WO2000000603A2

WO2000000603A2 - Modularly constructed rna molecules having two sequence region types

Info

Publication number: WO2000000603A2
Application number: PCT/DE1999/001867
Authority: WO
Inventors: Annemarie Poustka; Johannes Coy
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date: 1998-06-26
Filing date: 1999-06-25
Publication date: 2000-01-06
Also published as: AU5407699A; EP1090115A2; DE19828624C1; CA2332175A1; WO2000000603A3

Abstract

The invention relates to modularly constructed RNA molecules which can bind to a ligand and which are characterized by two sequence regions. Said first sequence region contributes to the maintenance of the three-dimensional structure of the RNA molecule, and a second sequence region is responsible for the specific bond of the ligand. These RNA molecules, e.g. the NINTROX-RNA, can be used for directly influencing the gene expression. The invention also relates to vectors containing the inventive RNA molecules, to medicaments and diagnostic compositions which contain said RNA molecules or vectors, to an antibody which specifically recognizes these RNA molecules or antisense RNA which specifically binds to these RNA molecules, or to ribozymes which split these RNA molecules. In addition, the invention relates to non-human mammals whose NINTROX gene is modified by inserting a heterologous sequence and to the cells obtained therefrom.

Description

Modular RNA molecules with two sequence region types

The present invention relates to RNA molecules which are characterized by two sequence region types, a first sequence region type which contributes to the maintenance of the three-dimensional structure of the RNA molecule and a second sequence region type which is responsible for the specific binding of a ligand. These RNA molecules are preferably useful for direct control of gene expression. The present invention further provides the DNA sequence derived for the RNA molecules according to the invention and vectors containing them. Furthermore, the present invention relates to medicaments and diagnostic compositions which contain the above RNA molecules or vectors, an antibody which specifically recognizes these RNA molecules or antisense RNA which specifically binds to these RNA molecules, or ribozymes which split these RNA molecules. The invention also relates to non-human transgenic mammals and cells obtained therefrom.

The regulation of gene expression in eukaryotes is usually carried out via proteins that usually bind specifically to certain regulatory sequences upstream of the gene to be expressed and have a characteristic effect (RNA polymerases, transcription factors, hormone-activatable receptors etc.). So far, only a few examples of the control of gene expression directly via RNA molecules are known. These include the RNA "XI ST"("X-Chromosome inactivation specific transcript") responsible for inactivating the entire X chromosome, an RNA labeled "IPprinted"("imprinted in Prader-Willi syndrome") and the RNA H19, which is a tumor suppressor and is involved in the control of certain development processes. The artificial control of gene expression is meanwhile achieved through the use of antisense RNAs that specifically bind to mRNAs or through the use of catalytically active RNA molecules, so-called ribozymes, which not only specifically bind to the target RNA, but these also split and thus inactivate. However, the possible uses for these antisense RNAs or ribozymes are limited, especially with regard to the ligand to be bound and inactivated, which in principle can only be RNA.

There is therefore a need for the provision of compounds which can recognize or deactivate universally different target molecules, for example DNA, RNA, proteins or low molecular weight substances, and for example for the control of gene expression and thus of course also the prevention and treatment of diseases associated with associated with a gene expression disorder are suitable.

The invention is therefore essentially based on the technical problem of providing those compounds which are useful, inter alia, for the prevention or therapy (and also diagnosis) of such diseases.

This technical problem has been solved by providing the embodiments characterized in the patent claims.

The inventors have identified an RNA molecule that has the desired properties described above. This RNA molecule is encoded by the gene "NINTROX" (No INTROns X-chromosome), which has no introns, on which the X-chromosome is located and which does not encode any protein. This RNA molecule is part of certain (longer) transcripts of the MeCP2 gene. The MeCp2 gene (methyl CpG binding protein 2) in Xq28 has a transcript of approximately 1.8 kb which codes for the MeCP2 protein. The RNA described above is part of longer MeCP2 transcripts that also encode the MeCP2 protein but have a different 3'-untranslated region. This 3'-untranslated region is of crucial importance for the MeCP2 gene and its function. In the following, the term "NINTROX" is synonymous with the longer transcripts of the MeCP2 gene mentioned.

