DE10350256A1 - PIM-1-specific siRNA compounds - Google Patents

Abstract

The present invention provides dsRNAs specific for PIM-1 kinase. Further, these containing host cells are provided. In a further aspect, the compositions containing them are disclosed, in particular for the treatment of pain and other PIM-1-related diseases.

Description

The Invention relates to small, in particular interference double-stranded RNA molecules (dsRNA), which are directed against PIM-1, and the ds RNA according to the invention containing host cells. The dsRNA according to the invention and corresponding Host cells are useful as drugs or for the production of Medicines, in particular for the treatment of pain and others associated with PIM-1 pathological conditions.

pain is according to the definition of the International Society for Study of Pain (IASP) an "unpleasant Sensory and emotional experience, associated with acute or potential tissue damage or with terms of such damages is described "(Wall and Melzack, 1999).

The Effective treatment of pain is a big challenge for the molecular Medicine. Acute and transitory pain is an important signal of the body, to protect people from severe damage from the environment or overload of the body to preserve. In contrast, the chronic pain that lasts longer than the cause of the pain and the expected timeframe of healing is no known biological function and affects hundreds of millions People worldwide. Around 7.5 million people suffer in the Federal Republic alone Germany with chronic pain. Unfortunately, the pharmacological Treatment of chronic pain is still unsatisfactory and therefore remains a challenge for the current medical research. The currently existing analgesics are common not sufficiently effective and have z. T. serious side effects.

numerous Proteins are known as starting points in pain research. Which also includes the family of PIM kinases, for example, among these Proteins described the PIM-1 as a starting point for the treatment of pain is. Thus, the PIM-1 kinase is a promising starting point for the Development of new pain medications dar. The cDNA sequence of human PIM-1 kinase is found in the databases under AN: NM 002648. The amino acid sequence of human PIM-1 kinase, is found in the databases under AN: NP_002639. The cDNA sequence of PIM1 kinase, rat, is found in the databases under AN: NM_017034. The amino acid sequence of PIM1 kinase, rat, is found in the databases under AN: NP_058730. The cDNA sequence of PIM-1 kinase, mouse, is found in the databases under AN: NM_008842. The amino acid sequence of PIM-1 kinase, mouse, is found in the databases under AN: NP_032868.

The Belongs to PIM-1 kinase to the family of serine / threonine kinases involved in evolution highly conserved in multicellular organisms. The kinase is one Enzyme, the phosphate groups of ATP or another nucleoside triphosphate transfers to another molecule. In the Control of cell growth, cell differentiation and apoptosis the PIM-1 plays a role. The following belong to the PIM kinase family PIM kinases: PIM-1, PIM-2, PIM-3, PIM-4 (Fox, CJ, et al., Al. Deneen, B., et. al. 2003, Fisher, K., et. al. 2001). A variety Growth factors may stimulate expression of PIM-1 and transcription, posttranscription, translation and posttranslation regulate. Also, the PIM-1 kinase an important role outside of the hematopoietic Systems assigned. PIM-1 phosphorylates HP1 and other targets, such as. cdc25 and c-myb (Koike, et al, 2000, Mochizuki, et al. 1999, Winn, et. al., 2003).

There Pain is multi-layered, there is a need for new targets, such. Eg PIM-1, as well as for Molecules that tie against these targets. For example, low molecular weight are known Drugs or other compounds such as antisense oligodeoxynucleotides (ODN), for the treatment of chronic pain in particular.

In the unpublished German patent application DE 10226702.2 such antisense oligonucleotides against PIM-1 are disclosed, as pain therapeutics. However, such antisense oligonucleotides have the disadvantage that they have poor cell permeability and low efficiency.

The Object of the present invention is to provide further substances for disposal nonetheless, to efficiently modulate the effect of PIM-1 but without further components possess cell permeability and efficient the Can modulate expression of PIM-1 and also galenically well can be used.

The The above object is characterized by that in the claims Objects of solved present invention. According to the invention to the solution task PIM-1-specific dsRNAs provided the phenomenon of To trigger RNA interference capital.

On the phenomenon The RNA interference was discovered during immunological research attentively.

The Immune system higher Eukaryotes has namely developed a set of defense mechanisms against pathogens. you So should assume that the genome before the complete adoption of such protects molecular invaders.

In In recent years, an RNA-based defense mechanism has been discovered both in the kingdom of mushrooms, as well as in the plant and animal kingdom occurs and how an "immune system of the genome "works. The system was originally independently from each other in different species, first in C. elegans (Fire et. al., 1998), before considering the underlying mechanisms identify as identical at certain levels of the processes could: RNA-mediated virus resistance in plants (Lindbo and Dougherty, 1992), PTGS (posttranscriptional gene silencing) Plants (Napoli et al., 1990) and RNA interference in eukaryotes are based on a common mode of operation (Plasterk, 2002).

The In vitro technique of RNA interference (RNAi) is based on double-stranded RNA molecules (dsRNA), which trigger the sequence-specific suppression of gene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5: 485-490: Hannon (2002) Nature 41: 244-251). The activation of protein kinase R and RNaseL caused the Transfection of mammalian cells with long dsRNA nonspecific effects, e.g. an interferon response (Stark et al (1998) Annu., Rev. Biochem 67: 227-264; He and Cat (2002) Viral Immunol. 15: 95-119). These non-specific Effects become shorter when using, for example, 21 to 23 merer, so-called dsRNA ("small interfering RNA ") bypassed, as nonspecific effects by dsRNA, which is shorter than 30 by is not triggered (Elbashir et al., (2001) Nature 411: 494-498). Recently, dsRNA molecules were also used in vivo (McCaffrey et al., (2002) Nature 418: 38-39; Xia et. al. (2002), Nature Biotech. 20: 1006-1010; Brummelkamp et. al. (2002), Cancer Cell 2: 243-247.

The double-stranded RNA (dsRNA) of the present invention contains a sequence of general structure 5 '- (N _17-25 ) -3', where N is any base and is nucleotides. The general structure consists of a double-stranded RNA with a macromolecule composed of ribonucleotides, the ribonucleotide consisting of a pentose (ribose), an organic base and a phosphate. The organic bases in the RNA consist of the purine bases adenine (A) and guanine (G) as well as the pyrimidine bases cytosine (C) and uracil (U). The dsRNA contains nucleotides with a directed structure with overhangs.

