmacia) .
  • any other plasmids and vectors 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 acetyl transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lacl, lacz, T3, T7, gpt, lambda p B and trc.
  • 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 cellj 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 coded by the recombinant sequence.
  • the encoded polypeptide 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, the 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) , of-factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • 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.
  • 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) .
  • Such commercial vectors include, for example, PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) . These 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.
  • DNase is recovered and purified from recombinant cell cultures by methods used theretofore with human, bovine, ovine, or porcine DNase, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation or exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using DNA or nucleotides on a solid support) , hydroxylapatite chromatography and lectin chromatography. Moreover, reverse-phase HPLC and chromatography using anti-DNase antibodies are useful for the purification of DNase. As noted previously (Price, et al., J. Biol. Chem.
  • DNase may be purified in the presence of a protease inhibitor such as PMSF.
  • 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.
  • Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more salting-outs, aqueous ion exchange or size exclusion chromatography steps. 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. 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.
  • 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) of a polynucleotide sequence of the present S 4/04954 invention.
  • 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 (at position 1) .
  • allelic forms of the polypeptide also embraces analogs and fragments thereof.
  • one or more of the amino acid residues of the polypeptide may be replaced by conserved amino acid residues.
  • Purified DNase of the present invention may be employed to enzymatically reduce the viscosity of mucus in the manner as described above. Based on this activity it would be of particular use for the treatment of patients with pulmonary disease who have abnormal or viscous or inspissated purulent secretions in conditions such as acute or chronic bronchial pulmonary disease (infectious pneumonia, bronchitis, or tracheobronchitis, bronchiectasis, cystic fibrosis, asthma, TB, or fungal infections) , atelectasis due to tracheal or bronchial impaction and complications due to tracheostomy.
  • a solution or finely divided dry preparation of human DNase is instilled in conventional fashion into the bronchi, e.g., by aerosolization of a solution of DNase.
  • DNase would have adjunctive treatment for the management of abscesses of closed spaced infections, emphysema, meningitis, peritonitis, sinusitis, otitis, periodontitis, pancreatitis, cholelithiasis, endocroditis, and septic arthritis, as well as in topical treatments in a variety of inflammatory and infected lesions, such as infected lesions of the skin and/or mucosal membranes, surgical wounds, ulcerative lesions and burns.
  • Human DNase finds utility in maintaining the flow in medical conduits communicating with a body cavity, including surgical drainage tubes, urinary catheters, peritoneal dialysis ports, and intratracheal oxygen catheters.
  • the DNase of the present invention may also improve the efficacy of antibiotics in infections.
  • the DNase may also be useful in degrading DNA contaminants in pharmaceutical preparations.
  • DNase may be useful in treating non- infected conditions in which there is an accumulation of cellular debris, including cellular DNA.
  • DNase will be useful after systemic administration in the treatment of pyelonephritis and tubulo-interstitial kidney disease.
  • polypeptide of the present invention 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 such as lung and heart cells may be transduced with a polynucleotide (DNA or RNA) encoding the polypeptide ex vivo, with the transduced cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cells may be transduced by procedures known in the art by use of a retroviral particle containing RNA encoding the polypeptide of the present invention.
  • tran ⁇ duction of cells may be accomplished 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 transduction in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for transducing cells may be other than a retroviral particle, for example, an adenovirus.
  • a polynucleotide encoding a polypeptide of the present invention is ligated into an adenoviral vector.
  • the adenoviral vector can then be packaged in a delivery vehicle, e.g., an aerosol spray, and administered to the patient by spraying through the nasal or oral cavity into the lungs.
  • Cells are then transfected in vivo with the DNA sequence encoding for the polypeptide of the present invention to produce DNase.
  • the DNase degrades the excessive secretions of mucous in the lungs, such as in pneumonia, and facilitates respiration.