The genomic sequence of the human NINTROX gene is shown in FIG. 1 and the genomic sequence of the murine NINTROX gene in FIG. 2. In FIG. 3 a sequence comparison between human and murine sequence was carried out. From this it can be seen that there are some very sequence-conserved areas which are characterized by high energy according to an energy analysis carried out by computer (cf. FIG. 4).

While the mechanism of action of the genes which were active at the RNA level and which were discussed above was completely unclear, the principle of action of such a gene, which is described in more detail below, could be determined for the first time by analyzing the NINTROX gene. The NINTROX gene contributes significantly to the maintenance of the functions of the CNS, especially the hippocampus. Defects in this gene lead to restrictions on the CNS functions, which range up to mental retardation. Furthermore, the NINTROX gene has an important function in the control of cell proliferation. Changes in this gene can lead to errors in the control of cell growth, such as cancer. Changes in this gene can lead to increased or reduced DNA methylation. Increased DNA methylation can, among other things, restrict or prevent the activity of growth-controlling genes (tumor suppressor genes) and thus lead to a generally increased cancer rate. Reduced DNA methylation can lead, among other things, to overexpression of genes and the associated developmental disorders of the cell or the whole organism. Further investigations showed that the expression pattern of the NINTROX gene is tissue and development specific. The Northem analysis showed an expression in all examined fetal and adult tissues. No sequence homologies with already known sequences could be found.

The strategy that led to the identification of this nucleic acid molecule is described below. As part of the systematic analysis of the q28 region of the human X chromosome, various expressed sequences could be detected and isolated. With the help of these expressed sequences, some previously unknown genes could be identified and characterized according to standard methods, including the NINTROX gene on which the present application is based. Interestingly, the NINTROX RNA molecules according to the invention have a modular structure, ie they are characterized by the presence of two different sequence region types. While one sequence area allows the three-dimensional structure to be maintained and, as can be seen by comparing the sequences from different species (human, hamster, kangaroo, macaque, orangutan, chimpanzee and rat; see FIG. 5), it is only partially preserved , the second sequence region, which is responsible for the specific binding to the target molecule, is sequence conserved. Due to this modular construction of the NINTROX-RNA it is possible to modify it in such a way that its effect is not only limited to the control of the gene expression described above, but can be used for a multitude of possibilities. In addition to controlling the gene expression, the structure (for example chromatin structure, nuclear scaffold) of chromosomal regions can also be changed with the aid of such modular RNA molecules. This results in the previously unknown possibility of being able to influence the expression of larger genomic regions in a targeted manner. For example, certain sequence regions of the two modules of the NINTROX gene can be replaced by other sequences or even artificial sequences, whereby (a) the interaction of this RNA with other binding partners (RNA, DNA, other macromolecules and low-molecular compounds) or their biochemical implementation (e.g. increase or lowering the turnover rate) can be changed in a targeted manner, so that the RNA molecule can be specifically adapted to new tasks, and / or (b) the three-dimensional structure of the NINTROX-RNA can be specifically adapted to specific requirements. A partially or completely new function of the N1NTROX-RNA molecule according to the invention can thereby be achieved.

Thus, one embodiment of the present invention relates to an RNA molecule that can bind to a ligand and comprises the following sequence regions: (a) a sequence region that maintains the three-dimensional structure of the RNA molecule, and (b) a sequence region for the specific binding of the ligand.

