Deoxyribonuclease, gene encoding same and use thereof Number:7,049,123 from the United States Patent and Trademark Office (PTO) owispatent This invention provides a novel acid DNase (DLAD) which is an endonuclease capable of cleaving DNA independently from divalent cations, under acidic conditions, which retains its activity in acidic to even neutral pH range, and which is not inhibited by G-actin. This invention also provides a DNA encoding the enzyme, an expression vector containing the DNA, and a host cell transformed with the expression vector. Furthermore, a pharmaceutical composition containing DLAD, DLAD expression vector or a host cell transformed with the expression vector as an active ingredient is provided. The pharmaceutical composition is useful as a therapeutic agent replacing DNase I for cystic fibrosis, and can provide a new approach for the prophylaxis and treatment of infectious diseases.<H Deoxyribonuclease, encoding, Deoxyribonuclea, 7049123.html, Patent, pto, 7049123

Title: Deoxyribonuclease, gene encoding same and use thereof

Abstract: This invention provides a novel acid DNase (DLAD) which is an endonuclease capable of cleaving DNA independently from divalent cations, under acidic conditions, which retains its activity in acidic to even neutral pH range, and which is not inhibited by G-actin. This invention also provides a DNA encoding the enzyme, an expression vector containing the DNA, and a host cell transformed with the expression vector. Furthermore, a pharmaceutical composition containing DLAD, DLAD expression vector or a host cell transformed with the expression vector as an active ingredient is provided. The pharmaceutical composition is useful as a therapeutic agent replacing DNase I for cystic fibrosis, and can provide a new approach for the prophylaxis and treatment of infectious diseases.

Patent Number: 7,049,123 Issued on 05/23/2006 to Tanuma, et al.

Inventors: Tanuma; Sei-ichi (Hachioji, JP); Shiokawa; Daisuke (Tokyo, JP)
Assignee: Sei-ichi Tanuma (Hachioji, JP)

Appl. No.: 670863

Filed: September 25, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09807784		6653118
PCT/JP00/02893	May., 2000

Foreign Application Priority Data


Aug 17, 1999 [JP]			11-230870

Current U.S. Class: 435/199 ; 435/252.3; 435/320.1; 536/23.2

Current International Class: C12N 9/22 (20060101); C12N 15/55 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


5821103	October 1998	Tanuma
6358723	March 2002	Eastman et al.

Foreign Patent Documents


0 853 121	Aug., 1998	EP
WO 97/28266	Aug., 1997	WO
WO 97/40134	Oct., 1997	WO

Other References

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Barry et al., "Identification of Deoxyribonuclease II as an Endonuclease Involved in Apoptosis," Archives of Biochemistry and Biophysics, 300 (1), 440-450, Academic Press, Inc. (Jan. 1993). cited by other .
Yasuda et al., "Human Urine Deoxyribonuclease II (DNase II) Isoenzymes: A Novel Immunoaffinity Purification, Biochemical Multiplicity, Genetic Heterogeneity and Broad Distribution Among Tissues and Body Fluids," Biochimica et Biophysica Acta, 1119, 185-193 (1992). cited by other .
Yasuda et al., "Molecular Cloning of the cDNA Encoding Human Deoxyribonuclease II," The Journal of Biological Chemistry, 273 (5), 2610-2616 (Jan. 30, 1998). cited by other .
Liao, "The Subunit Structure and Active Site Sequence of Porcine Spleen Deoxyribonuclease," The Journal of Biological Chemistry, 260 (19), 10708-10713, (Sep. 5, 1985). cited by other .
Shiokawa et al., "Cloning of cDNAs Encoding Porcine and Human DNase II," Biochemical and Biophysical Research Communication, 247, 864-869 (Article No. RC988839) (1998). cited by other .
Lesca, "Protein Inhibitor of Acid Deoxyribonucleases," The Journal of Biological Chemistry, 251 (1), 116-123 (Jan. 10, 1976). cited by other .
Baker et al., "Molecular Cloning and Characterization of Human and Murine DNase II," Gene, 215, 281-289 (1998). cited by other .
Dulaney et al., "Isolation of Deoxyribonuclease II of Rat Liver Lysosomes," The Journal of Biological Chemistry, 247 (5), 1424-1432 (Mar. 10, 1972). cited by other .
Harosh et al., "Mechanism of Action of Deoxyribonuclease II from Human Lymphoblasts," Eur. J. Biochem., 202, 479-484 (1991). cited by other .
Liao et al., "Deoxyribonuclease II Purified from the Isolated Lysosomes of Porcine Spleen and from Porcine Liver Homogenates. Comparison with Deoxyribonuclease II Purified from Porcine Spleen Homogenates," Biochimica et Biophysica Acta, 1007, 15-22 (1989). cited by other .
Slor, "Purification of .sup.14C-Labelled Deoxyribonuclease II from HeLa S3 Lysosomes and its Use as a Marker for the Study of Nuclear Deoxyribonuclease II," Biochem. J., 136, 83-87 (1973). cited by other .
Wagner et al., "Molecular Strategies for Therapy of Cystic Fibrosis," Annu. Rev. Pharmacol. Toxicol., 35, 257-276 (1995). cited by other .
Shak, "Aerosolized Recombinant Human DNase l for the Treatment of Cystic Fibrosis," Chest, 107 (2), 65-70 (Feb. 1995). cited by other .
Ulmer et al., "Engineering Actin-Resistant Human DNase l for Treatment of Cystic Fibrosis," Proc. Natl. Acad. Sci. USA, 93, 8225-8229 (Aug. 1996). cited by other .
Pan et al., "Improved Potency of Hyperactive and Actin-Resistant Human DNase I Variants for Treatment of Cystic Fibrosis and Systemic Lupus Erythematosus," The Journal of Biological Chemistry, 273 (29), 18374-18381 (Jul. 17, 1998). cited by other .
Laidlaw et al., "Fowlpox Virus Encodes Nonessential Homologs of Cellular Alpha-SNAP, PC-1, and Orphan Human Homolog of a Secreted Nematode Protein," Journal of Virology, 72 (8), 6742-6751 (Aug. 1998). cited by other .
Baron et al., "Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages," Gene, 215 (2), 291-301 (1998). cited by other .
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Primary Examiner: Patterson, Jr.; Charles L.
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.

