WO2002088345A2

WO2002088345A2 - Murine dnasex, medicament containing the same and non-human mammal comprising modified dnasex gene

Info

Publication number: WO2002088345A2
Application number: PCT/EP2002/004003
Authority: WO
Inventors: Annemarie Poustka; Johannes Coy
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date: 2001-04-11
Filing date: 2002-04-10
Publication date: 2002-11-07
Also published as: AU2002338549A1; EP1249495A1; WO2002088345A3

Abstract

The invention relates to a DNA sequence coding for murine DNAseX, the murine DNaseX protein and anti-DNaseX antibodies. The invention also relates to medicaments containing the above compounds, used preferably for preventing and/or treating diseases where apoptosis plays a role, and to diagnostic methods and kits, all based on these compounds. In addition, the invention relates to a non-human mammal in which the DNaseX gene is modified, preferably inactivated.

Description

Murine DNaseX, medicament containing the same and non-human mammal comprising modified DNaseX gene

The present invention relates to a DNA sequence coding for murine DNaseX and the accompanying control elements, the murine DNaseX protein and anti-DNaseX antibodies. The present invention also relates to medicaments containing the above compounds and used preferably for preventing and/or treating systemic lupus erythe atosus (SLE) or other diseases where apoptosis plays a role, and to diagnostic methods and kits, all based on these compounds. Finally, the present invention relates to a non-human mammal with modified, preferably inactivated, DNaseX gene. The non-human mammal according to the invention is suited for further studying diseases where apoptosis plays a role and/or for developing further therapy approaches .

Unlike the pathological death of cells (necrosis) apoptosis is a genetic program resulting in cell death. Certain external or internal factors may trigger this programmed cell death. Apoptosis is an essential process necessary to remove certain undesired or harmful cells. This process is of decisive significance inter alia for embryogenesis, for building up and maintaining the nervous system, for developing and maintaining tissues having a high division rate, such as the epidermis and epithelial cells (of the digestive system, for example) . Apoptosis also plays an important role in connection with programmed cell death occurring in physiological processes, e.g. in cells of the nervous system when neurotropic factors are withdrawn, when the prostate atrophies as a result of androgen deficiency (e.g. in the case of castration) and the death of tumor cells caused by cytotoxic T lymphocytes. Apoptosis also plays an important role in the case of pathologic phenomena, such as the death of thymocytes following irradiation, the viral death of cells after an infection with AIDS or influenzavirus, the death of cancer cells in malignant tissues, the cell death induced by medicaments or chemicals and following the administration of anti-tumor medicaments or bacterial toxins, and the death of tumor cells resulting from thermotherapy. Studying and understanding molecular mechanisms of apoptosis are an important step towards comprehending the significance and role of programmed cell death in the development of multicellular organisms and suppressing and controlling cancer. When the cells die in well-calculated fashion, the DNA of the cells is usually decomposed. A characteristic phenomenon usually occurring during apoptosis is the condensation of chromatin and the fragmentation of chromatin DNA. In particular the fragmentation of chromatin DNA into nucleosomal units is a characteristic change which occurs in apoptotic cells, irrespective of the differences existing between the triggering factors. It is assumed that DNases effect this decomposition. However, this decomposition process may also be disturbed. On the one hand, it is possible that the controlled occurrence of cell death as such does not function properly, which stimulates degeneration of the cells and thus development of a tumor. On the other hand, the DNA cannot be decomposed completely, the remaining DNA acting as an immunogen thus triggering autoimmune diseases, e.g. systemic lupus erythematosus. The remaining DNA may also occur as a mucus in the lungs, which is the case in patients suffering from the symptoms of cystic fibrosis. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease from which over one million people suffer in the U.S. alone, for example. The presence of anti-nucleus antibodies directed against the "naked" DNA and against nucleosomes, is specific for SLE. It is assumed that the resulting immune complexes accumulate and trigger a type III hypersensitivity reaction which manifests itself as glomerulonephritis, arthritis and general vasculitis. Although the etiology of SLE is unknown thus far there are many signs that an enhanced release or a disturbed decomposition of DNA protein complexes can trigger, or at least enhance, the disease following cell death. It was possible to show that DNase activity in the serum of SLE patients is generally reduced as compared to healthy humans. When the DNase activity is determined in patients ' serums it is not yet possible to distinguish between the four human DNase proteins of the DNase 1 family (DNase 1, DNaseX, DNase 3 and DNase 4) .