Preferably, dsRNAs of the invention have the general structure 5 '- (N _19-25 ) -3', more strongly 5 '- (N _19-24 ) -3', even more preferably 5 '- (N _21-23 ) -3' where N is any base. In this case, at least 90%, preferably 99% and in particular 100% of the nucleotides of a dsRNA according to the invention may be complementary to a section of the (m) RNA sequence of PIM-1. 90% complementary here means that, for example, given a length of 20 nucleotides of a dsRNA according to the invention, this is not complementary to the corresponding segment on the (m) RNA for a maximum of only 2 nucleotides. The sequence of the double-stranded RNA according to the invention, with its general structure, is preferably completely complementary to a section of the (m) RNA of PIM-1.

Basically an inventive dsRNA to any section on the mRNA or primary transcript of PIM-1, or against mRNAs or primary transcripts of other members be complementary to the PIM kinase family.

In A eukaryotic cell is used for the production of an mRNA the gene in its entire length, both introns and exons transcribed into a long RNA molecule, the primary transcript. The stability of mRNA is made by processing the primary transcript at the 5 'end with an addition of an untypical nucleotide with a methylated guanine and a Polyadenylation at the 3 'end. Before the RNA leaves the nucleus, be through RNA splicing the intron sequences removed and the exons joined together.

target sequences for dsRNA according to the invention can both the primary transcript as well as the processed mRNA. The primary transcript and the mRNA become collectively referred to below as (m) RNA.

In principle, everyone can 17 to 29, preferably 25, occurring in the coding region of the (m) RNA Base pairs long sections as the target sequence for a dsRNA according to the invention serve. Particularly preferred are also those target sequences for dsRNAs according to the invention, between positions 75 and 900 (calculated from the AUG start triplet the coding region of the (m) RNA of human PIM-1). Prefers are therefore such 17 to 25, in particular 19 to 25 and especially 21 to 23 base-pair sections on the (m) RNA, their initial nucleotide a nucleotide of position 50 to 890 of the coding region the PIM-1 (m) RNA, and its end nucleotide 17 to 25, preferably 19 to 25 and most preferably 21 to 23 nucleotides further downstream from the starting nucleotide.

Especially preferred are those dsRNAs which are coding against regions in the Area of PIM-1 (m) RNA are directed. In particular, such should be dsRNAs according to the invention be directed against PIM-1 (m) RNA sections in the central area of the coding region, preferably at least 50, 70, 100 Nucleotides removed from the AUG start triplet of the (m) RNA, or at least 50 nucleotides, preferably at least 70 even more preferred 100 nucleotides from the 3'-terminal coding region of the (m) RNA.

All Particular preference is given to those siRNAs which are resistant to portions in the coding region of the PIM-1 (m) RNA, which with Start the starting sequence AA. Here, siRNAs become very special preferred, the complementary and are thus directed against (m) RNA sections, the example. the sequence sequence AACGACCUGCA at the 5'-terminus, the sequence AAGCUGGCG, the Sequence AAGGAGAA, the sequence AAGGAGCCC, the sequence AACUUGCCGGUGG, the sequence AAACACGU, the sequence AAGGACCGGA, the sequence AAUGGCACUCG, the sequence AAGUGGUCC, the sequence AAGAAGGUGA, the sequence AAGAUCUCUUCGA, the sequence AAAGGGGAGC, the sequence AAGAGGAGCUGG, the sequence AACUGCGGGGU, the sequence AAGGACGAAAACA, the sequence AAAACAUCCUU, the sequence AACAUCCUUA, the sequence AAUCGCGGCGA, the sequence AAGCUCAUC, the Sequence AAGGACACCGUC, the sequence AAGAGAUCAUCA, the sequence AAUGUCAGCAUCU, the sequence AACCUUCGAAGA, the sequence AAGAAAUCCAG or the sequence AACCAUCCAU, preferably in the 5'-terminal Range of the target sequence, or in principle included in the target sequence.

nevertheless could dsRNAs according to the invention also against nucleotide sequences on the PIM-1 (m) RNA that are not in the be directed, in particular in the non-coding 5 'region of the (m) RNA, having the regulatory functions.

The boundaries at the 5 'end of the target sequence with, for example, AA of the nucleotide bonds are also reflected in the associated dsRNA according to the invention in a sequence sequence 5'-AAN _15-23 and the complementary strand 3'-TTN _15-2 3. This sequence can be directed in the translated and untranslated region and against all control elements. One strand of double-stranded RNA is thus complementary to the primary or processed RNA transcript of the PIM-1 gene.

Preferably however, it will be for effective blocking and cleavage of the (m) RNA of PIM-1 particularly achieved by that for the choice of the target sequence of dsRNA according to the invention certain selection rules considered become.

A particularly preferred embodiment In the present invention, a dsRNA having a GC content of at least 38%, in a stronger one preferred embodiment from to 38 to 60%, and in a more preferred embodiment from 38 to 50% (1). In a further particularly preferred embodiment According to the present invention, dsRNA molecules according to the invention have at most 2 consecutive Guanidine residues in their sequence (2). Another particularly preferred Embodiment of The present invention is a target sequence to be investigated in Genome only present in the target gene, but not elsewhere in the genome (3). Further particularly preferred are combinations of aforementioned properties in fiction, contemporary dsRNAs. So will the Target sequence of a dsRNA according to the invention particularly preferably occur only once in the target genes.

Various Combinations of the aforementioned properties are possible, eg. the properties of (1), (2) and / or (3).

The Target sequence of the PIM-1 (m) RNA is preferably selected from a group consisting of the following 5 sequences: (a) AAC GUG GAG AAG GAC CGG AUU; (b) AAC UCG AGU GCC CAU GGA AGU; (c) AAC CGC GAC AUC AAG GAC GAA, (d) AAU AUU CCU UUC GAG CAU GAC; (e) AAG GGU CUC UUC AGA AUG UCA.

at a dsRNA according to the invention For example, a modified nucleotide may preferably occur. The term "modified Nucleotide " according to the invention the respective nucleotide is chemically modified. The expert understands under the term "chemical Modification "that the modified nucleotide by replacing, adding or removing individual or more atoms or atomic groups compared to naturally occurring ones Nucleotides changed is. At least one modified nucleotide in dsRNA according to the invention On the one hand serves the stability and on the other hand prevention of dissociation.

Preferably are between 2 and 10, especially between 2 and 5 nucleotides modified.