  • compositions comprise a therapeutically effective amount of the protein, and a pharmaceutically acceptable carrier or excipient.
  • a 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 human DNase of the present invention is placed into therapeutic formulations, together with required co-factors.
  • the formulation of DNase may be liquid, lyophilized powder or the DNase may be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
  • the DNase of the present invention When used as a drug, as described above, it is given, for example, in appropriate doses of at least about 10 ⁇ g/kg body weight and in most cases it would be administered in an amount not in excess of about 8 mg/kg body weight per day, for example, in doses of about 2.5 mg/person/day, taking into account the routes of administration, symptoms, etc.
  • 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 polypeptide of the present invention may be employed in conjunction with other therapeutic compounds.
  • Each of the cDNA sequences identified herein or a portion thereof can be used in numerous ways as polynucleotide reagents.
  • the sequences can be used as diagnostic probes for the presence of a specific mRNA in a particular cell type.
  • these sequences can be used as diagnostic probes suitable for use in genetic linkage analysis (polymorphisms) .
  • 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 polymorphisms) are presently available for marking chromosomal location.
  • the mapping of cDNAs 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 cDNA 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 hybridization, pre ⁇ creening with labeled flowsorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clone from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • Verma et al. Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
  • 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 I megaba ⁇ e mapping resolution and one gene per 20 kb) .
  • Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the protein, its 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, monoclonal, chimeric, single chain, Fab fragments, or the product of an Fab expression library.
  • Various procedures known in the art may be used for the production of polyclonal antibodies.
  • Antibodies generated against the polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide. Moreover, a panel of such antibodies, specific to a large number of polypeptides, can be used to identify and differentiate such tissue.
  • 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 (Kohler 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).
  • the antibodies can be used in methods relating to the localization and activity of the protein sequences of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples and the like.
  • 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. In addition, 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.
  • plasmid or DNA fragment typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • 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 l hour or longer at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on an agarose gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using a 0.8- 2.0 percent polyagarose gel. (Maniatis)
  • 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 to T4 ⁇ DNA liga ⁇ e ("ligase") per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase DNA liga ⁇ e
  • the DNA sequence encoding for human DNase, pBLSKDNase (ATCC # 75515) is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end of the DNA sequence to synthe ⁇ ize in ⁇ ertion fragment .
  • the 5' oligonucleotide primer has the sequence GACGCCGGATCCC-ACTACCCAACTGCA, contains a BamHI restriction enzyme site followed by 15 nucleotides of DNase coding ⁇ equence following the initiation codon; the 3' sequence 5'- G G C T G C T C T A G A C A G C G T A G T C T G G G C A - GGTCGTATGGGTACTTCAGCTCCACCTCCACGGGGTAG-3 ' contains complementary sequences to Xbal site and is in the 3' untranslated region of the gene.
  • the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc.
  • the plasmid vector 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-histidine tag (6- His) and restriction enzyme cloning sites.
  • the pQE-9 vector was digested with BamHI and Xbal and the insertion fragment ⁇ were then ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS.
  • Figure 7 show ⁇ a schematic representation of this arrangement. The ligation mixture wa ⁇ then used to transform the E.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resi ⁇ tance (Kan r ) .
  • Tran ⁇ formant ⁇ are identified by their ability to grow on LB plates containing both Amp and Kan.
  • Clones containing the desired construct ⁇ were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ⁇ g/ml) and Kan (25 ⁇ g/ml) .
  • the O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells were grown to an optical density at 600nm (O.D. 600 ) 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 were grown an extra 2-4 hours. Cells were then harvested by centrifugation.
  • Expression of DNase was tested by solubilizing a portion of the E. coli and analyzing on a SDS polyacrylamide gel. The presence of a new protein corresponding to 33 kd following induction demonstrated expression of the DNase. ( Figure 2) .