Claims

What is claimed is:

1. An isolated DNA consisting of a nucleotide sequence of nucleotide Nos. 279 to 1274 of SEQ ID NO:2.

2. An isolated DNA consisting of a nucleotide sequence of nucleotide Nos. 213 to 1274 of SEQ ID NO:2.

3. A recombinant vector comprising the DNA of: (i) a nucleotide sequence of nucleotide Nos. 279 to 1274 of SEQ ID NO:2, or (ii) a nucleotide sequence of nucleotide Nos. 213 to 1274 of SEQ ID NO:2.

4. An expression vector comprising (a) the DNA of: (i) a nucleotide sequence of nucleotide Nos. 279 to 1274 of SEQ ID NO:2, or (ii) a nucleotide sequence of nucleotide Nos. 213 to 1274 of SEQ ID NO:2, and (b) a promoter operably linked to said DNA.

5. A transformant obtained by transforming a host cell with the expression vector of claim 4.

6. A method for producing the deoxyribonuclease, which comprises culturing the transformant of claim 5 in a medium and recovering the deoxyribonuclease from a resulting culture.

7. An isolated DNA encoding a deoxyribonuclease comprising: an amino acid sequence of amino acid Nos. 1 to 332 of SEQ ID NO: 1.

8. An expression vector comprising the DNA of claim 7 and a promoter operably linked to said DNA.

9. A transformant obtained by transforming a host cell with the expression vector of claim 8.

10. A method for producing a deoxyribonuclease, which comprises culturing the transformant of claim 9 in a medium and recovering the deoxyribonuclease from a resulting culture.

11. An isolated DNA consisting of a nucleotide sequence that hybridizes to the complement of the nucleotide sequence of nucleotides Nos. 279 to 1274 of SEQ ID NO:2 under highly stringent conditions, which encodes a deoxyribonuclease having an endonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6.

12. An isolated DNA consisting of a nucleotide sequence that hybridizes to the complement of the nucleotide sequence of nucleotides Nos. 213 to 1274 of SEQ ID NO:2 under highly stringent conditions, which encodes a primary translation product of a deoxyribonuclease which in mature protein form has an endonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6.

13. A recombinant vector comprising the DNA of: (i) a nucleotide sequence that hybridizes to the, complement of the nucleotide sequence of nucleotides Nos. 279 to 1274 of SEQ ID NO:2 under highly stringent conditions, which encodes a deoxyribonuclease having an endonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6, or (ii) a nucleotide sequence that hybridizes to the complement of the nucleotide sequence of nucleotides Nos. 213 to 1274 of SEQ ID NO:2 under highly stringent conditions, which encodes a primary translation product of a deoxyribonuclease which in mature protein form has an endonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6.

14. An expression vector comprising (a) the DNA of: (i) a nucleotide sequence that hybridizes to the complement of the nucleotide sequence of nucleotides Nos. 279 to 1274 of SEQ ID NO:2 under highly stringent conditions, which encodes a deoxyribonuclease having an endonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6, or (ii) a nucleotide sequence that hybridizes to the complement of the nucleotide sequence of nucleotides Nos. 213 to 1274 of SEQ ID NO:2 under highly stringent, conditions, which encodes a primary translation product of a deoxyribonuclease which in mature protein form has an endonuclease activity that cleaves DNA, at a pH range of from ca. 4.0 to ca. 7.6, and (b) a promoter operably linked to said DNA.

15. A transformant obtained by transforming a host cell with the expression vector of claim 14.

16. A method for producing the deoxyribonuclease, which comprises culturing the transformant of claim 15 in a medium and recovering the deoxyribonuclease from a resulting culture.

17. An isolated DNA comprising a nucleotide sequence of the nucleotide Nos. 279 to 1274 of SEQ ID NO: 2.

18. An isolated DNA comprising a nucleotide sequence of the nucleotide Nos. 213 to 1274 of SEQ ID NO: 2.

Description

TECHNICAL FIELD

This invention relates to a novel deoxyribonuclease capable of cleaving DNA independently from divalent cations under acidic conditions, a DNA encoding same and use of these for the prophylaxis and treatment of infectious diseases as well as for the treatment of cystic fibrosis.

BACKGROUND ART

The presence of various deoxyribonucleases (hereinafter referred to as DNase) in mammalian cells has been known. DNase II is one of the DNases studied most and catalyzes DNA hydrolysis reaction in the absence of divalent cations at acidic pH [in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 4, pp. 271 287 (1971), Academic Press, New York; Arch. Biochem. Biophys., 300: 440 450 (1993)]. While the acid DNase activities are widely found in various animal tissues [Biochim. Biophys. Acta, 1119: 185 193 (1992); J. Biol. Chem., 273: 2610 2616 (1998)], DNase II has been considered to be the sole enzyme responsible for the acid DNase activities. Because DNase II shows low organ specificity and is distributed ubiquitously, a possibility of DNase II playing an important biological role in the fundamental biological phenomena, such as DNA catabolism and apoptosis, has been suggested [The Enzymes (1971), supra; Arch. Biochem. Biophys., 300: 440 450 (1993)].

Even though the enzymological properties of the DNase II isolated from different organisms are very similar, their physicochemical properties and molecular structures are strikingly different. For example, it is known that porcine DNase II is a complex protein consisting of unidentical subunits derived from its precursor protein, but DNase II derived from other animals are mostly single polypeptides [J. Biol. Chem., 260: 10708 10713 (1985); Biochem. Biophys. Res. Commun., 247: 864 869 (1998); J. Biol. Chem., 251: 116 123 (1976); Gene, 215: 281 289 (1998)]. Furthermore, the apparent molecular weights of DNase II vary from 26.5 kDa to 45 kDa [J. Biol. Chem. (1976), supra; Gene, (1998), supra; J. Biol. Chem., 247: 1424 1432 (1972); Eur. J. Biochem., 202: 479 484 (1991)].

The diversity of acid DNases can be also appreciated from the subcellular localization. DNase II is considered to be localized in lysosomes [J. Biol. Chem. (1972), supra; Biochim. Biophys. Acta, 1007: 15 22 (1989)], but acid DNase activity is also found in nuclear fraction [Arch. Biochem. Biophys. (1993), supra; Biochem. J., 136: 83 87 (1973)].

The reason for such molecular diversity of DNase II still remains unclear, but the aforementioned findings suggest the existence of a different acid DNase distinguishable from DNase II. In fact, the present inventors have identified and partially purified novel acid DNases (DNase .alpha. and DNase .beta.) from the nuclear fraction of rat thymus (JP 8-187079 A). In view of the foregoing situation it is considered to be critical for the elucidation of the diversity of acid DNases to search other novel acid DNases and determine their characteristics.

