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Ep 1608732 B1 (19) & (11) EP 1 608 732 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/55 (2006.01) 12.05.2010 Bulletin 2010/19 (86) International application number: (21) Application number: 04749602.1 PCT/US2004/009955 (22) Date of filing: 31.03.2004 (87) International publication number: WO 2004/090114 (21.10.2004 Gazette 2004/43) (54) COMPOSITIONS AND METHODS OF USING A SYNTHETIC DNASE I ZUSAMMENSETZUNGEN UND VERFAHREN ZUR VERWENDUNG EINER SYNTHETISCHEN DNASE I COMPOSITIONS ET PROCEDES POUR UTILISER UNE DNASE I DE SYNTHESE (84) Designated Contracting States: (74) Representative: Muir, Benjamin M. J. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Beck Greener HU IE IT LI LU MC NL PL PT RO SE SI SK TR Fulwood House 12 Fulwood Place (30) Priority: 31.03.2003 US 404023 London WC1V 6HR (GB) 22.04.2003 US 420345 (56) References cited: (43) Date of publication of application: WO-A-94/15644 28.12.2005 Bulletin 2005/52 • OEFNER C ET AL: "CRYSTALLOGRAPHIC (60) Divisional application: REFINEMENT AND STRUCTURE OF DNASE I AT 10153371.9 2 ANGSTROM RESOLUTION" JOURNAL OF MOLECULAR BIOLOGY, vol. 192, no. 3, 1986, (73) Proprietor: Ambion, Inc. pages 605-632, XP008078567 ISSN: 0022-2836 Austin, TX 78744-1832 (US) • DATABASE EMBL [Online] 2 November 2000 (2000-11-02), "Bos taurus epithelial lens (72) Inventors: deoxyribonuclease I mRNA, partial cds." • LATHAM, Gary XP002432551 retrieved from EBI accession no. Austin, TX 78748 (US) EMBL:AF311922 Database accession no. • KEMPPAINEN, Jon AF311922 Austin, TX 78733 (US) • LIAO T-H ET AL: ’Bovine pancreatic deoxyribonuclease A. Isolation of cyanogen bromidepeptides; complete covalent structure of the polypeptide chain’ J. BIOL. CHEM. vol. 248, no. 4, 1973, pages 1489 - 1495, XP002040574 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 608 732 B1 Printed by Jouve, 75001 PARIS (FR) EP 1 608 732 B1 Description [0001] The present invention relates in general to the field of polydeoxyribonucleic acid hydrolases, and more partic- ularly, to nucleic acids encoding and methods of making a synthetic bovine DNase I. 5 BACKGROUND OF THE INVENTION [0002] This invention was made with government support under R44CA69967 awarded by the National Cancer Insti- tute. The government may own certain rights in the invention. Without limiting the scope of the invention, its background 10 is described in connection with polydeoxyribonucleic acid hydrolases, as an example. [0003] Deoxyribonuclease (DNase) is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid into individual 3’ or 5’-phosphate deoxynucleotides on hydrolysis of deoxyribonucleic acid (DNA). Based on their biochemical properties and enzymatic activities, DNase proteins have been classified as two types, DNase I and DNase II. DNase I proteins have a pH optimum near neutral and an obligatory requirement for divalent cations, and create 5’-phosphate 15 deoxynucleotide products. DNase II has an acid pH optimum, can be activated by divalent cations, and produces 3’- phosphate deoxynucleotides on hydrolysis of DNA. [0004] The nucleic acid encoding a human DNase I has been isolated, sequenced and the protein expressed in recombinant host cells, thereby enabling the production of human DNase I in commercially useful quantities. The DNA encoding other polypeptides having homology to human DNase I have also been identified. 20 [0005] Human DNase I has recently been used to reduce the viscoelasticity of pulmonary secretions (mucus) in such diseases as pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of respiratory airways. One such phar- maceutical composition is described by United States Patent No. 6,440,412, issued to Frenz, et al., for purified forms of DNase I in which the DNase is provided in a formulation for use in administering to patients suffering from pulmonary distress. 25 [0006] One such formulation is sold under the tradename Pulmozyme® (dornase alfa, Genentech, USA), in which the recombinant human deoxyribonuclease I (rhDNase) is provided to a patient in an inhaled solution that is sterile, clear, colorless, and contains a highly purified solution of rhDNase. The characteristics of the rhDNase were selected to attack pulmonary secretions of persons having such diseases are complex materials, which include mucus glycoproteins, mucopolysaccharides, proteases, actin and DNA. rhDNase I was found to be effective in reducing the viscoelasticity of 30 pulmonary secretions by hydrolyzing, or degrading, high-molecular-weight DNA that is present in such secretions. [0007] A hyperactive rhDNase is described by United States Patent No. 6,391,607, issued to Lazarus, et al. , for human DNase I hyperactive variants, in