  • step dialysis is utilized to remove the GnHCl.
  • the purified protein isolate from the Ni-chelate column can be bound to a second column over which a decreasing linear GnHCl gradient is run. The protein is allowed to renature while bound to the column and is ⁇ ub ⁇ equently eluted with GnHCl, pH 5.0.
  • ⁇ oluble protein i ⁇ dialyzed against a storage buffer containing 140mM NaCl, 20mM NaP0 4 and 10% w/v Glyconol. The purified protein was analyzed by SDS-PAGE. ( Figure 3) .
  • Example 2 Expression of DNase in COS Cells
  • SoCMVIN/Amp containing: 1) SV40 origin of replication, ⁇ globin intron, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenalytion site.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (MYPYDVPDYA) as previously described (I. Wilson, et al. Cell. 37:767 (1984).
  • MYPYDVPDYA influenza hemagglutinin protein
  • the DNA insert was con ⁇ tructed by PCR on the original EST u ⁇ ing two primer ⁇ : the 5' primer GACGCCGGATCCCACTACCCAACTGCA contain ⁇ a BamHI ⁇ ite followed by 15 nucleotide ⁇ of DNase coding ⁇ equence starting from the initiation codon; the 3' sequence GGCTGCTCTAGACAGCGTAGTCTGGCAGGTCGTATGGG- TACTTCAGCTCCACCTCCACGGGGTAG contains complementary ⁇ equences to the XBal site, translation stop codon, HA tag and the 23 nucleotides of the DNase coding sequence (not including the stop codon) that correspond to pancreatic DNase terminal re ⁇ idues.
  • the PCR product contains a BamHI site, DNase coding sequence, HA tag fused in frame, followed by a translation termination stop codon next to the HA tag, and a Xbal ⁇ ite.
  • the PCR amplified DNA fragment and the vector, SoCMVIN/amp were digested with BamHI and Xbal restriction enzyme and ligated.
  • the ligation mixture was tran ⁇ formed into E. coli ⁇ train available under the trademark SURE.
  • SUBSTITUTE Sh ⁇ ET (RULE 26) re ⁇ istant colonies were selected. Plasmid DNA was isolated from transformant ⁇ and examined by re ⁇ triction analy ⁇ i ⁇ for the presence of the correct fragment.
  • COS cells were transfected with the expres ⁇ ion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual. Cold Spring Laboratory Press, 1989.) The expres ⁇ ion of the DNa ⁇ e-HA protein wa ⁇ detected by radiolabelling and immunoprecipitation method.
  • RNAzol Total cellular RNA samples were isolated with RNAzol" ' B system (Biotecx Laboratories, Inc., 6023 South Loop East, Hou ⁇ ton, TX 77033.) About lOug of total RNA i ⁇ olated from each human ti ⁇ ue ⁇ pecified wa ⁇ separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, Frit ⁇ ch, and Maniati ⁇ , Molecular Cloning. Cold Spring Harbor Pre ⁇ , (1989)) . The labeling reaction wa ⁇ done according to the Stratagen Prime-It kit with 50 ng of the Eco Rl-Xhol fragment of pBL ⁇ le DNa ⁇ e DNA fragment.
  • the labeled DNA wa ⁇ purified with a Seclect-G-50 column. (5 Prime -- 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303) .
  • the filter wa ⁇ then hybridized with radioactive labeled full length thrombin inhibitor gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 and 7% SDS overnight at 65°C. After wash twice at room temperature and twice at 60°C with 0.5 x SSC, 0.1% SDS, the filters were then expo ⁇ ed at -70 ⁇ C overnight with inten ⁇ ifying ⁇ creen. ( Figure 4) .
  • the first assay measures the hydrolysi ⁇ of 32 P-labeled DNA.
  • Degradation of 32 P-DNA was prepared using a Random Prime Labeling Kit (Strategene' ⁇ Prime It II) u ⁇ ing 32 P-dCTP, non- radioactive dGTP, dTTP, dATP and Exo (-) Klenow Enzyme.
  • 1 ul of template (25 ng) , lOul of random oligonucleotide primers and 23 ul of water were mixed and incubated at 100'C for 5 minutes.
  • 10 ul of 5X dCTP primer buffer, 5 ul of - 32 PdCTP (3.000 Ci/mMole) and 1 ul Exo (-) Klenow Enzyme (5U/ul) were added to the reaction mixture and incubated at 37°C for 15 minutes.
  • the free nucleotides were separated from the radiolabled DNA by centrifuga ion through a Sephadex G-50 column.
  • DNase activity was then measured by incubating 5 X 10 5 cpm 32 P-DNA and 20 ul of protein in DNase buffer (40mM TRIS- HC1 pH 7.6, 50mM NaCL, 0.ImM DTT, 6mM MgCl 2 , ImM CaCl 2 ) in a total volume of lOOul. 20ul were removed for time point 0 and for each sub ⁇ equent time point.
  • DNase buffer 40mM TRIS- HC1 pH 7.6, 50mM NaCL, 0.ImM DTT, 6mM MgCl 2 , ImM CaCl 2
  • a ⁇ econd assay takes advantage of the ability of DNase to nick and digest double-stranded DNA.
  • Supercoiled plasmid DNA i ⁇ incubated with DNa ⁇ e and ⁇ ample ⁇ are removed at time point ⁇ following addition of the DNa ⁇ e.
  • the ⁇ ample ⁇ are electrophore ⁇ ed on agarose gel ⁇ and the conver ⁇ ion from supercoiled to relaxed DNA is ob ⁇ erved following addition of the DNase. ( Figure 5) .
  • a rapid plate DNase assay can also be used in which test agar contains both DNA and methyl green. Purified protein or supernatant ⁇ from tran ⁇ fected cell ⁇ are ⁇ potted on the plate ⁇ along with a standard amount of bovine DNa ⁇ e. DNa ⁇ e activity is measured by vi ⁇ ualizing the ⁇ ize of the cleared zone on the plate.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • AGCCATGCAC TACCCAAC G CACTCCTCTT CCTCATCCTG GCCAATGGGA CCCAGACCTT 60
  • GGCCAGCACC CACTGCACCT ATGACCGCGT CGTGCTGCAC GGGGAGCGCT GCCGGAGTCT 720
  • CCAGCCTCCC CCGTCCATCC AGCCCTGGGG CTGGGGGGCT TCAACTATAG TTGCCCTGTG 1020