Thus, the invention is substantially based on the technical problem of providing a product for preventing or treating SLE. Another purpose is to study how to utilize the activation of DNases, in particular DNaseX, to kill and remove undesired cells or degenerated cells, which comprise in particular tumor cells. The invention also focuses on the use of DNases for preventing or treating cystic fibrosis.

The solution to this technical problem has been obtained by providing the embodiments characterized in the claims.

The present invention describes a formerly unknown DNase catalyzing DNA fragmentation. In doing so, chromatin DNA is cleaved into mononucleosome units or oligonucleosome units. This is also a characteristic phenomenon of apoptosis. Based on the studies resulting in the present invention it was possible to show that DNaseX plays an important role in apoptosis and is expressed in cells having a major turnover. Hence it can be assumed that the absence of DNaseX activity or the insufficient expression of the corresponding gene is correlated with the various diseases where disturbed apoptosis plays a role. These diseases are e.g. cancer, AIDS, SLE or cystic fibrosis. Therefore, the prevention and/or therapy should be possible by administering DNaseX (as protein or via a gene therapy) . Finally, an animal model was developed (DNaseX knock-out mouse) , permitting another study regarding the significance of DNaseX, for example.

The subject matter of the present invention is thus a DNA sequence comprising the DNA sequence shown in figure 1 (genomic DNA) . Three splice variants (figures 2a, 2b, 2c) of murine DNaseX exist, of which splice forms 1 and 2 are most likely active, whereas splice form 3 has to be considered inactive. The present invention also relates to a DNA sequence coding for a protein having the biological properties of DNaseX, this DNA sequence being a fragment, an allelic variant or another variant of the above DNA sequences according to the invention. The present invention also relates to non-transcribed and transcribed control elements. The DNaseX promoter represents a non-transcribed control sequence. It can be used inter alia to ensure a DNase-specific expression of any protein. For example, it is thus possible to generate a genetically modified mouse line in which the Cre protein (Cre recombinase) is controlled by the DNaseX promoter. The present invention also relates to a transcribed, non-translated differentially spliced DNaseX sequence in the 5' region of the DNaseX transcript. This sequence can be used to control and activate the DNaseX protein. It is also possible to control any protein by this control element. Furthermore, it is possible to isolate proteins binding to the DNaseX transcript. As a result, the translation efficiency of the DNaseX transcript among other things can be influenced in well-calculated fashion by proteins binding to the transcript.

The terms "variant" and "fragment" used in the present invention comprise DNA sequences differing from the sequences indicated in figure 1 by deletion(s), insertion(s) or exchange (s) and/or other modifications known in the art or comprise a fragment of the original DNA molecule, the protein encoded by these DNA sequences still having the biological properties of the DNaseX protein and being biologically active in mammals. Allelic variants are also comprised. The above modifications in the DNA sequence yield a protein, 90 %, preferably 95 %, more preferably 98 %, and most preferably 99 %, of which being identical with that of the murine DNaseX. Modifications in the DNA sequence which only result in conservative amino acid exchanges are preferred the most. Methods of producing the above modifications in the DNA sequence are known to a person skilled in the art and described in standard manuals of molecular biology, e.g. in Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2 edition, Cold Spring Harbor Laboratory Press,. Cold Spring Harbor NY (1989) . The person skilled in the art can also determine whether a DNaseX encoded by a DNA sequence modified in this way still has the biological properties of DNaseX. All of these DNA sequences are generally referred to as "DNaseX sequences" hereinafter.

DNaseX sequences can also be inserted in a vector, preferably an expression vector. Thus, the present invention also comprises these vectors and/or expression vectors. The term "vector" refers to a plasmid (pUC18, pBR322, pBlueScript, etc.), to a virus or another suitable vehicle. In a preferred embodiment, the DNaseX sequence according to the invention is functionally linked in the vector with regulatory elements which permit the expression thereof in prokaryotic or eukaryotic host cells. Along with the regulatory elements, e.g. a promoter, such vectors typically contain a replication origin and specific genes which permit the phenotypic selection of a transformed host cell. The regulatory elements comprise the lac-, trp promoter or T7 promoter for expression in prokaryotes, e.g. E. coli , and the AOX1 or GALl promoter in yeast for expression in eukaryotes, and the CMV-, SV40-,RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells. Suitable regulatory sequences are also described in Goeddel : Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) . Further examples of suitable promoters are the metallothionein I and polyhedrin promoters. Suitable expression vectors for E. coli are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8. pYlOO and Ycpad 1 belong to the vectors suited for expression in yeast, and pMSXND, pKCR, pEFBOS, cDM8 and pCEV4 as well as vectors derived from pcDNA/amp, pcDNAl/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7 , pko-neo and pHyg are suited for expression in mammalian cells. The expression vectors according to the invention also comprise vectors derived from baculo virus for expression in insect cells, e.g. pAcSGHisNT-A.