In addition, DNase has been actively studied with the aim of applying same for the prophylaxis and treatment of various diseases. One of the clinical applications of DNase, which has been drawing particular attention in recent years, is an application to the treatment of cystic fibrosis (hereinafter sometimes to be also referred to as CF) [Annu. Rev. Pharmacol. Toxicol., 35: 257 276 (1995); Chest, 107: 65 70 (1995)]. CF is a lethal hereditary disease caused by abnormal chloride ion channel of exocrine glands. In the Caucasian population, one in 2500 newborns suffers from this disease and one in 25 Caucasians is a carrier. About 90% of the CF patients die of respiratory insufficiency caused by intractable infection with Pseudomonas aeruginosa in the inferior airway in their 20's and 30's [Curr. Opin. Pulm. Med., 6: 425 434 (1995)]. Phlegm that is accumulated in the airway to impair the respiratory function is caused by high concentration DNA released from the disrupted leukocytes infiltrating into the inflammatory site. Genentech, Inc. U.S. is selling a recombinant DNase I as a therapeutic agent for CF in Europe and America, which aims at removing the high molecular weight DNA accumulated in the lung, recovering the respiratory function and preventing infectious diseases [Annu. Rev. Pharmacol. Toxicol. (1995), supra; Chest (1995), supra]. DNase I not only degrades DNA, but also depolymerizes F-actin which is abundant in the sputum of CF patients. However, since the resulting monomeric G-actin strongly inhibits DNase I, DNase I is immediately inactivated. Actually, DNase I hardly shows any therapeutic effect. Some attempts have been made to produce a G-actin nonsensitive DNase I by genetic recombination, but satisfactory DNase has not been obtained yet [Proc. Natl. Acad. Sci. USA, 93: 8225 8229 (1996); J. Biol. Chem., 273: 18374 18381 (1998)]. Thus, there is a demand on the identification of a novel G-actin nonsensitive DNase effective for the treatment of CF.

A second interest in the clinical application of DNase is that for the prophylaxis and treatment of infectious diseases. Some DNases are considered to play an important role in the biological defense mechanisms against infection with bacteria and viruses, based on degradation of foreign genomic DNAs. Accordingly, identification of the DNase involved in the prevention of infection in mammals, such as human, and utilization thereof as a medicament are expected to open a new possibility in the prophylaxis and treatment of infectious diseases.

It is therefore an object of the present invention to provide a novel acid DNase and clarify the characteristics of the enzyme, thereby providing critical information for the study of the molecular diversity of acid DNases. It is another object of the present invention to provide a novel G-actin nonsensitive DNase that can be effectively used as a therapeutic agent of CF. It is yet another object of the present invention to provide a novel DNase useful for the prophylaxis and treatment of infectious diseases.

DISCLOSURE OF THE INVENTION

In an attempt to accomplish the above-mentioned objects, the present inventors have conducted intensive studies, and succeeded in isolating cDNA clones containing an ORF encoding a novel protein homologous to human DNase II, from RNA derived from the liver of human, mouse or rat. Furthermore, it has been confirmed that this protein has an endonuclease activity capable of cleaving the DNA independently from divalent cations under acidic conditions, like DNase II, but is a novel acid DNase distinguishable from DNase II in the capability of exerting the DNase activity even in the neutral pH range and the sensitivity against divalent metallic ion inhibitors, as a result of the analysis of the physicochemical and enzymological characteristics of the protein obtained by culturing a host cell transformed with an expression vector containing the cDNA clone and purifying the recombinant protein. Then, the present inventors have designated the novel acid DNase as DLAD (DNase II-Like Acid DNase). The present inventors have also demonstrated that this enzyme has a high possibility of making an effective therapeutic agent of CF by confirming that the DLAD activity is not inhibited by G-actin. Moreover, the present inventors have confirmed a high possibility of the DLAD having a preventive effect on viral infectious diseases, which resulted in the completion of the present invention.

Accordingly, the present invention provides the following. (1) A DNase which is an endonuclease capable of cleaving DNA independently from divalent cations under acidic conditions and having the following properties: (1) active pH range: ca. 4.0 to ca. 7.6 (2) DNA cleavage mode: 3'-P/5'-OH end forming type (3) sensitivity against inhibitors: (i) inhibited by Zn.sup.2+ (ii) not inhibited by G-actin (2) The DNase of (1) above, further having the following properties: (1) optimal pH: ca. 5.2 (2) molecular weight: ca. 55 kDa as a post-translational modification product (SDS-PAGE) (3) localization: present in cytoplasm and extracellularly, rich in cytoplasm (4) tissue specificity: specifically expressed in the liver. (3) A DNase having the following polypeptide (a) or (b): (a) a polypeptide consisting of an amino acid sequence of amino acid Nos. 1 to 332 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1 (b) a polypeptide having the same amino acid sequence of (a) above, except that one to several amino acids are deleted, substituted, inserted, added or modified, wherein a mature protein has an endonuclease activity capable of cleaving a DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6. (4) The DNase of any of (1) to (3) above, wherein a primary translation product contains an N terminal signal peptide sequence, preferably an amino acid sequence of the amino acid Nos. -22 to -1 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1. (5) The DNase of any of (1) to (4) above, which is derived from a mammal, preferably mouse. (6) A DNase having the following polypeptide (a) or (b): (a) a polypeptide consisting of an amino acid sequence of the amino acid Nos. 1 to 334 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3. (b) a polypeptide having the same amino acid sequence of (a) above, except that one to several amino acids are deleted, substituted, inserted, added or modified, wherein a mature protein has an endonuclease activity capable of cleaving a DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6. (7) The DNase of any of (1), (2) and (6) above, wherein a primary translation product contains an N terminal signal sequence, preferably an amino acid sequence of the amino acid Nos. -27 to -1 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3. (8) The DNase of (1), (2), (6) or (7) above, which is derived from a mammal, preferably human. (9) A DNA encoding the DNase of any of (1) to (8) above. (10) A DNA consisting of the following nucleotide sequence (a) or (b): (a) a nucleotide sequence of the nucleotide Nos. 279 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2 (b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a DNase having an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6. (11) A DNA consisting of the following nucleotide sequence (a) or (b): (a) a nucleotide sequence of the nucleotide Nos. 213 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2 (b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a primary translation product of a DNase whose mature protein has an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6. (12) The DNA of (10) or (11) above, which is derived from a mammal, preferably mouse. (13) A DNA consisting of the following nucleotide sequence (a) or (b): (a) a nucleotide sequence of the nucleotide Nos. 82 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4 (b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a DNase having an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6. (14) A DNA consisting of the following nucleotide sequence (a) or (b): (a) a nucleotide sequence of the nucleotide Nos. 1 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4. (b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a primary translation product of a DNase whose mature protein has an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6. (15) The DNA of (13) or (14) above, which is derived from a mammal, preferably human. (16) A recombinant vector containing the DNA of any of (9) to (15) above. (17) An expression vector containing the DNA of any of (9) to (15) above and a promoter operably linked to said DNA. (18) A transformant obtained by transforming a host cell with the expression vector of (17) above. (19) A method for producing the DNase of any of (1) to (8) above, which comprises culturing the transformant of (18) above in a medium and recovering said DNase from the resulting culture. (20) A pharmaceutical composition containing the DNase of any of (1) to (8) above, the expression vector of (17) above or the transformant of (18) above as an active ingredient. (21) The pharmaceutical composition of (20) above, which is for the prophylaxis and treatment of infectious diseases or for the treatment of cystic fibrosis.