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Abstract

L'invention concerne un polypeptide de déoxyribonucléase chez l'homme, ainsi que l'ADN (ARN) codant ledit polypeptide. On peut obtenir ce polypeptide de déoxyribonucléase (DNase) au moyen de techniques d'ADN recombinant et il présente une efficacité dans le traitement de différentes maladies où on l'administre avec un véhicule pharmaceutique approprié ou un diluant, afin de générer des effets thérapeutiques contre lesdites maladies.
EP94916012A 1994-05-05 1994-05-05 Deoxyribonuclease chez l'homme Withdrawn EP0817839A1 (fr)

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PCT/US1994/004954 WO1995030428A1 (fr) 1994-05-05 1994-05-05 Deoxyribonuclease chez l'homme

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US6265195B1 (en) * 1996-04-25 2001-07-24 Genentech, Inc. Human DNase II
CZ20023548A3 (cs) * 2000-04-27 2003-05-14 Smithkline Beecham Corporation Nové sloučeniny
GB201101794D0 (en) 2011-02-02 2011-03-16 Fermentas Uab Protein production
EP3214172B1 (fr) * 2014-10-31 2019-02-13 JCR Pharmaceuticals CO., LTD. Procédé pour la production de dnase i humaine recombinée

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Publication number Priority date Publication date Assignee Title
WO1993025670A1 (fr) * 1992-06-08 1993-12-23 Genentech, Inc. Formes purifiees de desoxyribonuclease
DE19521046C1 (de) * 1995-06-09 1996-08-08 Deutsches Krebsforsch Protein mit DNase-Aktivität

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DE68929551T2 (de) * 1988-12-23 2008-03-06 Genentech, Inc., South San Francisco Menschliche DNase

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025670A1 (fr) * 1992-06-08 1993-12-23 Genentech, Inc. Formes purifiees de desoxyribonuclease
DE19521046C1 (de) * 1995-06-09 1996-08-08 Deutsches Krebsforsch Protein mit DNase-Aktivität

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Title
See also references of WO9530428A1 *

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