The above described DNaseX sequences are preferably inserted in a vector suited for gene therapy, e.g. under the control of a tissue-specific promoter, and introduced into the cells. In a preferred embodiment, the vector containing the above-described DNaseX sequences is a virus, e.g. an adenovirus, vaccinia virus or retrovirus . Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. Vectors suited for gene therapy are also disclosed in WO 93/04701, WO 92/22635, WO 92/20316, WO 92/19749 and WO 92/06180. For use in gene therapy the above-described DNaseX sequences can also be transported to the target cells in the form of colloidal dispersions. They comprise e.g. liposomes or lipoplexes (Mannino et al . , Biotechniques 6 (1988), 682).

General methods known in the art may be used to design expression vectors containing DNaseX sequences and suitable control sequences. These methods comprise e.g. in vi tro recombination techniques, synthetic methods as well as in vivo recombination methods as described in Sambrook et al . , supra , for example. The DNaseX sequences according to the invention may also be inserted in combination with a DNA coding for another protein or peptide, so that DNaseX can be expressed in the form of a fusion protein, for example. The person skilled in the art is also familiar with methods for the recombinant production of DNaseX, i.e. the culturing of suitable host cells under conditions permitting the expression of the protein (or fusion protein) (preferably stable expression) and the collection of the protein from the culture or the host cells. Suitable purification methods (e.g. preparative chromatography, affinity chromatography, e.g. immunoaffinity chromatography, HPLC, etc.) are also generally known.

The present invention also relates to a murine DNaseX protein or a protein having its biological activity and encoded by the DNaseX sequences according to the invention. Three splice forms of DNaseX protein exist which are shown in figures 2a, 2b and 2c.

The DNA sequences and protein sequences according to the invention are also suited to find suitable binding partners assisting in making further statements about the function of the DNase gene. These binding partners can also be administered as a medicament, where appropriate.

In addition, the present invention relates to an antibody directed against a DNaseX protein encoded by the DNaseX sequences according to the invention. These antibodies may be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this connection, the term "fragment" refers to all parts of the monoclonal antibody (e.g. Fab, Fv or "single chain Fv" fragments (scFv) ) which have an epitope specificity the same as that of the complete antibody. The production of such fragments is known to the person skilled in the art . The antibodies according to the invention are preferably monoclonal antibodies. The antibodies according to the invention can be prepared according to standard methods, DNaseX encoded by the DNaseX sequences according to the invention or a (synthetic) fragment thereof serving preferably as an immunogen. Methods of obtaining monoclonal antibodies are known to the person skilled in the art. One of them is the isolation of the desired antibody, e.g. an scFv fragment, from complex antibody libraries via phage display.

It can be assumed that in the case of diseases where apoptosis plays a role, in particularly in the case of SLE, the concentration or activity of DNaseX is modified, i.e. usually reduced, as compared to the standard situation. Thus, the present invention also relates to a diagnostic method of detecting a modified expression or concentration of DNaseX, comprising contacting a sample with a probe suited for specific hybridization with an mRNA transcribed by a DNaseX sequence or a primer or a primer set or an anti- DNaseX antibody and subsequently determining directly or indirectly whether the concentration of DNaseX-mRNA or DNaseX protein in the sample differs from a control sample (from a healthy person) . The person skilled in the art is familiar with methods for proper sampling and also suitable control samples. The control sample is preferably a tissue corresponding to a tissue the same as that of SLE patients but originating from a healthy source. A DNaseX activity or concentration reduced as compared to the control sample is a diagnostic indication as to SLE or a predisposition for SLE. The same applies correspondingly to the other diseases. The probes usable for this diagnostic method comprise primers based on the DNaseX sequences according to the invention, e.g. for PCR, RT-PCR or aptamer diagnosis (SELEX method) . The probes (or primers) are preferably oligonucleotides comprising a nucleic acid region which under stringent hybridization conditions hybridizes with at least 13, preferably at least 20, successive nucleotides of the sequence of figure 2a or 2b or naturally occurring variants thereof. The probe (or the primer) may also bear a label, e.g. a radioisotope, fluorescent compound, enzyme or enzyme cofactor. The person skilled in the art also knows conditions permitting that only the DNaseX-mRNA but not the DNaseX-DNA is amplified in the PCR, etc., or a distinction can be made between DNA amplification products and mRNA amplification products, e.g. by treating the sample with DNase or by using primers resulting in the amplification of intron regions, so that an DNA amplificate is longer than the mRNA amplificate. Alternatively, an oligo (dT) -anchored primer can also be used for PCR, RT-PCR, etc.