Inasmuch as the DLAD of the present invention is an acid DNase that expresses the activity in a broad pH range of from acidic to neutral pHs independently from divalent cations, and is resistant to G-actin, it is useful for degrading a high concentration DNA contained in the sputum of CF patients, improving the respiratory function.

Furthermore, because the DLAD of the present invention can suppress the intracellular expression of foreign genes, it also provides a useful means for the prophylaxis and treatment of infectious diseases, such as viral infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an intracellular/extracellular (medium) presence ratio (% of the total) of each protein in HeLa S3 cells engineered to express DLAD, DNase II signal-DLAD chimeric protein or DNase II, wherein a black line shows intracellular presence and a white line shows extracellular presence.

FIG. 2 shows sensitivity of DLAD to various DNase inhibitors, wherein A (.smallcircle.): MgCl.sub.2, (.circle-solid.): MgSO.sub.4; B: aurintricarboxylic acid; C: G-actin; D (.box-solid.): CoCl.sub.2, (.DELTA.): NiCl.sub.2, (.circle-solid.): ZnCl.sub.2.

FIG. 3 shows GFP or .beta.-galactosidase activity in HeLa S3 cells co-transfected with a DLAD or DNase II expression vector and a GFP (closed column) or .beta.-galactosidase (open column) expression vector. The activities are shown as the ratio (%) to the activity in the HeLa S3 cells co-transfected with a control vector and a reportor gene expression vector. The data are the average values (column) of 3 independent experiments .+-. standard error (error bar).

BEST MODE FOR CARRYING OUT THE INVENTION

The DLAD of the present invention is similar to DNase II, which is a known acid DNase, in that it has an endonuclease activity that hydrolyzes DNA to generate 3'-P/5'-OH termini under acidic conditions independently from divalent cations. However, DLAD is extremely characteristic in that it exerts DNase activity over a wide pH range of from pH ca. 4.0 to ca. 7.6, whereas DNase II shows activity only in a pH range of not more than ca. 5.6. The pH range preferable for the DLAD activity is from ca. 4.4 to ca. 6.8, and the optimal pH is about 5.2.

DLAD is also characteristically different from DNase II in the sensitivity to divalent metal ions. To be specific, DLAD is significantly sensitive to Zn.sup.2+ as compared with Co.sup.2+, Ni.sup.2+ and the like, whereas Co.sup.2+, Ni.sup.2+, Cu.sup.2+, Zn.sup.2+ and the like influence DNase II activity to almost the same level.

The DLAD of the present invention is not inhibited by G-actin, unlike DNase I, etc.

The DLAD of the present invention is not particularly limited as long as it has the above-mentioned characteristics, and the origin of DLAD is not limited, either. Thus, it encompasses not only those originated from naturally occurring organisms but also spontaneous or artificial mutants or those derived from transformants obtained by introducing a heterologous (i.e., foreign) DLAD gene. Preferably, it includes DLAD derived from mammals, such as human, bovine, porcine, horse, monkey, sheep, goat, canine, feline, rabbit, mouse, rat, guinea pig and the like. Those derived from human, bovine, porcine, mouse and rat are particularly preferable.

The DLAD of the present invention can have various molecular weights by changing the amino acid composition or by glycosylation, and preferably has a molecular weight of about 38 to 39 kDa (calculated) when it is an unglycosylated mature polypeptide chain, and about 55 kDa (SDS-PAGE) when it is a glycosylated mature protein (post-translational modification product). When DLAD is translated as a precursor containing a signal peptide sequence, it preferably has a molecular weight of about 41 to 42 kDa when it is a primary translation product.

In an embodiment of the present invention, DLAD is distributed both extracellularly and in cytoplasm. More specifically, DLAD is present mainly in cytoplasm, and shows a presence ratio different from that of DNase II. In this embodiment, as is expected from additional extracellular secretion, the primary translation product of DLAD contains a signal peptide sequence at its N terminus. The signal peptide is not particularly limited as long as it is recognized and cleaved by a signal peptidase in endoplasmic reticulum, resulting in a mature DLAD protein. Examples thereof include an amino acid sequence of the amino acid Nos. -22 to -1 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1, an amino acid sequence of the amino acid Nos. -27 to -1 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3, and an amino acid sequence obtained by deleting, substituting, inserting or adding one to several amino acids of these amino acid sequences as long as the property of a signal sequence is generally understood to be retained. Signal sequences of other secretory proteins, such as DNase II, are also preferable.

It is not particularly limited where in the substructure of cytoplasm a cytoplasmic DLAD is localized, but it is preferably localized in one of more organelles in an acidic environment, such as lysosomes and peroxysomes.

The expression of the DLAD of the present invention is highly restricted to that in the liver, making a sharp contrast with DNAse II which is low in organ specificity.

In a preferable embodiment of the present invention, DLAD is a polypeptide consisting of an amino acid sequence of the amino acid Nos. 1 to 332 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1 or a polypeptide consisting of an amino acid sequence of the amino acid Nos. 1 to 334 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3, or a polypeptide which consists of the same amino acid sequence of these, except that one to several amino acids are deleted, substituted, inserted, added or modified, and which has an endonuclease activity capable of cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6 independently from divalent cations.

The DLAD of the present invention can be obtained by appropriately employing (1) a method including extraction and purification from the cells or tissues, that produce this enzyme, as a starting material, (2) a method including chemical synthesis or (3) a method including purification from the cells engineered to express DLAD by genetic recombination techniques, or the like.