The expression "a sequence region maintaining the three-dimensional structure of the RNA molecule" used here has the following content. Three-dimensional RNA structures are created by base pairing different bases within of the RNA molecule. Structures such as "stars" or "loops" are formed. Many of these structures thus form the overall structure of the RNA molecule. A sequence change within the RNA molecule can have no consequences for the spatial structure if the sequence change does not change the base pairings or if the sequence change is compensated for by a second sequence change. If, for example, the base pair AT is destroyed by mutating the A to the G, this mutation can be compensated for by the further mutation of the T to the C. This changes the sequence, but the spatial structure remains the same. As a result, the same RNA structure can be formed by an extremely large number of different RNA sequences. The analysis of the energy contained therein provides clues to certain RNA structures. This analysis can be carried out using commercially available computer programs (for example "FOLD"; Michael Zuker and P. Stiegler: Optimal Computer Folding of Large RNA Sequences using Thermodynamics and Auxiliary Information, Nucleic Acids Research (81), 9 (1), p. 133) become. The lower the energy content of a certain sequence, the more stable the three-dimensional RNA structures. Analysis of the NINTROX gene showed a conserved distribution of these low-energy structures (see FIG. 4). The base sequence of these RNA regions are very different in different species, but the energy content is extremely conserved. In Fig. 3 these are the sequence areas that are not identified by a black bar on the edge. This means that the sequence region which maintains the three-dimensional structure of the RNA molecule is not sequence-conserved but energy-conserved. So modifications of this sequence area do not depend on the base sequence, but on the preservation of the determined energy content.

The term "a sequence region for the specific binding of the ligand" as used herein refers to a sequence region which is designed in such a way that it can specifically bind the desired ligand. These sequence areas are very sequence conserved. In Fig. 3, these areas are identified by a black bar on the edge and have a high energy content (see. Fig. 4). This coincides with the observation that these sequence regions are not "packaged" but are directed outwards and are responsible for the binding of the ligand, enzymatic reactions or the binding to other RNA or DNA sequences. If the ligand to be bound is an RNA molecule or a DNA If this is a molecule, this sequence region will have complementarity with a corresponding, sufficiently long section of the RNA molecule or DNA molecule. If the ligand to be bound is a protein, the sequence region (b) can be replaced or supplemented in whole or in part by a DNA sequence which is known to specifically bind the desired protein.

The two sequence types described above occur several times within the NINTROX RNA. The exchange or modification of individual such modules enables the targeted modification of the NINTROX-RNA. When modifying the module that maintains the three-dimensional structure, attention must be paid to the energy content determined so that it maintains a minimum value. The modification of the other sequence area, although this is currently considered to be sequence conserved, is subject to only minor restrictions. This area can be omitted in whole or in part or can contain insertions. For example, sequences can also be integrated into the NINTROX RNA molecule that have known biochemical properties or bind certain DNA, RNA molecules or proteins. In addition, random sequences of different lengths can be inserted at different locations in the NINTROX gene and then selected for specific properties such as biochemical conversion, specific binding, etc.

In a preferred embodiment of the RNA molecule according to the invention, the sequence region (a) comprises the sequence regions not marked at the edge in FIG. 3 or sequences related thereto, which likewise allow the three-dimensional structure of the RNA molecule to be maintained and from the sequence region (a) in FIG 3 deviates. These deviations relate to the addition, deletion and / or insertion of bases, the energy content determined for the sequence of FIG. 3 being retained at least 80%, preferably at least 85% and more preferably at least 90%. With these changes introduced, the original three-dimensional structure is preferably retained.

In a particularly preferred embodiment, the sequence region (b) of the RNA molecule according to the invention comprises the sequences shown in FIG Border with black bars.

In a further preferred embodiment of the RNA molecule according to the invention, the ligand to be bound is a DNA molecule or a protein or enzyme, for example DNA polymerase I. The RNA molecule according to the invention preferably contains a poly (A) sequence on 3 ^' end, which can contribute to stability in a desired host cell.