Preferably can the ends of the double-stranded ones RNA (dsRNA) can be modified to cause degradation in the cell or counteract a dissociation into the single strands, in particular to avoid premature degradation by nucleases.

A regularly not desired Dissociation of single strands of dsRNA occurs especially when using low concentrations or short chain lengths on. For particularly effective inhibition of dissociation by the the nucleotide pairs brought cohesion of the double-stranded structure of inventive dsRNA by at least one, preferably more chemical linkage (s) increase. An inventive dsRNA, their dissociation is reduced, has a higher stability against enzymatic and chemical degradation in the cell or organism or ex vivo on.

The chemical linkage the single strands a dsRNA according to the invention is expediently by a covalent or ionic bond, hydrogen bonding, hydrophobic interaction, preferably van der Waals or stacking interactions, or formed by metal ion coordination. She can after one particularly advantageous embodiment feature on at least one, preferably at both ends. It has continued proved to be advantageous that the chemical linkage means one or more connection groups is formed, wherein the Compound groups preferably poly (oxyphosphinicooxy-1,3-propanediol) - and / or polyethylene glycol chains are. The chemical linkage can also by in the double-stranded Structure instead of purines used purine analogues are formed. It is also advantageous that the chemical linkage by in the double-stranded structure introduced Azabenzene units is formed. It can also by in the double-stranded structure formed in place of nucleotides used branched nucleotide analogues become.

It has proved to be useful at least one of the following to produce the chemical linkage Groups used: methyl blue; bifunctional groups, preferably Bis (2-chloroethyl) -amine; N-acetyl-N '- (p-glyoxybenzoyl) -cystamine; 4-thiouracil; Psoralen. Furthermore, the chemical linkage by at the ends of the double-stranded Area attached thiophosphoryl groups are formed. Preferably becomes the chemical linkage at the ends of the double-stranded Area by triple helix bonds produced. The chemical linkage may suitably be induced by ultraviolet light.

The Modification of the nucleotides of the dsRNA leads to deactivation one at double-stranded RNA dependent Protein kinase (PKR), in the cell. PKR induces apoptosis. advantageously, is at least one 2'-hydroxy group the nucleotides of the dsRNA in the double-stranded structure by a chemical group, preferably a 2'-amino or a 2'-methyl group replaced. At least one Nucleotide in at least one strand of the double-stranded structure can also be a so-called "locked nucleotide "with one, preferably by a 2'-O, 4'-C-methylene bridge, chemical be modified sugar ring. Advantageously, several Nucleotides "locked nucleotides ".

modifications the nucleotides of dsRNA according to the invention concerns especially the dissociation of the nucleotides by amplification of the nucleotides Hydrogen bonding. The stability the nucleotide is increased and against attack by RNA sen protected.

A another possibility to prevent premature Dissociation of dsRNA according to the invention in the cell exists through the formation of the hairpin loop. In a particular embodiment has a dsRNA according to the invention a hairpin structure to slow the dissociation kinetics. In such a structure is preferably at the 5 'and / or 3' end a loop structure educated. Such a loop structure has no hydrogen bonds.

Moreover, by modifying the backbone of the dsRNA according to the invention, premature degradation can be prevented. Especially preferred is dsRNA which is (phosphorothioate, 2'-O-methyl-RNA, LNA, LNA / DNA gapmer) and therefore has a longer half-life in vivo.

A dsRNA according to the invention is preferably against the (m) RNA of the PIM kinases, in particular the PIM-1 kinase, of mammals, like human, ape, rat, dog, cat, mouse, rabbit, guinea pig, hamster, Beef, pork, sheep and goat, derived.

Preferably repressed the dsRNA the expression of PIM-1 in the cell at least 50%, 60%, 70%, at least 90%; it is thus dsRNA according to the invention then so-called siRNA, which triggers the phenomenon of RNA interference. The Measurement of suppression can over Northern blot, quantitative Real-time PCR or at the protein level with PIM-1 specific antibodies.

Farther can dsRNAs according to the invention, in particular human dsRNAs according to the invention, both having so-called "blunt ends" or overhanging ends.

Basically, overhanging end at least two overhanging nucleotides have, preferably 2 to 10, in particular 2 to 5 overhanging nucleotides at the 3'-terminus, if appropriate, however, alternatively at the 5'-terminus.

at the overhanging one Ends are dTdT, each at the 3'-terminus the double-stranded one siRNA according to the invention prefers.

The overhanging Nucleotides can, as mentioned above, dT (desoxy thymidine) or else uracil, in principle can have any overhanging ends to the mRNA of PIM-1 complementary dsRNA according to the invention duplexes attached become.

A preferred embodiment the dsRNA according to the invention is directed against a target sequence of the PIM1 mRNA which is the general Structure 5'-AAG UGU ACU UUA GGC AAA GGG-3 '(rat) having. A particularly preferred dsRNA of the present invention is therefore a duplex molecule, its sense strand the sequence 5'-GUG UAC UUU AGG CAA AGG GdTdT-3 'and whose antisense strand has the sequence 5'-CCC UUU GCC UAA AGU ACA CdTdT-3 '. This is directed against the aforementioned portion of the PIM-1 mRNA.

The dsRNA is prepared by methods known to those skilled in the art, thereby be nucleotides, especially oligonucleotides, for example in the manner of the Merryfield synthesis, on an insoluble carrier (H. G. Gassen, Chemical and Enzymatic Synthesis of Gene Fragments (Verlag Chemie, Weinheim 1982)) or otherwise synthesized (Beyer / Walter, Lehrbuch of Organic Chemistry, 20th Edition, (S. Hirzel Verlag, Stuttgart 1984), p. 816 ff.). The recovery of the mRNA of PIM-1 can by Hybridization can be achieved by means of genome and cDNA databases. siRNA molecules can, for example. be prepared synthetically various providers, for example IBA GmbH (Göttingen, Germany) become.

Double-stranded RNA according to the invention (dsRNA) may be included in micellar structures, which are the Influence separation of substance groups in vitro and in vivo. In this case, the dsRNA is preferably present in liposomes. The liposomes are artificial, Globular self-contained membranes, phospholipids, in the encapsulating both hydrophilic substances in the aqueous interior, as well as lipophilic substances in the interior of the lipid membrane can be incorporated. The requirement for the use of liposomes for experimental or therapeutic purposes is their compatibility with cells and tissues. The dsRNA, preferably in the liposomes can be present with a peptide sequence, preferably with a lysine and arginine-rich sequence, for example, a sequence from the viral TAT protein (eg containing AS 49-57) may be modified in order then as a transporter peptide to overcome the cell membrane easier.