Finally, the present invention relates to a diagnostic kit for carrying out the diagnostic method according to the invention, which contains a probe suited for the specific hybridization with an mRNA transcribed by a DNaseX-DNA sequence or a primer/primer set and/or a an anti-DNaseX antibody or a fragment thereof. Depending on the arrangement of the diagnostic kit, the sample, probe, primer or antibody or the fragment thereof may be immobilized. In immunoassays, for example, the antibodies may also be present in liquid phase. The antibodies may here be labeled in different ways. Suitable markers and labeling methods are known in the art. Examples of immuoassays are ELISA and RIA.

The present invention also relates to a medicament which contains a DNaseX sequence according to the invention, an expression vector according to the invention or a DNaseX protein according to the invention. The medicament according to the invention enables preventive or therapeutic measures to be carried out with respect to diseases where apoptosis plays a role, in particular as regards SLE. The medicament is preferably combined with a suitable carrier. Suitable carriers and the formulation of such medicaments are known to the person skilled in the art. Suitable carriers are e.g. phosphate-buffered common salt solutions, water, emulsions, e.g. oil/water emulsions, wetting agents, sterile solutions, etc. The medicaments can be administered orally or parenterally. The methods of parenteral administration comprise the topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration.

Finally, the present invention also relates to a non-human mammal in which the DNaseX gene containing the DNaseX sequences according to the invention is modified, preferably inactivated, in ubiquitous or in tissue-specific manner ("knock-out") . Such a non-human mammal, preferably a mouse, can be provided by common methods . A method comprising the following steps is of advantage:

(a) producing a DNA fragment, in particular a vector, containing a modified murine DNaseX gene, the DNaseX gene having been modified by inserting a heterologous sequence, in particular a selectable marker;

(b) preparing embryonic stem cells from a non-human mammal (preferably mouse) ;

(c) transforming the embryonic stem cells from step (b) with the DNA fragment from step (a) , the DNaseX gene in the embryonic stem cells being modified by homologous recombination with the DNA fragment from (a) ;

(d) culturing the cells from step (c) ;

(e) selecting the cultured cells from step (d) for the presence of the heterologous sequence, in particular the selectable marker;

(f) producing chimeric non-human mammals from the cells from step (e) by administering these cells into mammalian blastocysts (preferably murine blastocysts) by injection; and

(g) transferring the blastocysts obtained in step (f) into pseudo-pregnant female mammals (preferably mice) and analyzing the resulting offspring as regards a modified, in particular inactivated, DNaseX gene.

In step (c) , the mechanism of the homologous recombination (cf. R.M. Torres, R. Kiihn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997) is utilized to transfect embryonic stem cells. The homologous recombination between the DNaseX sequences existing in a chromosome and new cloned DNA sequences added enables a cloned gene, instead of the original gene, to be inserted in the genome of a living cell. Using embryonic germ cells this method serves for obtaining, via chimeras, animals which are homozygous for the desired gene or the desired gene portion or the desired mutation. The preparation of transgenic mammals is also described in A.L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.

The term "embryonic stem cells" relates to any embryonic stem cells of a non-human mammal suited for mutation of the DNaseX gene. The embryonic stem cells are preferably derived from a mouse, in particular cells E14/1 or 129/SV.

In this connection, the term "vector" comprises any vector which by recombination with the DNA of embryonic stem cells enables the DNaseX gene to be modified. The vector preferably has a marker by means of which it is possible to select for existing stem cells in which the desired recombination has taken place. Such a marker is e.g. the loxP/tk neo-cassette which can be removed from the genome again by means of the Cre/loxP system (Sauer et al . , Proc. Natl. Acad. Sci. U.S.A. 85 (14), pages 5166-5179, 1988). The person skilled in the art also knows conditions and materials to carry out steps (a) -(g).