In a further preferred embodiment, the RNA molecule according to the invention is used to control gene expression. For this purpose, the sequence region (b) is modified in such a way that it binds a protein responsible for gene expression, or to a specific DNA region of the target gene, as a result of which, for example, the attachment of proteins which have an inhibiting or promoting effect on gene expression is hindered or prevented is, or directly to the mRNA of the target gene, whereby, for example, translation is hindered or prevented. The person skilled in the art can readily modify the RNA molecule according to the invention by appropriate modifications of the sequence region (b) and possibly also of the sequence region (a) in such a way that it binds the desired ligand and thus controls the gene expression to the desired extent.

The present invention also relates to a DNA sequence encoding the RNA molecule according to the invention and a gene with the following features: It contains a promoter which allows transcription in a desired host cell and a DNA which is functionally linked and encodes the RNA molecule according to the invention - sequence. The gene preferably additionally contains a termination signal and a polyadenylation site.

In a preferred embodiment, the gene according to the invention comprises the sequence shown in FIG. 1 or 2.

The DNA sequences or genes encoding the RNA molecule according to the invention can also be inserted into a vector. Thus, the present invention also includes vectors containing these DNA sequences or genes. The term "vector" refers to a plasmid (eg pJC18, pBR322, pBlueScript) Virus or other suitable vehicle. In a preferred embodiment, the sequence coding the DNA molecule according to the invention is functionally linked in the vector to regulatory elements which allow its expression in prokaryotic or eukaryotic host cells. In addition to the regulatory elements, for example a promoter, such vectors typically contain an origin of replication and specific genes which allow the phenotypic selection of a transformed host cell. The regulatory elements for expression in prokaryotes, for example E. coli, include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOX1 or GAL1 promoter in yeast, and the CMV, SV40 , RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells. Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter. Suitable vectors include, for example, T7-based expression vectors for expression in bacteria (Rosenberg et al., Gene 56 (1987), 125), pMSXND for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263 (1988) , 3521) and baculovirus-derived vectors for expression in insect cells.

In a preferred embodiment, the vector containing the sequences encoding the RNA molecules according to the invention is a viral vector, for example a vaccinia virus or adenovirus, which is useful in gene therapy. RNA viruses, especially retroviruses, are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. For the purposes of gene therapy, the RNA molecules according to the invention can also be transported to the target cells in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682).

General methods known in the art can be used to construct expression vectors containing the sequences encoding the RNA molecules of the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as are described, for example, in Sambrook et al.

The present invention also relates to the vectors described above containing host cell. These host cells include bacteria, yeast, insect and animal cells, preferably mammalian cells. Preferred mammalian cells are CHO, VERO, BHK, HeLa, COS, MDCK, 293 and WI38 cells. Methods for transforming these host cells, for phenotypically selecting transformants and for expressing the nucleic acid molecules according to the invention using the vectors described above are known in the art.

The present invention also relates to antibodies which specifically recognize the RNA molecule according to the invention. The antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof, for example Fab, Fv or scFv fragments. It is preferably a monoclonal antibody. The antibodies according to the invention can be produced according to standard methods, the RNA molecule according to the invention or a fragment thereof serving as an immunogen. Monoclonal antibodies can be produced, for example, by the method described by Köhler and Milstein (Nature 256 (1975), 495) and Galfre (Meth. Eπzymol.73 (1981), 3), mouse myeloma cells being fused with spleen cells derived from immunized mammals . These antibodies can be used, for example, to inhibit the activity of the RNA molecules according to the invention, for example to influence gene expression. The antibodies can also be used in diagnostic assays, for example, in order to be able to demonstrate whether there is a dysregulation of gene expression, for example due to a loss or deficiency of the responsible NINTROX RNA. The antibodies can be present in liquid phase in immunoassays or can be bound to a solid support. The antibodies can be labeled in different ways. Suitable markers and labeling methods are known in the art. Examples of immunoassays are ELISA and RIA.