The dsRNA can also be found in viral natural capsids or in chemical or enzymatic route produced artificial capsids or thereof be included in derived structures. The features mentioned enable introducing the dsRNA into predetermined target cells.

According to a further particularly advantageous embodiment, it is provided that the dsRNA is bound to, associated with or surrounded by at least one virus-derived, derived or synthetically produced viral coat protein. The coat protein can be derived from the polyomavirus. The coat protein may contain the virus protein 1 (VP1) and / or the virus protein 2 (VP2) of the polyomavirus. The use of such envelope proteins is for example from DE 19618797 A1 known. The before mentioned features significantly facilitates the introduction of dsRNA into the cell.

In a preferred embodiment the expression of the dsRNA according to the invention takes place in that the 1. Template (sense dsRNA) and the 2nd template (antisense dsRNA) under the control of two identical or different promoters stand. Here, the expression takes place in vivo and is gene therapy via vectors brought into the cells.

One Another object of the present invention is a pharmaceutical containing at least one dsRNA according to the invention and / or one of these containing cell, and optionally suitable auxiliary and / or Additives.

• 1. Krankheiten, Leiden, Körperschäden oder krankhafte Beschwerden zu heilen, lindern, zu verhüten oder zu erkennen,
• 2. die Beschaffenheit, den Zustand oder die Funktionen des Körpers oder seelische Zustände erkennen zu lassen.
• 3. vom menschlichen Körper erzeugte Wirkstoffe oder Körperflüssigkeiten zu ersetzen,
• 4. Krankheitserreger, Parasiten oder körperfremde Stoffe abzuwehren, zu beseitigen oder unschädlich zu machen oder
• 5. die Beschaffenheit, den Zustand oder die Funktionen des Körpers oder seelische Zustände zu beeinflussen.

Medicines: a substance corresponds to the definition in Article 1 § 2 of the German Law on Marketing of Medicinal Products (AMG). That is, substances or preparations of substances intended for use on or in the human or animal body

• 1. To cure, alleviate, prevent or recognize diseases, ailments, physical injuries or pathological complaints
• 2. To identify the nature, condition or functions of the body or mental states.
• 3. replace active substances or body fluids produced by the human body,
• 4. to ward off, eliminate or neutralize pathogens, parasites or foreign substances, or
• 5. to influence the nature, condition or functions of the body or mental states.

The medicaments of the invention can as liquid Dosage forms in the form of injectable solutions, drops or juices, as semi-solid dosage forms in the form of granules, tablets, pellets, Patches, capsules, patches or aerosols are administered and contained in addition to the at least one article of the invention depending on the galenic form optionally carrier materials, fillers, Solvent, diluent, Dyes and / or binders. The selection of excipients as well the quantities to be used depends on whether the medicine oral, parenteral, intravenous, intraperitoneal, intradermal, intramuscular, intranasal, buccal, rectal or locally, For example, on infections of the skin, mucous membranes and on the eyes, to be applied. Suitable for oral administration preparations in the form of tablets, dragees, capsules, granules, Drops, juices and syrups, for parenteral, topical and inhalative administration, solutions, Suspensions, easily reconstitutable dry preparations as well Sprays. Inventive objects in one Depot in dissolved Form or in a plaster, optionally with the addition of the Promoting skin penetration Agents are suitable percutaneous application preparations. Oral or Percutaneously applicable preparation forms can release the articles according to the invention with a delay. The amount of drug to be administered to the patient varies dependence the weight of the patient, the type of application, the indication and the severity of the disease. Usually 2 to 500 mg / kg applied at least one article of the invention. When the medicine is used in particular for gene therapy should recommend as suitable auxiliaries or additives, for example a physiological saline solution, Stabilizers, proteinase, DNase inhibitors etc.

One Further subject matter of the present invention relates to host cells, excluding human germ cells and human embryonic stem cells, which are transformed with at least one dsRNA according to the invention. DsRNA molecules according to the invention can be used in the respective host cell via conventional methods, eg transformation, transfection, transduction, electroporation or particle gun are introduced. In the transformation will be at least two of the different dsRNAs are introduced into the cell, wherein a strand of each dsRNA is at least partially complementary to (m) RNA of PIM-1. The complementary Range of dsRNA to (m) RNA from PIM-1 contains less than 25 consecutive Nucleotide pairs.

When Host cells are all cells of pro- or eukaryotic nature Consider, for example, of bacteria, fungi, yeasts, plant or animal cells. Preferred host cells are bacterial cells, such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic Microorganisms, such as Aspergillus or Saccharomyces cerevisiae or the ordinary baker's yeast (Stinchcomb et al (1997) Nature 282: 39).

In a preferred embodiment, however, cells from multicellular organisms are selected for transformation by means of dsRNA constructs according to the invention. In principle, any higher eukaryotic cell culture is available as a host cell, albeit mammalian cells, such as monkeys, rats, hamsters, mice or humans, are particularly preferred. A variety of established cell lines are known to those skilled in the art. In a non-exhaustive list, the following cell lines are named: 293T (embryonic kidney cell line) (Graham et al., J. Gen. Virol. 36: 59 (1997), BHK (baby hamster kidney cells), CHO (cells from the hamster ovaries, holiday and chasin, Proc Natl Accad Sci USA 77: 4216, (1980)), HeLa (human carcinoma cells) and other cell lines, eg HEK293, SF9 or COS cells, especially established for laboratory use human cells, in particular neuronal stem cells and cells of the "pain pathway", preferably primary sensory neurons.Human cells, in particular autologous cells of a patient, are suitable for (especially ex vivo) transformation with dsRNA molecules according to the invention, especially as medicaments for For example, gene therapy purposes, ie after performing a cell collection, possibly ex vivo expansion, transformation, selection and final retransplantation in the patient.

One Another preferred object is also the use of at least a dsRNA according to the invention or pharmaceutical composition and / or a cell according to the invention for the preparation of a medicament or analgesic, for treatment pain, especially chronic pain, tactile allodynia, thermally triggered Pain, and / or inflammatory pain.

The Objects of the invention are suitable as a drug, eg. For the inhibition of nociception, eg. due to the reduction in expression of PIM1 kinase by means of dsRNA according to the invention.