A non-human mammal, preferably a mouse, having a modified DNaseX gene is provided by the present invention. This modification may be embodied by an elimination of the function, for example. The function of the DNaseX protein can be studied selectively by such a mammal or cells therefrom. It also serves for finding substances, medicaments and therapy approaches selectively influencing the function of DNaseX. Therefore, the present invention provides a basis for influencing diseases where apoptosis plays a role (e.g. SLE) . Due to the mammal having modified DNaseX function according to the invention it is also possible to conduct studies as to whether an attenuated course of the disease or a healing resulting from the application of various compounds is observed in these animals. This animal is thus a new disease model for the development of new or other therapy approaches. By means of the non-human mammal according to the invention it is possible to study the effects of a modification, which is also tissue-specific, for example, or inactivation of the DNaseX gene, e.g. as regards a possible modification of the expression of other genes which can be connected with the development of SLE, for example. The knowledge of these genes and their function then also permits the development of further starting points serving for pharmacologically influencing e.g. SLE or cystic fibrosis or processes resulting in these diseases, e.g. by providing compounds specifically influencing the expression of these genes or the activity of the gene products.

The production of the aforementioned mice having a modified Dnase X gene/protein has shown that the missing DNase X gene or the missing Dnase X proteins or mutations within the DnaseX gene or changes within the Dnase X protein leads to

- fertility problems,

- increased lethality of female mice (the following generations show a higher ratio of male to female mice) ,

- disorders of the internal organs and/or internal bleeding,

- growth malfunction and increased lethality,

- increased state of anxiety and behavioral disorders,

- developmental disruption and functional disorders of the gastrointestinal tract

- increased sensitivity to pathogens.

By influencing the Dnase X gene or the Dnase X protein it is possible to have a positive influence on the negative effects which occur during heart or brain infarction. In certain cases it is possible to inhibit the expression of the Dnase X gene or the function of the Dnase X protein so that apoptotic processes are partially or completely reduced.

Hence the present invention also relates to the use of the non-human mammal according to the invention for further studying the diseases or for characterizing genes and/or testing substances, medicaments and/or therapy approaches correlated with the diseases where apoptosis plays a role or processes resulting in the diseases, in particular SLE.

The present invention is of major use for the development of an agent for the diagnosis, prevention or therapy of cancer, autoimmune diseases, AIDS and diseases where apoptosis plays a role. Activating the present DNaseX enables undesired cells to be removed by triggering apoptosis in well- calculated fashion. These cells comprise cancer cells and neuronal cells causing neurodegenerative processes. By the inhibition of the present DNase or the processes controlled by DNase it is possible to protect cells in well-calculated fashion from programmed cell death. They can then exert the desired effect on the organism. By applying murine DNaseX it is possible to remove and decompose mucus in the lungs of patients suffering from cystic fibrosis. DNaseX can be applied to patients suffering from systemic lupus erythematosus, and the immunogenic DNA complexes can be decomposed so as to prevent an immune response.

Brief description of the figures:

Figure 1 : Genomic sequence of the murine DNaseX

Figure 2: (a) splice form 1 of murine DNaseX, (b) splice form 2 of murine DNaseX, (c) splice form 3 of murine DNaseX.

Figure 3 : Gene structure of the DNaseX gene

The illustrated distribution of exons and introns represents the DNaseX wild-type allele. Three coding exons were removed for the production of the target vector and replaced by the neo-cassette. A DNaseX null allele was generated by homologous recombination in ES cells, essential parts of the coding region of which are deleted.

Figure 4: Immunohistochemical analysis of the murine DNaseX protein

A: expression of the DNaseX protein in a subpopulation of cells in the brain of a mouse embryo (E17,5) .

B+C: expression of the DNaseX protein in the intestinal epithelium of a mouse embryo (E17,5)

Figure 5: Immunohistochemical analysis of the murine DNaseX protein

Figure 6 : Northern blot

Expression of the DNaseX gene in murine adult tissue

SMu = sceletal muscle

A murine transferrin sample was hybridized for the purpose of comparison. It shows the amount of RNA applied per lane.

The following examples explain the invention. Example

As regards the methods used, reference is also made to Sambrook, J., Fritsch, E.F., and Maniatis, T. (Molecular Cloning: a laboratory manual; second edition; Cold Spring Harbor, Laboratory Press, 1989) and Current Protocols in Molecular Biology (John Wiley and Sons, 1994-1998), the techniques listed below, in particular the preparation of DNA or RNA or Northern blot being sufficient known to, and mastered by, the person skilled in the art.

Example 1 Isolation and characterization of the murine DNaseX-coding cDNA or genomic DNA

Human DNaseX cDNA (JFC4, accession number X90392) was labeled radioactively and hybridized with a murine fetal brain-cDNA library (Stratagene company, La Jolla) and a total genomic murine cosmid library (resource center Berlin, RZPD) . The stringency of hybridization was reduced to 50°C.