The invention further relates to antisense RNAs which bind specifically to an RNA molecule according to the invention and which can be used in vitro or in vivo to reduce the expression of genes which are under the direct control of RNA, for example NINTROX-RNA. The administration of the antisense RNA according to the invention to a target cell leads to a reduced gene expression and is particularly useful for the treatment of diseases which are characterized by a too high gene expression of the gene under direct RNA control are (for example, cancer). The antisense RNAs can be administered directly or inserted as DNA encoding them, preferably in a suitable vector. Suitable vectors include all of the vectors already described above in connection with the RNA molecules according to the invention.

The antisense RNAs according to the invention comprise an antisense sequence with at least 7 to 10 or more nucleotides which hybridize specifically with a sequence of the RNA molecule according to the invention, for example NINTROX RNA. The antisense RNA according to the invention preferably has a length of about 10 to about 50 nucleotides or of about 14 to about 35 nucleotides. In further embodiments, the antisense RNAs according to the invention are RNAs that are shorter than approximately 100 nucleotides or shorter than approximately 200 nucleotides. In general, the antisense RNAs should be long enough to form a stable double helix, but short enough (depending on the type of delivery) to be administered in vivo if desired. In general, the antisense sequence to ensure specific hybridization is essentially complementary to the target sequence. In certain embodiments, the antisense sequence is exactly complementary to the target sequence. However, the antisense RNAs can also contain nucleotide substitutions, additions, deletions, transitions, transpositions or modifications, as long as the specific binding to the relevant target sequence is retained as a functional property of the antisense RNA. The antisense RNAs can also contain additional sequences in addition to the antisense sequences. The antisense RNAs (and also the RNA molecules according to the invention) can be produced using any method suitable for producing nucleic acids, for example by chemical synthesis de novo or by cloning. An antisense RNA can also be produced, for example, by inserting a sequence of the target RNA or a fragment thereof in the reverse orientation, functionally linked to a promoter, into a vector (for example a plasmid). Provided that the promoter and preferably termination and polyadenylation signals are correctly positioned, the strand of the inserted sequence corresponding to the non-coding strand is transcribed and this acts as an antisense RNA. The present invention also relates to ribozymes which specifically cleave the RNA molecules according to the invention and are therefore also useful for inhibiting gene expression. Useful ribozymes can include 5 'and 3' terminal sequences that are complementary to the target RNA, and these can be constructed by one of ordinary skill in the art (see, for example, PCT publication WO 93/23572). The ribozymes according to the invention include, for example, ribozymes with the features of the group I intron ribozymes (Cech, Biotechnoiogy 13 (1995), 323) and "hammerhead" ribozymes (Edgington, Biotechnoiogy 10 (1992), 256).

In one embodiment, the ribozymes according to the invention are used per se as medicaments. In another embodiment, gene therapy methods are used to express ribozymes in a target cell ex vivo or in vivo. The methods for administering the ribozymes or for expressing the ribozymes in vivo correspond to the methods described above in connection with the RNA molecules according to the invention.

The isolation and characterization of the human NINTROX gene and in particular the mouse homolog of the NINTROX gene allows the establishment of an animal model that allows the provision of therapies and drugs for the diseases discussed above. By providing the sequence of the NINTROX gene, both diagnosis (post- or prenatal) and therapy of diseases in which gene expression is characterized by the lack of NINTROX-RNA or an excess of NINTROX-RNA is possible. However, the therapeutic or diagnostic application is not only limited to diseases which are associated with a malregulation of the expression of a gene which is under the control of the NINTROX-RNA, but the RNA molecules modified according to the possibilities described above also offer the possibility of completely new ones Therapeutics.

The present invention thus further relates to medicaments which contain the above-described RNA molecules, vectors, antibodies, antisense RNAs or ribozymes. These drugs may also contain a pharmaceutically acceptable carrier. Suitable carriers and the formulation of such medicaments are known to the person skilled in the art. Suitable carriers include Wise phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions etc. The drugs can be administered orally or parenterally. Methods for parenteral administration include topical, intra-arterial (e.g., directly to a tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The appropriate dosage is determined by the attending physician and depends on various factors, for example the age, gender, weight of the patient, the stage of a tumor, the type of administration, etc.