Furthermore is the use of at least one dsRNA according to the invention, containing dsRNA according to the invention, and / or a cell according to the invention for the manufacture of a medicament for the treatment of urinary incontinence; also of neurogenic bladder symptoms; Pruritus, tumors, inflammations; especially PIM-1 kinase-associated inflammation with symptoms such as asthma; as well as all disease symptoms associated with PIM-1.

One Another object of the invention is a method of treatment, especially pain treatment, of a non-human mammal or people, the or the treatment of pain, in particular chronic pain, needed by administration of a medicament according to the invention, in particular those containing a dsRNA according to the invention. Another item are also appropriate procedures for treatment of pruritus and / or Urinary incontinence.

One Another preferred object is also the use of at least an siRNA according to the invention and / or a cell according to the invention for the Gene therapy, preferably in vivo or in vitro gene therapy.

One Another preferred object is also the use of at least a dsRNA according to the invention and / or a cell according to the invention for the Gene therapy, preferably in vivo or in vitro gene therapy. Under Gene therapy is a form of therapy in which the introduction of Nucleic acids in Cells, for example, an effector gene, usually a protein, is expressed, In the present case, in particular, dsRNA according to the invention. In principle, a distinction is made between in vivo and in vitro methods. In-vitro method Cells are removed from the organism and ex vivo with vectors transfected, then again to be introduced into the same or another organism. In in vivo gene therapy, vectors such as for fight of tumors, systematically (e.g. the bloodstream) or directly into the tumor. According to one preferred embodiments a vector is administered which contains both the transcriptional element for the Sense dsRNA, as well as the transcriptional element for the antisense dsRNA under the control of suitable promoters. According to another preferred embodiment can the two transcription elements lie on different vectors.

One Another preferred object is also a diagnostic containing at least one dsRNA and / or a cell according to the invention and optionally suitable additives. Diagnostic means herein a compound or method that can be used to to diagnose a disease.

• (a) gentechnische Manipulation mindestens einer Zelle (Testzelle) mit mindestens einem erfindungsgemäßen dsRNA-Konstrukt,
• (a') parallele gentechnische Manipulation mindestens einer identischen Zelle (Kontrollzelle) entweder • unterbleibend, • unter Durchführung der Manipulation parallel ohne dsRNA oder dsRNA-Konstrukt oder • mit einer veränderten nicht mehr erfindungsgemäßen dsRNA.
• (b) parallele Inkubation einer zu testenden Substanz unter geeigneten Bedingungen mit mindestens einer Test-Zelle und mindestens einer Kontrollzelle und/oder einer Präparation aus einer solchen Zelle, die mindestens ein Protein, ausgewählt aus der PIM-1-Familie, vorzugsweise der PIM-1-Kinase, synthetisiert hat.
• (c) Messung der Bindung der Testzubstanz an dem von den Zellen synthetisierten Protein oder Messung mindestens eines der durch die Bindung der Testsubstanz and das Protein veränderten funktionellen Parameter.
• (d) Identifizierung der Substanzen über das Ausmaß der Differenz zwischen dem Messwert bei der Testzelle und dem bei der Kontrollzelle.

A further preferred subject matter is also a method for identifying pain-modulating substances with the following method steps:

• (a) genetic manipulation of at least one cell (test cell) with at least one dsRNA construct according to the invention,
• (a ') parallel genetic manipulation of at least one identical cell (control cell) either • non-existent, • while performing the manipulation in parallel without dsRNA or dsRNA construct or • with an altered dsRNA no longer according to the invention.
• (b) parallel incubation of a substance to be tested under suitable conditions with at least one test cell and at least one control cell and / or preparation from such a cell containing at least one protein selected from the PIM-1 family, preferably the PIM 1-kinase, synthesized.
• (c) measuring the binding of the test sample to the protein synthesized by the cells or measuring at least one of the functional parameters altered by the binding of the test substance to the protein.
• (d) Identification of the substances by the extent of the difference between the measured value in the test cell and that in the control cell.

there the term refers to a potential to modulate pain regulating influence on the physiological pain event, in particular to an analgesic effect. The term substance includes each as a drug ingredient suitable compound, in particular so low molecular weight drugs, but others like nucleic acids, Fats, sugars, peptides or proteins such as antibodies. Incubation under suitable Conditions here are to be understood that the to be examined Substance with the cell or the appropriate preparation in an aqueous Medium can react a defined time before the measurement. there can the watery Medium tempered, for example, between 4 ° C and 40 ° C, preferably at Room temperature or at 37 ° C. The incubation time can be between a few seconds and several hours be varied, depending on the interaction of the substance with the Protein. However, cells are preferred between 1 min and 60 min. The aqueous Medium may contain suitable salts and / or buffer systems, so that during incubation, for example, a pH between 6 and 8, preferably pH 7.0-7.5 in the medium prevails. The medium can further suitable substances such as coenzymes, nutrients, etc. are added. The suitable conditions can by the expert in dependence from the interaction of the substance with the protein to be investigated because of his experience, the literature or less, a fold Preliminary tests can be easily determined in order to maximize the process to obtain a clear reading. A cell that synthesizes a protein has is a cell that already endogenously expresses this protein or has one that has been genetically engineered, so they do this Protein expressed and correspondingly from the beginning of the method according to the invention contains the protein. The cells can Cells from evt. Immortalisierten cell lines be or native Tissues derived from and isolated from these cells, the Cell structure mostly dissolved is. The preparation from these cells in particular comprises homogenates from the cells, the cytosol, a membrane fraction of cells with membrane fragments, a suspension of isolated cell organelles etc.

• – genetisch manipuliert: Manipulation von Zellen, Geweben oder Organismen derart, dass hier genetisches Material eingebracht wird.
• – endogen exgrimiert: Expression eines Proteins, die eine Zellinie unter geeigneten Kulturbedingungen aufweist, ohne das dieses entsprechende Protein durch gentechnische Manipulation zur Expression veranlasst wurde.

The yardstick by which the method allows the discovery of interesting substances is either the binding to the protein, which can be detected, for example, by displacement of a known ligand or the extent of bound substance, or the alteration of a functional parameter by the interaction of the substance with the protein , This interaction may in particular be in the regulation, inhibition and / or activation of receptors, ion channels and / or enzymes and modified functional parameters may be, for example, gene expression, the ion milieu, the pH or the membrane potential, or the change in the enzyme activity or the concentration of the 2 ^{nd be} messenger. It means:

• - genetically manipulated: manipulation of cells, tissues or organisms such that genetic material is introduced here.
• Endogenously expressed: expression of a protein which has a cell line under suitable culture conditions, without this corresponding protein being induced by genetic manipulation for expression.