For example, the isolation and purification of DLAD from a naturally occurring DLAD-producing tissue can be carried out as follows. A mammalian tissue (e.g., a liver tissue section from human, mouse, rat, etc.) is homogenized in a suitable extraction buffer, ultrasonicated or treated with a surfactant to give a cell extract, and purified by a suitable combination of separation techniques conventionally utilized for separation and purification of proteins. Examples of the separation technique include methods utilizing difference in solubility, such as salting out and solvent precipitation, methods utilizing difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, non-denatured polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), methods utilizing charge, such as ion exchange chromatography and hydroxyapatite chromatography, methods utilizing specific affinity, such as affinity chromatography, methods utilizing difference in hydrophobicity, such as reversed phase high performance liquid chromatography, methods utilizing difference in isoelectric point, such as isoelectric focusing, and the like.

Alternatively, DLAD can be obtained by culturing mammal-derived cultured cells, for example, cultured cells derived from liver cells of human, mouse, rat and the like, in a suitable liquid medium and purifying from the obtained culture supernatant by the above-mentioned conventional protein separation techniques.

The production of DLAD by chemical synthesis can be carried out by, for example, synthesizing the whole or partial sequence based on an amino acid sequence (amino acid Nos. 1 to 332) shown in Sequence Listing, SEQ ID NO: 1 or an amino acid sequence (amino acid Nos. 1 to 334) shown in Sequence Listing, SEQ ID NO: 3 using a peptide synthesizer and renaturing the obtained polypeptide under suitable renaturation conditions.

The DLAD of the present invention is preferably produced by cloning a DNA encoding the protein, and isolating and purifying from the culture of a transformant containing an expression vector carrying the DNA.

The cloning of an enzyme gene is typically performed as follows. First, a desired enzyme is completely or partially purified from cells or tissues producing said enzyme using the above-mentioned means, followed by Edman method to determine its N terminal amino acid sequence. Furthermore, the enzyme is partially degraded with proteases or chemical substances that cleave a peptide in a sequence specific manner, and the amino acid sequence of the obtained oligopeptide is determined in the same manner by Edman method. The oligonucleotides having the nucleotide sequences corresponding to the determined amino acid sequences are synthesized, and using them as probes, a DNA encoding said enzyme is cloned from a cDNA or genomic DNA library prepared from the cells or tissues that produce said enzyme by colony (or plaque) hybridization method.

Alternatively, an antibody against the enzyme is produced according to a conventional method using, as an antigen, the entirety or a part of the completely of partially purified enzyme, and a DNA encoding said enzyme can be cloned by antibody screening method from a cDNA or genomic DNA library prepared from the cells or tissues, that produce this enzyme.

When a gene encoding an enzyme, whose enzymological properties are similar to those of the enzyme of interest, is known, a DNA encoding said enzyme can be cloned by searching EST (Expressed Sequence Tag) clones of mammals, such as human, mouse and rat, registered on generally available databases, such as EMBL and GenBank, extracting a clone that shows homology to the nucleotide sequence of the known gene, producing probes as mentioned above, based on the nucleotide sequence of the extracted EST clone, and carrying out colony (or plaque) hybridization. In the case of the DLAD of the present invention, an EST clone, which is a fragment of cDNA encoding DLAD, can be found by a homology search using a nucleotide sequence encoding DNase II derived from mammals such as human.

Alternatively, RACE method can be used to obtain a cDNA clone more rapidly and easily. To be specific, an EST clone corresponding to a part of the DLAD gene is extracted as mentioned above, oligonucleotides homologous to the partial nucleotide sequences of sense and antisense strands of said EST clone are respectively synthesized. Using each oligonucleotide and an appropriate adaptor primer as a pair of PCR primers, 5' and 3' RACE reactions are carried out, and each amplification fragment is ligated by a method using a restriction enzyme and a ligase to give a full length cDNA clone.

The nucleotide sequence of the DNA obtained as mentioned above can be determined using known sequencing techniques such as Maxam-Gilbert method and dideoxy termination method.

The DNA encoding DLAD of the present invention is preferably a DNA encoding an amino acid sequence of the amino acid Nos. 1 to 332 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1 or an amino acid sequence of the amino acid Nos. 1 to 334 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3, or the same amino acid sequences of these, except that one to several amino acids are deleted, substituted, inserted, added or modified, wherein a protein consisting of said mutated amino acid sequence has an endonuclease activity capable of cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6 independently from divalent cations. More preferably, the DNA encoding DLAD of the present invention is one which consists of a nucleotide sequence of the nucleotide Nos. 279 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2 or a nucleotide sequence of the nucleotide Nos. 82 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4, or a nucleotide sequence capable of being hybridized to these nucleotide sequences under stringent conditions, wherein the mutated nucleotide sequence encodes a protein having an endonuclease activity capable of cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6, independently from divalent cations.

The DNA encoding DLAD of the present invention is preferably one further containing a nucleotide sequence encoding a signal peptide sequence at the 5' terminus of the nucleotide sequence as mentioned above. More preferably, the DNA encoding DLAD of the present invention consists of the nucleotide sequence of the nucleotide Nos. 213 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2 or the nucleotide sequence of the nucleotide Nos. 1 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4, or a nucleotide sequence capable of being hybridized to these nucleotide sequence under stringent conditions, wherein the mutated nucleotide sequence encodes a primary translation product of a protein having an endonuclease activity capable of cleaving DNA in a pH range of from ca. 4.0 to ca. 7.6, independently from divalent cations.

In the context of the present invention, the "stringent conditions" means those under which a DNA having not less than about 60% homology to the nucleotide sequence can be hybridized. The stringency can be controlled by appropriately varying salt concentrations and temperatures of the hybridization reaction and washing.

The DNA encoding DLAD of the present invention can be a DNA chemically synthesized based on the nucleotide sequence of the nucleotide Nos. 279 to 1274 or the nucleotide Nos. 213 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2, or the nucleotide sequence of the nucleotide Nos. 82 to 1083 or the nucleotide Nos. 1 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4.