which amino acid sequence variants of human DNase I that have increased DNA- hydrolytic activity are disclosed. The patent disclosure describes nucleic acid sequences encoding such hyperactive variants, thereby enabling the production of these variants in quantities sufficient for clinical use. The invention also 35 relates to pharmaceutical compositions and therapeutic uses of hyperactive variants of human DNase I. SUMMARY OF THE INVENTION [0008] According to a first aspect, the present invention provides an isolated nucleic acid comprising a nucleotide 40 sequence that encodes an amino acid sequence of SEQ ID NO.: 2 for synthetic bovine DNase I (sbDNase I) or an amino acid sequence of SEQ ID NO:2 having E13R and/or N74K mutations. [0009] In one preferred embodiment, the DNase I comprises a synthetic bovine DNase I comprising an E13R and N74K mutation. In another preferred embodiment, the nucleic acid comprises SEQ ID No.: 1. [0010] The nucleic acid may further comprise a nucleic acid segment encoding a leader sequence, for example a pelB 45 leader sequence or an alpha mating factor leader sequence. [0011] The nucleic acid may be operably linked to a promoter recognized by a host cell transformed with a vector. Thus, the invention according to the first aspect also encompasses an expression vector that includes said nucleotide sequence operably linked to a promoter recognized by a host cell transformed with the vector. The host cell may be a yeast cell, e.g. Pichia pastoris. 50 [0012] According to a second aspect, the present invention provides a host cell transformed with a nucleic acid according to the first aspect. The host cell may be a yeast cell, e.g. Pichia pastoris. [0013] According to a third aspect, the present invention provides a process for making a protein with DNase activity comprising the steps of: transforming a host cell with a nucleic acid according the first aspect; and culturing the host cell under conditions such that the recombinant DNase protein is produced. The host cell may be a yeast cell, e.g.Pichia 55 pastoris. The yeast cell may produce at least 1 mg/l protein with DNase activity, at least 1 mg/l DNase I protein, or at least 7.5 mg/l bovine DNase I protein. [0014] The sbDNase I encoded by the nucleic acids of the first aspect is a versatile enzyme that eleaves DNA non- specifically to release 5’-phosphorylated di-, tri-, and oligonucleotide products. The sbDNase I may be used for a wide 2 EP 1 608 732 B1 range of molecular biology applications, including: degradation of contaminating DNA after RNA isolation; "clean- up" of RNA prior to RT-PCR and after in vitro transcription; removal of DNA prior to protein sample loading onD 2-gels; identification of protein binding sequences on DNA (DNase I footprinting); prevention of clumping when handling cultured cells; and creation of a fragmented library of DNA sequences for in vitro recombination reactions. 5 BRIEF DESCRIPTION OF THE DRAWINGS [0015] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures in which corresponding numerals 10 in the different figures refer to corresponding parts and in which: Fig. 1 shows the nucleotide coding sequence of a synthetic bovine DNase I gene of the present invention (SEQ ID NO.: 1); Fig. 2 shows another nucleotide coding sequence for a synthetic bovine DNase I gene of the present invention (SEQ 15 ID NO.: 3); Fig. 3A is the sequence of the pPicZαA_sbDNase I, 4315 bp that includes the Xho I and Not I restriction sites that were used to insert the sbDNase I gene are shown underlined (SEQ ID NO.:19); Fig. 3B is the amino acid sequence for a recombinant DNase I, optimized for expression and including the alpha mating factor leader sequence and mature protein (SEQ ID NO.: 20); 20 Fig. 4 is an alignment of the naturally-occurring bovine DNase I coding sequence (SEQ ID NO.: 21) with a codon- optimized synthetic bovine DNase I gene sequence of the present invention (SEQ ID NO.: 1); Fig. 5 is a graph that shows a >20- fold increase in DNA cleaving activity when E. coli culture fluid is compared before and after sbDNase I induction; Figs. 6A and 6B are graphs that compare the Km of E13R;N74K sbDNase I, against wild-type DNase I; 25 Fig. 7 is a three dimensional graph that shows the interrelationship between Ca 2+ and monovalent salt in modulating E13R;N74K sbDNase I activity; Fig. 8 is a graph that compares the salt tolerance of E13R, N74K, E13R; N74K, and wild-type DNase I in DNaseAlert Buffer; and Fig. 9 is a graph that demonstrates the residual DNA contamination remaining as a function of time using E13R; 30 N74K DNase I, a 1:1 blend of E13R;N74K DNase I and wild-type DNase I alone.
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