A further preferred embodiment This method provides that the cell already before the process steps (a) and (a ') are genetically engineered is manipulated.

A further preferred embodiment This procedure provides that the genetic manipulation the measurement of at least one of the modified by the test substance functional parameters allowed.

A further preferred embodiment This procedure provides that through genetic manipulation a non-endogenously expressed form of a member in the cell the PIM kinase family, preferably PIM-1 kinase or a reporter gene introduced becomes.

A further preferred embodiment of this method provides that the measurement of the binding via the displacement of a known labeled ligand of a member PIM kinase family, vorzugswei the PIM-1 kinase.

A further preferred embodiment This method provides that between the parallel process steps (a) and (a ') and the method step (b) ≥ 8 h, preferably ≥ 12 h, in particular ≥ 24 h, pass away.

The Introducing the objects according to the invention in the Cell can be done in the manner outlined above.

The Figures show:

1A and B shows fluorescence micrographs (A) and corresponding Western blot analyzes (B) of cells used to compare the activities of PsiR4 for inhibition of expression of PIM-1 with a PIM-1-GFP-encoding plasmid and various concentrations of PsiR4 were cotransfected. PsiR4 was used in concentrations between 1.5 and 50 nM. PsiR4inv served as a control representing the inverted sequence of PsiR4. As shown by the fluorescence microscopic analysis in (A), the siRNA of the present invention suppresses PIM-GFP expression (reduces the number of visible cells) even at a concentration of 5 nM. Western blot analysis (B) confirms the fluorescence microscopy experiments.

1C shows a Northern blot analysis of the specific degradation of PIM-1 (m) RNA in cotransfection experiments. As control for the amount of RNA served an actin specific probe. C stands for the PIM-1-GFP control, + stands for the effect of 50 nM PsiR4 on PIM-1 mRNA and - stands for the effect of 50 nM PsiR4inv on PIM-1 (m) RNA. PsiR4inv is an inverted control sequence.

2A shows the effect of 5 'sense Cy3 labeling of PsiR4 on PIM-1-GFP expression. A shows in the upper row the GFP fluorescence of COS-7 cells. The second row shows the Cy3 fluorescence of COS-7 cells. Depicted are cotransfection experiments with 10 nM or 50 nM PsiR4-Cy3 as indicated. The controls were performed alone with PIM-1-GFP.

2 B Figure 3 shows a Western blot analysis of the down-regulation of PIM-1-GFP with PsiR4 (-) and PsiR4-Cy3 (+) at concentrations of 0 nM, 5 nM and 50 nM.

3 shows a fluorescence microscopic image of the colocalization of PIM-1-GFP and PsiR4-Cy3 24 hours after cotransfection of COS-7 cells. The labeled arrow # 1 identifies cells expressing PIM-1 GFP in the absence of detectable levels of siRNA. The labeled arrow # 2 identifies cells that do not express PIM-1 GFP but a visible portion of siRNA. The labeled arrow # 3 indicates cells expressing PIM-1 GFP and containing a detectable amount of siRNA. The over-housings were carried out with Adobe Photoshop 7.0.

4 shows a fluorescence microscopic image of the location of PsiR4-Cy3 24 hours after lipofection in live F-11 cells. The left image shows F-11 cells in phase contrast, while the right one shows the same cells that contain PsiR4-Cy3.

5 shows fluorescence micrographs of F-11 cells with neutritic-like structures transfected with PsiR4-Cy3. The localization of siRNA in the neurite-like structures is indicated by arrows.

6A shows a graphical representation of a quantitative evaluation of an intrathecally administered PIM-1 siR4 in the Bennet model (rat). Shown are the number of paw lifts on a 4 ° C cold plate on 4 consecutive days. As a control served the administration of a NaCl solution. In the illustration, the days are plotted on the X axis and the paw lifts per 2 minutes on the Y axis. The points each show the mean of two independent experiments with the given standard deviation.

6B shows a graphical representation of a quantitative evaluation of an intrathecally administered PIM-1siR4KTR in Bennetmodell (rat). Shown are the number of paw lifts on a 4 ° C cold plate on 4 consecutive days. As a control served the administration of a NaCl solution. In the illustration, the days are plotted on the X axis and the paw lifts per 2 minutes on the Y axis. The points each show the mean of two independent experiments with the specified Standard deviation.

Table 1 Characteristic siRNAs against PIM-1

siRNA against PIM-1

at both sequences is first the sequence segment of PIM-1 (m) RNA lists from which the PIM-1 siRNA was derived.

The Duplex sequences are the actual ones in vivo and in vitro tested double-stranded RNA molecules.

example 1

Generation of PIM-1 more specific siRNA

A set of 5 siRNAs was constructed according to the criteria of the Dharmacon's homepage (Www.dharmacon.com).

These siRNAs were transfected into COS-7 cells at various concentrations, and the effect on PIM-1-GFP expression was examined by fluorescence microscopy and Western blot analysis. The siRNAs PsiR1, PsiR2 and PsiR3 showed no significant down-regulation of the PIM-1-GFP reporter gene in concentrations up to 100 nM. Only PsiR4 resulted in strong PIM-1 downregulation at low concentrations and therefore PsiR4 was studied in detail. The down-regulation efficiency of PsiR4 was examined in comparison to a control siRNA consisting of the inverted PsiR4 sequence in a concentration range of 1-50 nM. At the fluorescence level, a reduction of cells with a green fluorescent phenotype at a concentration of 5 nM was observed, whereas the inverted control showed no detectable downregulation effect up to a concentration of 50 nM (FIG. 1A ) showed. The Western Blot experiments confirmed the effects obtained by fluorescence imaging ( 1B ) in an independent method. PIM-1 undergoes autophosphorylation and is therefore in 1B represented by double bands representing different phosphorylation states of the protein. At a concentration of 1 nM PsiR4 showed no effect, however, the PIM-1-GFP expression was clearly reduced at a concentration of 5 nM. Higher siRNA concentrations led to more than 90% extinction of PIM-1-GFP, while the control siRNA showed no significant effect.

To prove that the observed siRNA extinction phenotypes are based on the degradation of PIM-1 (m) RNA, Northern blot analysis was performed with a PIM-1 specific probe. PIM-1 (m) RNA was degraded in the presence of PsiR4, but was unchanged in the presence of the inverted control siRNA at a concentration of 50 nM ( 1C ).