The present invention also provides a recombinant vector containing a DNA encoding DLAD of the invention. The inventive recombinant vector is not particularly limited as long as it can be maintained by replication or autonomously replicated within various host cells, such as prokaryotic cells and/or eukaryotic cells, and encompasses plasmid vectors, viral vectors and the like. The recombinant vectors can be easily prepared by ligating the above-mentioned DNA encoding DLAD to known cloning vectors or expression vectors available in this technical field, using suitable restriction enzymes and a ligase, and further, linkers or adaptors as necessary. Examples of such vectors include pBR322, pBR325, pUC18, pUC19 etc. as a plasmid derived from Escherichia coli; pSH19, pSH15 etc. as a plasmid derived from yeast; and pUB110, pTP5, pC194 etc. as a plasmid derived from Bacillus subtilis. Examples of the viruses include bacteriophages such as .lamda. phage, and animal and insect viruses such as parvovirus (SV40, bovine papilloma virus (BPV) etc.), retrovirus (Moloney murine leukemia virus (MoMuLV) etc.), adenovirus (AdV), adeno-associated virus (AAV), vacciniavirus, vaculovirus, and the like.

Particularly, the present invention provides a DLAD expression vector in which a DNA encoding DLAD is placed under the control of a promoter functional in a desired host cell. The vector to be used is not particularly limited as long as it contains a promoter region, which is capable of functioning in various host cells such as prokaryotic and/or eukaryotic cells and regulating the transcription of a gene located at its downstream (e.g., when the host is Escherichia coli, trp promoter, lac promoter, leca promoter, etc., when the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter, etc., when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc., and when the host is mammalian cell, viral promoters such as SV40 early promoter, MoMuLV long terminal repeat, adenovirus early promoter, etc.), and a termination signal of the transcription of said gene, i.e., terminator region, wherein the promoter region and the terminator region are ligated via a sequence containing at least one restriction enzyme recognition site, preferably unique restriction site that cleaves the vector only at this site. However, it is preferable that it further contain a selectable marker gene for selecting transformants (e.g., a gene imparting resistance to a drug such as tetracycline, ampicillin, kanamycin, hygromycin and phosphinothricin, a gene complementing auxotrophic mutation etc.). Moreover, when the DNA encoding DLAD to be inserted does not contain an initiation codon or a termination codon, a vector, which contains an initiation codon (ATG or GTG) and a termination codon (TAG, TGA or TAA) at the downstream of the promoter and the upstream of the terminator, respectively, is preferably used.

When bacteria is used as a host cell, in general, the expression vector needs to contain a replicable unit which allows autonomous replication in the host cell, in addition to the above-mentioned promoter region and terminator region. The promoter region also contains an operator and Shine-Dalgarno (SD) sequence near the promoter.

When a yeast, animal cell or insect cell is used as a host cell, the expression vector preferably further contains enhancer sequences, non-translated regions on the 5'-side and 3'-side of DLAD mRNA, a polyadenylation site, and the like.

When DLAD is secreted into a culture medium of the transformant or proper glycosylation of the mature DLAD protein is desired but DNA encoding DLAD to be inserted does not have a sequence encoding signal peptide, a secretory expression vector, further containing a suitable signal codon following an initiation codon, is preferably used as a vector.

When the DNA encoding DLAD of the present invention is isolated from a genomic DNA and obtained together with its native promoter and terminator regions, the expression vector of the present invention can be prepared by inserting the DNA into a suitable site of a known cloning vector which can be maintained by replication or which can be autonomously replicated in a desired host cell. Since DLAD is expressed in a liver-specific manner in a preferable embodiment of the present invention, the expression vector constructed as mentioned above can be preferably employed when a tissue- or organ-specific expression of DLAD is desired (e.g., in the treatment of a CF patient with hepatic duct occlusion).

The present invention also provides a transformant obtained by transforming a host cell with the above-mentioned DLAD expression vector.

The host cell to be used in the invention is not particularly limited as long as it is capable of adapting to the above-mentioned expression vector and can be transformed therewith, and is exemplified by various cells such as naturally occurring cells or artificially established mutant or recombinant cells conventionally used in the technical field of the present invention [e.g., bacteria (Escherichia coli, Bacillus subtilis, lactobacillus etc.), yeast (Saccharomyces, Pichia, Kluyveromyces etc.), animal cell and insect cell]. In view of the use of DLAD as a medicament to be mentioned below, the host cells are preferably mammalian cells, particularly the cells derived from human, monkey, mouse, rat, hamster etc., especially human-derived cells. To be specific, exemplified are mouse-derived cells (COP, L, C127, Sp2/0, NS-1, NIH3 and T3), rat-derived cells, hamster-derived cells (BHK and CHO), monkey-derived cells (COS1, COS3, COS7, CV1 and Vero) and human-derived cells (HeLa, diploid fibroblast-derived cells, myeloma cells and Namalwa).

The expression vector can be introduced into a host cell using a method conventionally known. For example, the method of Cohen et al. [Proc. Natl. Acad. Sci. USA., 69, 2110 (1972)], protoplast method [Mol. Gen. Genet., 168, 111 (1979)] and competent method [J. Mol. Biol., 56, 209 (1971)] can be used for bacteria; the method of Hinnen et al. [Proc. Natl. Acad. Sci. USA., 75, 1927 (1978)] or Lithium method [J. Bacteriol., 153, 163 (1983)] can be used for yeast; the method of Graham [Virology, 52, 456 (1973)] can be used for animal cell; and the method of Summers et al. [Mol. Cell. Biol., 3, 2156 2165 (1983)] can be used for insect cell, for transformation.

The DLAD of the present invention can be produced by culturing a transformant containing the DLAD expression vector prepared as mentioned above in a medium, and recovering DLAD from the resulting culture.

The medium to be used preferably contains carbon source and inorganic or organic nitrogen source necessary for the growth of host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch and sucrose; examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean lees, potato extract solution and the like. Where desired, other nutrient sources such as inorganic salts (e.g., calcium chloride, sodium dihydrogenphosphate and magnesium chloride), vitamins and antibiotics (e.g., tetracycline, neomycin, ampicillin and kanamycin) may be added.

Culture is performed by a method known in this field. Specific examples of the medium and culture conditions to be used depending on the host cell are shown in the following, which should not be construed as limiting the culture conditions of the invention.

When the host is bacteria, actinomyces, yeast or fungus, a liquid medium containing the aforesaid nutrient sources is suitable, with preference given to a medium having a pH of 5 to 8. When the host is Escherichia coli, preferable medium includes LB medium and M9 medium [Miller. J., Exp. Mol. Genet, p.431, Cold Spring Harbor Laboratory, New York (1972)]. In this case, culture can be typically performed at 14.degree. C. to 43.degree. C. for about 3 to 24 hr with aeration and agitation as necessary. When the host is Bacillus subtilis, culture can be typically performed at 30.degree. C. to 40.degree. C. for about 16 to 96 hr with aeration and agitation as necessary. When the host is yeast, examples of the medium include Burkholder minimum medium [Bostian. K. L. et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], and pH is preferably 5 to 8. Culture can be typically performed at 20.degree. C. to 35.degree. C. for about 14 to 144 hr with aeration and agitation as necessary.