The medicament according to the invention is preferably used for the prevention or treatment of diseases which are related to a disturbance in the control of the gene expression. The medicament according to the invention is particularly preferably used for the treatment of tumor diseases or diseases of the CNS. The medicinal product can be used in therapy, the methods or vectors described above being used to introduce the nucleic acids according to the invention. On the other hand, the RNA molecule according to the invention can be administered directly, so as to restore normal expression of the gene in cells which no longer have functional copies of the RNA molecule.

The present invention further relates to a diagnostic composition which contains the RNA molecule according to the invention, the DNA sequence encoding it or a fragment thereof, the antibody according to the invention or a fragment thereof, or the antisense RNA according to the invention or a fragment thereof or combinations thereof, if necessary together with a suitable means of detection. This diagnostic composition can be used to determine whether the RNA which directly controls gene expression, for example NINTROX-RNA, is present or is present in too high or low concentration or with a different length compared to a control. The antibody or a fragment thereof is preferably used in the assays described above or the antisense RNA or a fragment thereof as a probe in hybridization experiments. For this purpose, the probe preferably has a length of at least 10, particularly preferably at least 15 bases. Suitable on hybridization based detection methods are known to the person skilled in the art. Suitable labels for the probe are also known to the person skilled in the art and include, for example, labeling with radioisotopes, bioluminescence, chemiluminescence, fluorescence labels, metal chelates, enzymes etc. In this method, methods known to the person skilled in the art regarding the preparation of total RNA or poly (A) + RNA from biological samples, the separation of the RNAs on size-separating gels, for example denaturing agarose gels, the production and labeling of the probe and the detection of the hybrids, for example via "Northem blot"; be applied. The diagnosis of diseases as described above in connection with the medicaments according to the invention is preferably carried out.

A diagnosis can also be made at the DNA level. The nucleic acid molecules described above are used to examine the intactness of the gene encoding the RNA directly involved in the regulation of gene expression, for example NINTROX-RNA (for example with regard to presence, length or mutations). In this method, methods known to the person skilled in the art regarding the preparation of DNA from biological samples, the restriction digestion of the DNA, the separation of the restriction fragments on size-separating gels, for example agarose gels, the preparation and labeling of the probe and the detection of the hybridization, for example via " Southern blot "; be applied. The above detection can also be carried out using PCR. Primers are used to flank the coding sequence. Diagnostically important are amplification products of DNA from the tissue in question, which differ, for example in terms of their length or sequence, from the amplification products of DNA from healthy tissue.

The present invention further relates to a non-human mammal whose NINTROX gene is modified, e.g. by inserting a heterologous sequence, in particular a selection marker sequence.

The term "non-human mammal" includes any mammal whose NINTROX gene may be altered. Examples of such mammals are mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, with mouse Rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, with mouse being preferred.

The expression "NINTROX gene that is altered" means that the NINTROX gene naturally occurring in the non-human mammal is deleted by approximately 1-2 kb by standard methods. If desired, a heterologous sequence, e.g. a construct for mediating antibiotic resistance (e.g. a "neo-cassette") is inserted. This method is generally described in Schwartzberg et al., Proc. Natl. Acad. Be. USA, Vol. 87, pp. 3210-3214, 1990, to which reference is made here.

Another object of the present invention are cells obtained from the above non-human mammal. These cells can be in any form, e.g. in a primary or long-term culture.

A non-human mammal according to the invention can be provided by conventional methods. A method is favorable which comprises the following steps:

(a) production of a DNA fragment, in particular a vector, containing an altered NINTROX gene, the NINTROX gene being altered by deleting a homologous sequence and / or inserting a heterologous sequence, in particular a selectable marker;

(b) preparation of 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 NINTROX gene in the embryonic stem cells being changed by homologous recombination with the DNA fragment from (a),

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

(e) Selection of the cultured cells from step (d) for the lack of homologous I

Sequence or presence of the heterologous sequence, in particular the selectable marker,

(f) Generation of chimeric non-human mammals from the cells of step (e) by injection of these cells into mammalian blastocysts (preferably mouse blastocysts), transferring the blastocysts into pseudo-pregnant female mammals (preferably mouse) and analysis of the offspring obtained on a change in the NINTROX gene.