Example 2

Effectiveness of a Cy3-labeled PIM-1 siRNA

The labeling of siRNAs, for example, with a fluorescent dye is possible at 4 different positions: at the 5 'and 3' ends of the sense or antisense strand. Here, labeling was performed at the 5 'end of the sense strand with Cy3 to label PsiR4. To study cellular uptake, distribution, and localization, the effect of PsiR4-Cy3 labeling on siRNA function was first investigated. For this purpose, the reporter gene PIM-1-GFP was cotransfected with different concentrations of PsiR4-Cy3 and the efficacy of the labeled and unlabelled siRNA on PIM-1 expression was compared. Cellular uptake at low nanomolar concentrations of PsiR4-Cy3 was observed and this uptake continued to increase to a concentration of 50 nM ( 2A ). Western blot analysis confirmed extinction of PIM-1 with PsiR4-Cy3 on protein levels ( 2 B ). Compared to unlabelled PsiR4, we observed only a small but reproducible negative effect of dye labeling on siRNA efficiency. At low nanomolar concentrations, the unlabelled siRNA was more effective, but the downregulation effect for both began at a concentration of 5 nM.

Example 3

Cellular uptake of PsiR4

The cellular uptake of PsiR4-Cy3 in COS-7 cells was examined by lipofection. Cells with Cy3 fluorescence were observed as early as 15 minutes after transfection. The number of transfected cells increased up to 4 hours after lipofection and then plateaued (data not shown). During this period, more than 95% of the cells took up PsiR4-Cy3. Furthermore, PIM-1-GFP and a low concentration of PsiR4-Cy3 (10nM) were cotransfected to ensure that a significant number of cells continue to express PIM-1-GFP in the equivalent of siRNA. Under these conditions, it was observed that most cells containing PsiR4-Cy3 did not show a GFP phenotype ( 3 , labeled arrow no. 2 denotes a few examples) and conversely, most cells which had a distinct GFP phenotype showed no PsiR4-Cy3 fluorescence ( 3 , marked arrow No. 1). Some of the cells contain Cy3-labeled siRNA and a GFP phenotype ( 3 marked arrow No. 3).

Example 4

Localization of PsiR4 in F-11 cells

To study the intracellular localization of PsiR4-Cy3 in detail, F-11 cells were transfected. These cells are larger than COS-7 cells and allow the siRNA to be localized by fluorescence microscopy in living cells. A punctate perinuclear localization of PsiR4-Cy3 was observed after lipofection ( 4 ). This staining pattern is probably due to the endosomal uptake of siRNA by F-11 cells. The accumulation around the nucleus may be an indication that siRNAs are being imported into the nucleus to perform functions in heterochromatin formation. PsiR4-Cy3 was localized not only at the nucleus but also in the neurite-like structures ( 5 ) found.

Example 5

Efficacy of PIM-1 siRNA and the PIM-1 control siRNA in pain treatment in vivo

pain model the rat after Bennett

neuropathic Pain occurs, i.a. after injury peripheral or central nerves and can therefore be subjected to animal experiments through targeted lesions induce and observe single nerves. An animal model is the nerve lesion according to Bennett (Bennett and Xie (1988) Pain 33: 87-107). In the Bennett model is the sciatic nerve unilaterally provided with loose ligatures. It will to observe the development of signs of neuropathy pain and can be quantified by thermal or mechanical allodynia become.

For this purpose, male Sprague-Dawley rats (Janvier, France) weighing from 140 to 160 grams initially anesthetized with pentobarbital (50 mg per kg body weight of the rat Nembutal ^®, ip, Sanofi, business cooperative German veterinarians eG, Hannover, Germany) mixture. Subsequently, unilateral multiple ligatures were performed on the right main sialic nerve of the rats. To this end, the sciatic nerve was exposed at the level of the mid-thigh and four loose ligatures (SoftCAT ^® chrome USP 4/0, metric2, Braun Melsungen, Germany) have been tied around the sciatic nerve that epineural blood flow was not interrupted. The day of surgery was day 1.

The Allodynia was tested on day 2 on a metal plate with Was a water bath at a temperature of 4 ° C tempered. The rats were before the intravenous Application of the respective solution divided into groups of 9 or 10 animals. To check the allodynia, the Rats placed on the cold metal plate, which was in a plastic cage. Then it was over counted a period of two minutes before applying a solution, such as often the animals with their damaged Paw from the chilled metal plate to pull back violently (Zero value). Then the solutions became containing 3.16 μg (5 μl) siRNA according to the invention in 15 μl NaCl or 1 ng (5 μl) Control RNA (sense strand of siRNA) in 15 μl NaCl i.t. applied, and the number of pull-out reactions was in each case after 60 minutes again for 2 minutes counted (Test value). The measurements were made on 4 consecutive days (Days 2 to 5). Animals that are pure NaCl solution was applied both in the experiments with siRNA as well as with control RNA as a comparison group.

The PIM-1 siRNA according to the invention (1 ng) showed a strong analgesic effect in this pain model, as shown by the reduction in the withdrawal reactions of up to about 1/3 compared to the NaCl control on days 2 to 4 ( 6A ). In comparison, the PIM-1 control siRNA was ineffective ( 6B ).

Effect of intrathecal PIM-1siRNAs in the Bennett model (rat) Measurement of cold allodynia (number of paw lifts on a 4 ° C cold plate per 2 minutes).

Effect of intrathecal PIM-1 control siRNA in the Bennett model (rat). Measurement of cold allodynia (number of paw lifts on a 4 ° C cold plate per 2 minutes).