The DNA samples were labeled with α-³²P-dATP and -³²P-dCTP (3000 ci/mmol) in a randomly primed reaction (Feinberg and Vogelstein, Anal. Biochem. 132, pp. 6-13 (1983)). The hybridizations were carried out at 50°C overnight in 0.5 M Na phosphate, 7 % SDS, 0.2 % BSA, 0.2 PEG 6,000, 0.05 % polyvinyl pyrrolidone 360,000, 0.05 % Ficoll 70,000, and 0.5 % dextran sulfate. The unbound sample was removed in a wash step at 50°C in 40 mM Na phosphate, pH 7.2, 1 % SDS for 60 minutes. The signals on the membranes were detected at 80°C by X-ray films (XAR-5, Kodak company) .

A clone comprising the entire murine genomic sequence of the DNaseX gene was deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German-type collection of microorganisms and cell cultures] , Mascheroder Weg lb, Braunschweig, Germany) in accordance with the Budapest Treaty on March 2, 2001:

E. coli JFC-C491 DSM 14141

Example 2 Preparation of antibodies

Animals for the production of antibodies against murine DNaseX are immunized as follows using a synthetically prepared peptide of the sequence "CAS LTK KRL DKL ELR TEP GF" .

Immunization protocol for polyclonal antibodies in rabbits

600 μg purified KLH-coupled peptide in 0.7 ml PBS and 0.7 ml complete or incomplete Freund's adjuvant are used per immunization:

Day 0: 1^st immunization (complete Freund's adjuvant)

Day 14: 2 immunization (incomplete Freund's adjuvant; icFA) Day 28: 3 immunization (icFA) Day 56: 4 immunization (icFA) Day 80: bleeding to death.

The rabbit serum is tested in an immunoblot. For this purpose, the peptide used for the immunization is subjected to SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10 (1984), 203-209). The Western blot analysis was carried out as described in Bock, C.-T. et al., Virus Genes 8, (1994), 215-229. For this purpose, the nitrocellulose filter is incubated with a first antibody at 37°C for one hour. This antibody is the rabbit serum (1:10,000 in PBS). After several wash steps using PBS, the nitrocellulose filter is incubated with a second antibody. This antibody is an alkaline phosphatase-coupled monoclonal goat anti-rabbit IgG antibody (Dianova company) (1:5,000) in PBS. 30 minutes of incubation at 37°C are followed by several wash steps using PBS and subsequently by the alkaline phosphatase detection reaction with developer solution (36 μM 5 ' -bromo-4-chloro-3-indolylphosphate, 400 μM nitro blue tetrazolium, 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl ) at room temperature until bands become visible.

It shows that polyclonal antibodies according to the invention can be prepared.

Immunization protocol for polyclonal antibodies in chickens

100 μg purified KLH-coupled peptide in 0.8 ml PBS and 0.8 ml complete or incomplete Freund's adjuvant are used per immunization.

Day 0: 1^st immunization (complete Freund's adjuvant)

Day 28: 2 immunization (incomplete Freund's adjuvant; icFA) Day 50: 3 immunization (icFA)

Antibodies are extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention are detected.

Immunization protocol for monoclonal antibodies of rats

250 μg purified KLH-coupled peptide in 0.25 ml PBS and 0.25 ml complete or incomplete Freund's adjuvant are used per immunization. The peptide is dissolved in 0.5 ml (without adjuvant) in the 4 immunization.

Day 0: 1^st immunization (complete Freund's adjuvant)

Day 28: 2^nd immunization (incomplete Freund's adjuvant; icFA) Day 56: 3 immunization (icFA) Day 84: 4^th immunization (PBS) Day 87: fusion. Supernatants of hybridomas are tested in a Western blot. Monoclonal antibodies according to the invention are identified. One of these antibodies is the monoclonal rat antibody JFC-DNX6 used below.

Example 3 DNaseX is overexpressed in cells having a high turnover

In order to study the distribution of the DNaseX protein in mice, embryos were removed at various stages (E13,5; E 15,5; E17,5) and fixed in 4 % paraformaldehyde overnight. Thereafter they were fixed in 75 % ethanol overnight. The other fixation steps were as follows: in 85 % ethanol for 2 hours, in 96 % ethanol for 2 hours, in absolute ethanol for 2 hours, then in xylene overnight, then in xylene for 2 hours twice and thereafter in paraffin (58°C) overnight, then in paraffin for 2 hours twice. Thereafter, the embryos are embedded in plastic dishes, placed on a cooling plate to cool down, and then 6 μm sections are prepared. The sections are deparaffined in a descending alcohol sequence (2 x 10 minutes in xylene, 2 x 5 minutes in 100 % EtOH, in each case 1 x 4 minutes in 95 %, 80 %, 60 %, 30 % ethanol and then in water) .