When the host is animal cell, examples of the medium include minimum essential medium (MEM) containing about 5 to 20% fetal calf serum [Science, 122, 501 (1952)], Dulbecco's modified minimum essential medium (DMEM) [Virology, 8, 396 (1959)], RPMI1640 medium [J. Am. Med. Assoc., 199, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] and the like. The pH of the medium is preferably about 6 to 8. Culture is typically performed at 30.degree. C. to 40.degree. C. for about 15 to 72 hr with aeration and agitation as necessary.

When the host is an insect cell, examples of the medium include Grace's medium containing fetal calf serum [Proc. Natl. Acad. Sci. USA, 82, 8404 (1985)], and pH is preferably about 5 to 8. Culture is typically performed at 20.degree. C. to 40.degree. C. for about 15 to 100 hr with aeration and agitation as necessary.

The DLAD can be purified by an appropriate combination of various separation techniques conventionally used, according to the fractions having DLAD activity. In a preferable embodiment of the invention, DLAD is present both in cytoplasm and extracellularly (i.e., in medium).

The DLAD present in the medium in the culture can be obtained by centrifuging or filtering the culture to give a culture supernatant (filtrate) and applying the culture supernatant to known separation methods (e.g., salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denatured PAGE, SDS-PAGE, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reversed-phase high performance liquid chromatography and isoelectric focusing), as appropriately selected.

The DLAD present in the cytoplasm can be isolated and purified by centrifuging or filtering the culture to harvest cells, suspending the cells in a suitable buffer, disrupting (lysing) the cells and organelle membranes by, for example, ultrasonication, lysozyme treatment, freeze-thawing, osmotic shock and/or treatment with surfactant such as Triton X-100, removing the debris by centrifugation or filtration to give a soluble fraction, and treating the soluble fraction according to the methods mentioned above.

As a means for obtaining the recombinant DLAD rapidly and easily, preferably exemplified is a method which comprises adding a DNA sequence encoding an amino acid sequence capable of adsorbing to a metal ion chelate (e.g., a sequence consisting of basic amino acids such as histidine, arginine or lysine, preferably histidine) to a certain region (preferably C terminus) of the DLAD coding sequence by gene manipulation, allowing expression within a host cell, and recovering DLAD from the DLAD active fraction in the cell culture by separation utilizing its affinity for a carrier immobilizing said metal ion chelate. The DNA sequence encoding an amino acid sequence capable of adsorbing to a metal ion chelate can be introduced into the DLAD coding sequence by, for example, performing PCR amplification using a hybrid primer comprising said DNA sequence linked to the nucleotide sequence encoding the C terminal amino acid sequence of DLAD, in the process of cloning DNA encoding DLAD, or by inserting the DNA encoding DLAD in frame into an expression vector containing said DNA sequence before the termination codon. The metal ion chelate adsorbent to be used for purification is prepared by bringing a solution containing a transition metal (e.g., divalent ion of cobalt, copper, nickel or iron, or trivalent ion of iron or aluminum, preferably divalent ion of cobalt or nickel) into contact with a ligand (e.g., a matrix onto which iminodiacetate (IDA) group, nitrilotriacetate (NTA) group or tris(carboxymethyl)ethylenediamine (TED) group is attached) to allow binding thereof with the ligand. The matrix part of the chelate adsorbent is not particularly limited as long as it is a conventional insoluble carrier.

The present invention provides a pharmaceutical composition containing the inventive DLAD, DLAD expression vector or transformant expressing DLAD as an active ingredient, specifically an agent for the treatment of chronic obstructive diseases (in particular cystic fibrosis) caused by the accumulation of high concentration DNA and an agent for the prophylaxis and treatment of infectious diseases caused by viruses and the like.

DNase I conventionally used for treating CF requires divalent cations for the expression of activity. However, it is speculated that the concentration of divalent cations in lung cysts is not sufficiently high to allow expression of activation of DNase I. It is also considered that the pH in the inflammatory lesions in lung cysts inclines from neutral to acidic, though the optimal pH of DNase I is about 7.1. Furthermore, due to the fatal property that DNase is inhibited by G-actin present in large amounts in the sputum of CF patients, DNase I is almost ineffective as an agent for treating CF. In contrast, the DLAD of the present invention is capable of cleaving DNA under acidic conditions independently from divalent cations. Furthermore, the DLAD of the invention has excellent properties for exhibiting a high DNase activity in the inflammatory lesions in lung cysts of CF patients, in that it is not inhibited by G-actin, it does not require any cofactor for the expression of its activity, and it can exhibit its activity even in the neutral pH range.

DLAD has a high homology to FP-CEL1 [J. Virol., 72: 6742 6751 (1998)], which is a DNase II-related protein derived from fowlpox virus (FWPV). It is considered that, when a virus enters a cell infected with FWPV, the FWPV-derived DLAD homolog cleaves a DNA of the virus, thereby to exclude the competitive virus [J. Virol. (1998), supra]. Therefore, DLAD can also enhance the defensive function of a body against infections with virus etc., and is effective for the prophylaxis and treatment of infectious diseases. The infectious diseases that can be prevented or treated are not particularly limited, and exemplified by those caused by hepatitis A, B and C viruses, human immunodeficiency virus, influenza virus and herpes virus.

The administration subject of the inventive pharmaceutical composition is not particularly limited as long as it is an animal in need of the treatment of a chronic obstructive disease caused by the accumulation of high concentration of DNA, or the prophylaxis and treatment of an infectious disease caused by a virus or the like. It is preferably a mammal, more preferably a mammal such as human, monkey, bovine, horse or porcine, especially human.

The pharmaceutical composition of the present invention containing a DLAD protein as an active ingredient can be formulated by admixing DLAD with a pharmaceutically acceptable carrier to give a liquid preparation, powder, granule, tablet, capsule, syrup, injection, aerosol or the like, and can be administered orally or parenterally.