In step (c), the mechanism of homologous recombination (cf. R.M. Torres, R. Kühn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997) is used to transform embryonic stem cells. The homologous recombination between the DNA sequences present in a chromosome and new, added cloned DNA sequences enables the insertion of a cloned gene into the genome of a living cell instead of the original gene. With this method, embryonic germ cells can be used to obtain via chimeras animals that are homozygous for the desired gene or the desired gene part or the desired mutation.

The term "embryonic stem cell" refers to any embryonic stem cells from a non-human mammal that are suitable for mutating the NINTROX gene. The embryonic stem cells are preferably from the mouse, in particular the cells E14 / 1 or 129 / SV.

The term "vector" encompasses any vector which, by recombination with the DNA of embryonic stem cells, enables the NINTROX gene to be changed. The vector preferably has a marker which can be used to select for existing stem cells in which the desired recombination has taken place. Such a marker is e.g. the loxP / tkneo cassette, which can be removed from the genome again using the Cre / loxP system.

Furthermore, the person skilled in the art knows conditions and materials in order to carry out steps (a) - (f). The present invention provides a non-human mammal whose NINTROX gene is altered. This change can be a switching off of the gene expression regulating function. With such a mammal or cells from it, the gene expression-controlling function of NINTROX can be investigated selectively. It is also possible to find substances, drugs and therapeutic approaches that can be used to selectively influence the controversial function of NINTROX. The present invention therefore provides a basis for acting on a wide variety of diseases. Such diseases are, for example, restrictions on the CNS functions, which extend to mental retardation, or the induction of cancer due to errors in the control of cell proliferation. It should also be possible to investigate and characterize the role of the hippocampus in more detail.

The following clones were deposited on May 4, 1998 with the DSMZ, German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig:

DSM 12153: E.coli JFC-484, partial sequence of the human NINTROX cDNA DSM 12154: E.coli JFC-622, partial sequence of the murine NINTROX cDNA DSM 12155: E.coli JFC-8D3, sequence of the human genomic NINTROX-

DNA DSM 12156: E. coli JFC-P1-165, sequence of the murine genomic NIN-

TROX DNA

The figures show:

Figure 1: Human sequence of the NINTROX gene

Figure 2: Murine sequence of the NINTROX gene

FIG. 3: sequence comparison of human (top) and murine (bottom) sequence of solid bars: sequence-conserved areas (b)

FIG. 4: energy diagram of the sequences from FIG. 3 Figure 5: Homology comparison of NINTROX from different species

Fig. 5a: Partial sequence from the hamster Fig. 5b: Partial sequence from the kangaroo Fig. 5c: Partial sequence from the macaque Fig. 5d: Partial sequence from the orangutan Fig. 5e: Partial sequence from the rat Fig. 5f: Partial sequence from the chimpanzee

The following example explains the invention:

Example 1: Identification and characterization of the NINTROX gene

To identify transcribed sequences from the region Xq2-7.3 to Xqter, total RNA was first isolated from various pig tissues (kidney, heart, spleen, liver, brain, etc.) and overwritten with the help of oligo-dT in first-strand cDNA. These complex cDNA samples, which represent all genes transcribed in the respective tissue, were then radioactively labeled and hybridized with the Xq27.3-Xqter-specific cosmid library. The cosmid library was analyzed in the form of cosmid clones systematically arranged on nylon membranes. The cosmid clones, which had positive hybridization signals with the complex cDNA samples, were then isolated, digested with EcoRI, separated by gel electrophoresis and transferred to nylon membranes. The restriction fragments, which then had a positive hybridization with the complex, radioactively labeled cDNA samples, were then isolated and radioactively labeled and used to search a fetal human cDNA library. As a result, positive cDNA clones could be isolated, which represented the transcript of the NINTROX gene.