The DNaseX epitopes are exposed by boiling the paraffin sections in citrate buffer. For this purpose, lOx citrate buffer (Antigen Retrival Citra, BioGenex company, San Ramon, CA 94583) is diluted 1:10 and the sections are sorted into a heat-resistant plastic box. In this box, the sections are heated in a microwave oven to a maximum for 2 minutes and to 180°C for 8 minutes. Thereafter, the sections are allowed to cool down for 30 minutes and then placed in PBS solution for 5 minutes. 400 ml peroxidase block solution are poured onto each slide and incubated in a chamber at room temperature for 40 minutes. For the purpose of rinsing the slides are incubated with PBS for 10 minutes and then wiped dry. The first antibody (JFC-DNX6) is diluted 1:100 with PBS, and 300 ml of this antibody solution are placed on each slide and incubated in a refrigerator in a chamber overnight. The slides are rinsed with PBS for 10 minutes. They are wiped dry, and 300 ml of the second antibody diluted in PBS at 1:100 (Alkaline Phosphatase conjugated AffiniPure Goat Anti- Rat IgG+IgM (H+L) , Jackson ImmunoResearch Laboratories, Inc.) are placed onto each slide. Thereafter, incubation is carried out at room temperature in a chamber for 45 minutes, followed by rinsing with PBS, and the slides are bathed in PBS for 10 minutes. The slides are wiped dry. Thereafter, staining is carried out using freshly prepared staining buffer A (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂ x 6 H₂0) . 10 ml of staining buffer A are used for staining. 33 μl BZip (5-bromo-4-chloro-3indolyl-phosphatase) (50 mg/2 ml dimethylformamide) and 66 μl NBT (NitroBlue tetrazolium) (10 mg/200 μl dimethylformamide) are then added. The resulting solution is blended thoroughly and placed in the dark. 300 μl liquid are used per slide. The slides are incubated for 2 minutes, then rinsed with water and placed one by one in freshly filtered hematoxylin for 30 seconds. Thereafter they are rinsed one by one with water until there is no more stain in the water. The slides are subsequently covered with Eukitt in bubble-free manner, and the analysis is carried out using a light microscope.

The result in shown in figures 4 A, B, C.

Another immunohistochemical study was carried out using adult murine tissue. The experiment was conducted analogously to that described above. It was shown that murine DNaseX protein occurs in murine kidneys, muscles, liver, brain and pancreas (figure 5) . Example 4 Northern Blot

Multiple tissue Northern blots were purchased from CLONTECH company (Palo Alto, California, U.S.A.) and used in accordance with the manufacturer's instructions. The DNA samples of DNaseX (BP 300 to Bp 760 of splice form 1, see figure 2a) was labeled radioactively and hybridized with the Northern blots. The random priming method was used to label double-stranded DNA.

a) Random priming:

100 ng DNA were dissolved in a volume of 12 μl for a typical labeling preparation. 10 minutes of heating to 95°C effects the denaturation of the DNA into single strands. The batch is stored on ice to prevent reassociation of the two complementary DNA strands. Complete the reaction batch by 4 μl OLB, 1 μl Klenow (1 U) and 2.5 μl a-³²P-dCTP and 2.5 μl a-³²P-dATP. Incubate at room temperature overnight. During this period, the complementary strand forms, based on the hexanucleotides attached to a single strand, by the Klenow fragment of E. coli DNA polymerase I. The DNA is labeled radioactively by incorporating α-³²P-dCTP and the α-³²P-dATP.

The non-incorporated nucleotides were separated by means of a home-made sephadex G-50 column. The separation principle of the column is based on exclusion chromatography. The relatively small nucleotides which are not incorporated fit into small pores of the column material whereas the DNA remains excluded therefrom. The volumes within which the nucleotides may range is thus greater than the volume available to the DNA. If a mixture consisting of DNA and nucleotides is placed on the column, the DNA will run faster through the column than the nucleotides. This permits the separation of the non-incorporated nucleotides. For this purpose, a Pasteur pipette is closed using a small glass bead. The Pasteur pipette is filled with sephadex G-50 dissolved in water ("Fine") until the filling material is located 5 cm below the top edge of the Pasteur pipette. The column is rinsed twice using TE. The radioactive labeling batch is applied, and 320 μl TE are added. The solution which has passed through the column is discarded. An Eppendorf tube is placed below the column. 350 μl TE are added. The radioactive solution that has passed through the column is discarded.

b) Hybridization:

The Northern blots were hybridized as described below. First, the Northern blots were prehybridized at 65°C in 10 ml hybridization solution (350 ml 20 % SDS, 500 ml 1 M phosphate buffer, pH 7.2; 150 ml distilled water). For this purpose, the Northern blots were incubated in a glass tube in a roll-over type hybridization furnace at 65°C for a period of 6 h.