The pharmaceutically acceptable carrier may include, but not limited to, excipients (e.g., sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate etc.), binding agents (e.g., cellulose, methyl cellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch etc.), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl-starch, sodium glycol-starch, sodium bicarbonate, calcium phosphate, calcium citrate etc.), lubricants (e.g. magnesium stearate, aerosil, talc, sodium laurylsulfate etc.), flavors (e.g., citric acid, mentol, ammonium salt of glycyrrhizin, glycine, orange powders etc.), preservatives (e.g., sodium benzoate, sodium bisulfite, methylparaben, propylparaben etc.), stabilizers (e.g., citric acid, sodium citrate, acetic acid, etc.), suspending agents (e.g., methyl cellulose, polyvinylpyrrolidone, aluminum stearate etc.), dispersing agents (e.g., surfactant etc.), diluents (e.g., water, physiological saline, orange juice etc.) and base waxes (e.g., cacao butter, polyethylene glycol, white kerosine etc.).

Preferably, the pharmaceutical composition containing DLAD protein as an active ingredient is a preparation for oral preparation, an injection or an aerosol preparation.

Preparations suitable for oral administration are liquid obtained by dissolving an effective amount of DLAD in diluents such as water, physiological saline and orange juice, capsule, sachet or tablet containing an effective amount of DLAD as solid or granule, suspension containing an effective amount of DLAD suspended in an appropriate dispersion medium, and emulsion prepared by suspending a solution containing an effective amount of DLAD dissolved in an appropriate dispersion medium and emulsifying the suspension.

The aerosol preparation may include one in which DLAD is compressed with dichlorodifluoromethane, propane or nitrogen and a non-compressed preparation such as nebulizer and atomizer, and can be administered by inhalation or spraying into airways and the like.

Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable liquids, which can contain antioxidant, buffer, bacteriostat and isotonicity agent and the like, and aqueous and non-aqueous sterile suspensions, which can contain suspending agent, solubilizer, thickener, stabilizer, preservative and the like. The DLAD preparations can be sealed in unit-dose or multi-dose containers such as ampoules or vials. It is also possible to lyophilize (freeze-dry) DLAD with a pharmaceutically acceptable carrier and preserved in the form that requires dissolving or suspending in an appropriate sterile vehicle immediately prior to use.

The dose of the pharmaceutical composition containing the DLAD protein of the invention varies depending upon the kind of disease to be prevented or treated, the progress of the disease, and the animal species, drug-tolerance, weight and age of the administration subject, which is typically 1 to 10,000 I.U./kg body weight, preferably 10 to 1,000 I.U./kg body weight, daily for an adult, which can be administered in a single dose or several doses.

The present invention also provides a pharmaceutical composition containing the DLAD expression vector of the present invention as an active ingredient. Since the treatment of CF using DNases is not fundamental but suppressive, continuous supply of DLAD to the inflammatory lesions in the lung cysts is required. Accordingly, a gene therapy, in which the DLAD expression vector is introduced into cells at or around the inflammatory lesion, is effective as a sustainable therapeutic method for CF. For the purpose of preventing viral infection of livestock and the like, a transgenic animal having enhanced preventive function against infection, can be produced by introducing the DLAD expression vector into the embryonic cells.

The vector to be used can be selected according to the administration subject, and examples of vectors preferably administered to human include viral vectors such as retrovirus, adenovirus and adeno-associated virus. Adenovirus is particularly preferable as a DLAD gene transfer vector for the treatment of CF, because it has a very high gene transfer efficiency, can be introduced even into non-dividing cells, and is trophic for the respiratory epithelium. However, since the integration of the introduced gene into the host chromosome is extremely rare, the gene expression is transient and typically lasts for about 4 weeks. In view of the sustainability of the therapeutic effect, the use of an adeno-associated virus is also preferable, which has a relatively high gene transfer efficiency, can be introduced even into non-dividing cells and can be integrated into chromosome via inverted terminal repeats (ITRs).

Examples of pharmaceutically acceptable carriers contained in the pharmaceutical composition containing a DLAD expression vector as an active ingredient may be those for the above-mentioned pharmaceutical composition containing the DLAD protein.

The vector can be introduced by either an ex vivo method, which comprises isolating the target cells from the administration subject, culturing, transferring the vector thereto and implanting the cells back into the subject, or an in vivo method, which comprises directly transferring the vector into the body of the administration subject. When the in vivo method is used, the administration of the vector via intravenous injection or the like may raise a problem of the antigenicity of the viral vector, but the undesired effects caused by the presence of the antibody can be reduced by topically injecting the vector into the organ/tissue containing the target cells (in situ method).

When a non-viral vector is used as a vector, the DLAD expression vector can be introduced using macromolecule carriers such as liposome and polylysine-DNA-protein conjugate.

The present invention also provides a pharmaceutical composition, which comprises a host cell containing the DLAD expression vector of the invention as an active ingredient. The host cells to be used may include autogenous cells, which are isolated as target cells from the administration subject in the ex vivo method of the gene therapy using the above-mentioned DLAD expression vector, cells isolated from the syngeneic or allogeneic individuals, or established cell lines derived from these cells by subculture. In another embodiment, a transformant obtained by transforming a host cell, which is normally present in the nasal cavity, pharynx, oral cavity, intestinal tract, skin, vagina and the like of the administration target animal, with a DLAD expression vector according to a conventional method, can be delivered to the site where the host cell is normally present in the administration subject.

The dose of the pharmaceutical composition containing the DLAD expression vector or the host cell, which expresses this vector of the present invention, as an active ingredient, is preferably one capable of expressing DLAD in the body of an animal to which it is administered, the dose corresponding to an amount suitable for allowing the expression to be achieved when the DLAD protein itself is administered.

The present invention is further explained in detail by way of Examples in the following. These are mere examples, which in no way limit the scope of the present invention.

EXAMPLE 1

Cloning and Sequence Analysis of Mouse DLAD cDNA

The EST subdivision of the NCBI GenBank database was screened for EST encoding an amino acid sequence homologous to the deduced amino acid sequence of human DNase II (GenBank AF060222) using the tblastn program. As a result, a mouse EST clone (GenBank AI048641) was identified. Based on the sequence of the EST clone, the following two oligonucleotide primers (GSP2/mD and GSP1/mD) were synthesized. Furthermore, the following oligonucleotide (AP1) was synthesized as a linker primer.

GSP2/mD:

5'-AATGAATATGGTGAAGCTGTGGACTGG-3' (Sequence Listing, SEQ ID NO: 5) (sequence identical to the nucleotide sequence of the nucleotide Nos. 300 to 326 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2) GSP1/mD: 5'-CCATCGTTGTATATTAGATAGGCTGTG-3' (Sequence Listing, SEQ ID NO: 6) (sequence complementary to the nucleotide sequence of the nucleotide Nos. 509 to 535 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2) AP1: 5'-CCATCCTAATACGACTCACT