Claims (36)

Double-stranded RNA containing a sequence of general structure 5 '- (N _17-25 ) -3' that is at least 90%, preferably 99% and 100% complementary to a portion of the (m) RNA sequence of PIM-1 where N is any base. Double-stranded RNA according to claim 1, containing a sequence with the general structure 5 '- (N _19-25 ) -3'. Double-stranded RNA according to claim 1 or 2, containing a sequence with the general structure 5 '- (N _21-23 ) -3'. double RNA according to one of the claims 1 to 3, wherein the sequence having the general structure is completely complementary to a Section of the (m) RNA of PIM-1. double RNA according to a the claims 1 to 4, wherein the double-stranded RNA (dsRNA) against nucleotide sections between position 50 and position 890 in the coding region of PIM-1 (m) RNA. double RNA according to claim 1 to 5, wherein dsRNA against nucleotide portions of the coding region of PIM-1 (m) RNA containing at least 50, 70, 100 nucleotides from the AUG start triplet the coding region of the (m) RNA are removed. double RNA according to claim 5 or 6, wherein the dsRNA against nucleotide portions of the coding Region of PIM-1 (m) RNA containing at least 50 nucleotides, preferably at least 70 even stronger preferably at least 100 nucleotides from the 3'-terminal coding region of (m) RNA is remote, is complementary. double RNA according to a the claims 1 to 7, with the dsRNA against sections in the coding region the PIM-1 (m) RNA, complementary is containing the sequence AA. double RNA according to a the claims 1 to 8, with the dsRNA at the 5'-terminus complementary to the sequence AA. Doppelsträngie RNA according to a the claims 1 to 4, wherein the dsRNA against nucleotide segments in non-coding 5 'region of PIM-1 (m) RNA, is complementary. double RNA according to a the claims 1 to 10, wherein the dsRNA has at least one of the following properties has: (a) the GC content is at least 38%, (b) the dsRNA is not directed against regions that are at most 50 nucleotides away from the start or stop codon, (c) at most 2 consecutive guanidine residues, (d) the target sequence is in to be examined genome only in the target gene. double RNA according to a the claims 1 to 11, wherein the target sequence of the PIM-1 (m) RNA is selected from the group consisting of: AAC GUG GAG AAG GAC CGG AUU; AAC UCG AGU GCC CAU GGA AGU; AAC CGC GAC AUC AAG GAC GAA; AAU AUU CCU UUC GAG CAU GAC; AAG GGU CUC UUC AGA AUG UCA. double RNA according to any one of the preceding claims 1 to 12, wherein the dsRNA is chemically modified. double RNA according to one of the claims 1 to 13, where the double-stranded RNA suppresses the expression of PIM-1 in the cell by at least 50% (dsRNA) is. double RNA according to a of the preceding claims 1 to 14, wherein the dsRNA complementary to nucleotide sections of (m) mammalian PIM-1 RNA, especially of man, is. double RNA according to a of the preceding claims 1 to 15, where the dsRNA is "blunt ends" or overhanging ends having. double RNA according to a the claims 1 to 16, wherein the 3'-terminus or am 5'-terminus, respectively the overhanging Ends, in particular dTdT, which has double strands linked single strands. double RNA according to claim 17, with the overhanging Ends at least 2 overhanging nucleotides, preferably 2 to 10, in particular 2 to 5, overhanging nucleotides at the 3'-terminus or on 5'-terminus. double RNA according to claim 18, with the overhanging Nucleotides, preferably deoxidized thymidines or uracils attached to the (m) RNA sequence of PIM-1 complementary double strand. double The RNA according to claims 1 to 4, wherein the target sequence complementary to the dsRNA sequence 5'-AAGUGUACUUUAGGCAAAGGG-3 '. double An RNA according to claim 20, wherein the sense strand has the sequence 5'-GUGUACUUUAGGCAAAGGGdTdT-3 'and whose Antisense strand the sequence has 5'-CCCUUUGCCUAAAGUACACdTdT-3 '. Cell containing at least one dsRNA according to one the claims 1 to 21. Medicaments containing at least one dsRNA according to a the claims 1 to 21 and / or a cell according to claim 22, and optionally suitable auxiliaries and / or additives. Diagnostic agent containing at least one dsRNA according to a the claims 1 to 21 and / or a cell according to claim 22, and optionally suitable additives. Use of a dsRNA according to one of claims 1 to 22 and / or a cell according to claim 22 for the manufacture of a medicament or analgesic for Treatment of pain, especially of chronic pain, more tactile Allodynia, thermally triggered Pain and / or inflammatory pain. Use of a dsRNA according to one of claims 1 to 21 and / or a cell according to claim 22 for the manufacture of a medicament for the treatment of urinary incontinence, also of neurogenic bladder symptom, pruritus, tumors, inflammation; in particular, PIM-1 kinase associated inflammation with symptoms such as asthma; as well as all disease symptoms related to the physiological function of PIM-1. Use of a dsRNA according to one of claims 1 to 21 and / or a cell according to claim 22 for the Gene therapy, preferably in vivo or in vitro gene therapy. Method for inhibiting the expression of PIM-1 in a cell, wherein a dsRNA according to any one of claims 1 to 21 introduced into the cell is, wherein a strand of the dsRNA rich to the mRNA of PIM-1 Be rich characterized in that the complementary region less than 25 consecutive nucleotide pairs. The method of claim 28, wherein the dsRNA is in micellar Structures, preferably liposomes, is included. The method of claims 28 or 29, wherein the dsRNA in viral natural Capsids or in manufactured by chemical or enzymatic means Kapside or derived structures is included. A method of identifying dsRNA having pain modulating function, characterized net, that the binding constant of the binding of a preferably labeled dsRNA, according to claims 1 to 21, as a test substance in a binding assay to a (m) RNA of PIM-1 is measured. Method for identifying a pain modulating Substance with the following process steps: (a) overexpression a PIM kinase, preferably PIM-1 in a test cell, (B) genetic manipulation of at least one cell (test cell) with at least one dsRNA according to one of claims 1 to 21, (B ') parallel genetic manipulation of at least one identical Cell (control cell) either • remaining, • by carrying out the Manipulation in parallel without dsRNA or • with an altered, no longer the claims 1 to 21 corresponding dsRNA, (c) parallel incubation of a substance to be tested under suitable conditions with at least a test cell and at least one control cell and / or in a test cell according to (a) becomes. (d) measuring the binding of the test substance to that of the Cells synthesized protein or measuring at least one of the changed by the binding of the test substance to the protein functional parameters. (e) identification of the substances via the Extent of Difference between the measured value at the test cell and at the control cell. Method according to claim 32, characterized in that the cell in the process step (a) the cell is genetically engineered. Method according to claim 33, characterized that genetic engineering involves the measurement of at least one which changed by the test substance functional parameters allowed. Method according to claim 32 to 34, characterized in that by the genetic engineering Manipulation of a not endogenously expressed in the cell form of a Member of the PIM kinase family, preferably the PIM-1 kinase expressed or a reporter gene introduced becomes. Method according to one the claims 32 to 35, characterized in that between the parallel Process steps (b) and (b ') and the method step (c) ≥ 8 h, preferably ≥ 12 h, in particular ≥ 24 h, pass away.