The prehybridization solution was discarded. The radioactively labeled sample was placed on the filters with 10 ml hybridization solution (65°C) .

Hybridization was carried out overnight at 65°C. The filters were then washed twice with about 500 ml wash buffer (80 ml 1 M phosphate buffer, pH 7.2; 100 ml 20 % SDS, 1820 ml distilled water) in a shaking bath at 65°C for 30 minutes.

c) Autoradiography: The filters were welded into a plastic sheet. Autoradiography was carried out at -80°C in an X-ray cassette containing a reinforcing sheet made of calcium tungstate. The exposure period was 1 to 4 days depending on the intensity of the signal.

The results of the Northern blots carried out are shown in figure 6.

Claims

1. A DNA sequence which codes for murine DNaseX having the amino acid sequence shown in figure 2 a, b or c .

2. The DNA sequence, comprising the DNA sequence shown in figure 1.

3. The DNA sequence which codes for a protein having the biological properties of DNaseX which is a fragment, an allelic variant or another variant of the DNA sequence of claim 1 or 2.

4. An expression vector containing a DNA sequence according to any of claims 1 to 3.

5. A murine DNaseX protein or a protein having its biological activity, which is encoded by the DNA sequence according to any of claims 1 to 3.

6. An antibody which is specific for the protein of claim 5.

7. A diagnostic method of detecting a modified expression or concentration of DNaseX, comprising contacting a sample with a probe suited for specific hybridization with an mRNA transcribed by the DNA sequence according to any of claims 1 to 3 , a primer or primer set or an antibody according to claim 6 and subsequently determining directly or indirectly whether the concentration of DNaseX-mRNA or DNaseX protein in the sample differs from that of a control sample.

8. A diagnostic kit for carrying out the method according to claim 7, containing a probe suited for the specific hybridization with an mRNA transcribed by the DNA sequence according to any of claims 1 to 3 , a primer or primer set and/or an antibody according to claim 6.

9. A medicament containing a DNA sequence according to any of claims 1 to 3 , an expression vector according to claim 4 or a DNaseX protein according to claim 5.

10. Use of a compound defined in any of claims 1 to 5 for preventing and/or treating diseases where apoptosis plays a role .

11. Use according to claim 10, wherein the diseases are systemic lupus erythematosus (SLE) , AIDS, cancerous diseases, cystic fibrosis or atrophy of the prostate.

12. A non-human mammal, characterized in that DNaseX gene comprising a DNA sequence according to any of claims 1 to 3 is modified in ubiquitous or tissue-specific fashion.

13. The non-human mammal according to claim 12, wherein the DNaseX gene is inactivated.

14. A method of producing the non-human mammal according to claim 11 or 12, comprising the steps of:

(a) preparing a DNA fragment, in particular a vector, which contains a modified murine DNaseX gene, the DNaseX gene having been modified by inserting a heterologous sequence, in particular a selectable marker;

(b) preparing embryonic stem cells from a non-human mammal;

(c) transforming the embryonic stem cells from step (b) with the DNA fragment from step (a) , the DNaseX gene being modified in the embryonic stem cells by homologous recombination with the DNA fragment from (a) ;

(d) culturing the cells from step (c) ,

(e) selecting the cultured cells from step (d) for the presence of the heterologous sequence, in particular the selectable marker,

(f) producing chimeric non-human mammals from the cells from step (e) by administering these cells into mammal blastocysts by injection; and

(g) transferring the blastocysts obtained in step (f) into pseudo-pregnant female mammals and analyzing the resulting offspring with respect to a modified, in particular inactivated, DNaseX gene.

15. Use of the non-human mammal according to claim 12 or 13 or the non-human mammal obtainable according to a method as defined in claim 14 for further studying SLE, AIDS, tumor diseases and cystic fibrosis or for characterizing genes and/or testing substances, medicaments and/or therapy approaches correlated with these diseases or processes resulting in these diseases.

16. A non-human mammal according to claim 12 or 13, method according to claim 14 or use according to claim 15, wherein the non-human mammal is a mouse.