Claims

1 patent claims

1. RNA molecule which can bind to a ligand and comprises the following sequence regions:

(a) a sequence region maintaining the three-dimensional structure of the RNA molecule; and

(b) a sequence region for the specific binding of the ligand.

2. RNA molecule according to claim 1, wherein the sequence region (a) comprises the derived DNA sequence shown in Figure 3 without a bar at the edge or a sequence related thereto, which also allows the three-dimensional structure of the RNA molecule to be maintained.

3. RNA molecule according to claim 1 or 2, wherein the sequence region (b) comprises the derived DNA sequence shown in Figure 3 with bars on the edge.

4. RNA molecule according to one of claims 1 to 3, wherein the ligand is a DNA molecule or a protein.

5. RNA molecule according to one of claims 1 to 4, which additionally contains a poly (A) sequence at the 3 ^' end.

6. RNA molecule according to one of claims 1 to 5 for controlling gene expression.

7. DNA sequence encoding an RNA molecule according to any one of claims 1 to 6.

8. gene comprising the sequence shown in Figure 1 or 2.

9. Vector containing the DNA sequence according to claim 7 or the gene according to claim 8. iy

10. The vector of claim 9, wherein the vector is a plasmid.

11. The vector of claim 10, wherein the vector is a viral vector.

12. The vector of claim 11 which is an RNA virus.

13. The vector of claim 12 which is a retrovirus.

14. Host cell containing the vector according to any one of claims 9 to 13.

15. The host cell of claim 14, wherein the host cell is a mammalian cell.

16. Antibodies or a fragment thereof which specifically bind an RNA molecule according to one of claims 1 to 6.

17. The antibody of claim 16, wherein the antibody is a monoclonal antibody.

18. Antisense RNA which specifically binds to an RNA molecule according to one of claims 1 to 6.

19. Ribozyme that specifically cleaves an RNA molecule according to one of claims 1 to 6.

20. Use of the RNA molecule according to one of claims 1 to 6, the vector according to one of claims 9 to 13, the antibody or the fragment thereof according to claim 16 or 17, the antisense RNA according to claim 18 or the ribozyme according to claim 19 for the manufacture of a medicament for the prevention or treatment of diseases related to a disorder in the control of gene expression.

21. Use of the RNA molecule according to one of claims 1 to 6, the DNA sequence according to claim 7 or a fragment thereof, the antibody or the fragment thereof according to claim 16 or 17, or the anti sense-RNA according to claim 18 or a fragment thereof for the diagnosis of diseases which are associated with a disturbance in the control of gene expression.

22. Use according to claim 20 or 21, wherein the disease is a tumor disease or a disease of the central nervous system.

23. Non-human mammal whose NINTROX gene is altered by deleting a homologous sequence and / or inserting a heterologous sequence.

24. The non-human mammal of claim 23, wherein the heterologous sequence is a selection marker sequence.

25. The non-human mammal of claim 23 or 24, wherein the selection marker sequence mediates resistance to neomycin.

26. A method for producing a non-human mammal according to any one of claims 23-25, characterized by the following steps:

(b) preparation of embryonic stem lines from a non-human mammal (preferably mouse);

(c) transforming the embryonic stem cells from step (b) with the DNA fragment from step (a), the NINTROX gene in the embryonic stem cells being changed by homologous recombination with the DNA fragment from (a), (d) culturing the cells of step (c),

(e) selection of the cultured cells from step (d) for the absence of the homologous sequence and / or the presence of the heterologous sequence, in particular the selectable marker,

(f) Generating chimeric non-human mammals from the cells of step (e) by injecting these cells into mammalian blastocysts (preferably mouse blastocytes), transferring the blastocysts into pseudopregnant female mammals (preferably mouse) and analyzing the progeny obtained for one Change in the NINTROX gene.