DE102010022937A1 - Cell-specific activatable biologically active molecules based on siRNA, methods for their activation and application kit for administration - Google Patents

Abstract

The task was to create biological active substances which can be transfected both in vitro and in vivo in living beings, plants and fungi in or on target cells and only there highly effectively inhibit the expression of genes, without the drug-specific expression of the target gene in other cells of the organism and thus affect the formation of proteins. According to the invention, the siRNA (1) has at least one covalent bond (6) with one or more oligosaccharides (3), each of which has one or more specific chemical structures selected for target cell-typical enzymes. As a result of this binding (6), which is broken up exclusively by the target cell-specific enzyme (s) for biological activation of the active substance, the molecules remain biologically inactive in cells other than the target cells. These molecules are used, for example, to influence the gene expression of preferably diseased organs, cells or plants which are infected and infected with parasites or fungi.

Description

The invention relates to special biologically active molecules based on "short interfering RNA" (siRNA), a method for cell-specific activation after transfection into or on a cell by the occurrence of a specific enzyme, and an application kit for their use, in particular for administration in conjunction with a transfection system. The said biologically active molecules interact after activation with the mRNA of the target gene and, together with specific endoribonucleases, form an RNA-protein complex called "RISC" (RNA-induced silencing complex). The RISC complex binds to the target mRNA, with endonucleases cleaving the target mRNA. In this way, gene expression is prevented and thus the emergence of target proteins is inhibited.

The cell-specifically activatable, biologically active molecules can be used for example for controlling and inhibiting the growth of cells, in particular in the treatment of fungal infections and insect infestation. In general, the cell-specifically activatable, biologically active molecules can be used to modulate the gene expression of the target cells.

The inhibition of gene expression by introducing short (19-23 bp), double-stranded RNA molecules (siRNA) or PNA molecules into eukaryotic cells which is specific for a sequence segment of the mRNA of a target gene has already been described ( Elbashir SM et al .: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001 May 24, 411 (6836), 494-8 ; Liu Y et al .: Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 Feb 24, 43 (7), 1921-7 ; US 5,898,031 ; US 7,056,704 ).

Such molecules do not prevent the reading of a gene and the production of an mRNA, but in the case of siRNA, a cell-specific mechanism is initiated which degrades the target mRNA. Finally, as described above, the formation of a specific protein is suppressed without affecting the expression of other genes (post-transcriptional gene silencing).

In order to suppress the expression of a gene, the siRNA molecules can be introduced directly into the cell, in particular via transfection reagents and electroporation ( Zhang M et al .: downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells, RNA Biol. 2004 May, 1 (1), 74-7 ; Gilmore IR et al .: Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22, Curr Drug Deliv. 2006 Apr, 3 (2), 147-5 ; US 6,506,559 ).

The disadvantage here is that the siRNA is relatively unstable, which can be improved by chemical modifications ( US 6,107,094 ).

A special problem of the therapeutic application of biologically active molecules is an application in vivo. For such an application possibilities have been developed to stabilize the siRNA in order to reduce the degradation ( Morrissey et. al .: "Chemical Modifications of Synthetic siRNA", Pharmaceutical Discovery, May 1, 2005 ), and transfection reagents, for example nanoparticles, have been developed in vivo jetPEI ^™ (Polyplus), which also introduce the siRNA into cells in vivo ( Vernejoul et al .: Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models, Cancer Research 62, 2002, 6124-31 ; Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting through systemic application of polyethyleneimine (PEI) -complexed siRNA in vivo, Gene Ther 12 (5), 2005, 461-6. ).

Also, methods have been developed to transfect cells of a target tissue with siRNA in vivo ( Ikeda et. al., "Ligand-Targeted Delivery of Therapeutic siRNA", Pharmaceutical Research, Vol. 8, August 2006 ).

The administration of biologically active substances in vivo, however, is often problematic due to the systemic effect. The introduction of these substances selectively in target cells is not sufficiently specific. This is particularly disadvantageous for siRNA molecules which are intended to be selective and exclusive in target cells. By tissue- or cell-specific-labeled transfection reagents (eg antibody / antigen-labeled nanoparticles, TAT protein flanking, etc.), no sufficiently large cell specificity is achieved. Miscar transfections are the result.

The same problem concerns the use of siRNA molecules for the targeted influencing of parasites or pests (eg fungi) in human organs (eg lungs) or in plants.

Furthermore, it is known to inactivate siRNA molecules by binding fluorochromes in their biological action and to reconvert them into their active form by irradiation with light of a specific wavelength ( QN Nguyen et al.: Light controllable siRNAs regulate gene suppression and phenotypes in cells, Biochim Biophys Acta, 2006 ). This activation is initiated from the outside and is in no way cell-specific. As a result, the said siRNA molecules act after their activation again unintentionally in all other transfected cells and not only in the intended target cells.

Moreover, even this mechanism is difficult to apply in vivo or in plants.

Furthermore, it is known ( DE 10 2007 008 596 A1 ) that can be coupled to a siRNA peptides for the purpose of their inhibition and target cell-specific cleavage by specifically active peptidases. To do this, however, it is necessary that target peptides for such peptidases be coupled to the siRNA that are not present in the non-target cells. In practice, it has been shown that peptidases that are not available for all target cells are available. On the other hand, due to the chemical structure of the peptides and the siRNA, it is necessary that a so-called linker molecule be used between the two components, which remains after the activation of the siRNA, or cleavage of the peptide on the siRNA and affect their activity can.

The invention is based on the object to provide biological active substances which are transfected both in vitro and in vivo not only in living things and their tissues, but also in plants and fungi in or on a target cell and only there the expression of genes highly effective inhibit without influencing the drug-specific expression of the target gene and thus the formation of proteins in other cells of the organism.

For the exemplified treatment of fungal infections in plants, this means to selectively inhibit the expression of the target gene and thus the protein formation in fungal cells, without the fungus surrounding cells (for example infested plant tissue), which can also be reached by the active ingredients, and their survival affect.

According to the invention, the biologically active molecules are covalently bound to oligosaccharides, for example cellulose, based on siRNA, thereby inactivating the biologically active molecules. There is thus no inhibition of specific gene expression during and after transfection into cells, as long as, for example, the cellulose remains due to the absence of the cellulose-cutting enzyme (cellulase) on the siRNA molecules. For the purpose of said biological inactivation, the biologically active molecules have at least one covalent bond with at least one oligosaccharide which can be disrupted only by the cell-specific enzyme (s) of that target cell, each one or more significant and / or significant for the disruptible covalent bond to the one or more target cell-type enzymes having chemical structure.

By means of a suitable transfection system, for example nanoparticles, polyethyleneimines or liposomes, the inactivated active substance molecules can be transfected into the target cells. In each case, said inactivating covalent bonds are broken down by the specific enzyme, for example cellulase, exclusively in cells which express this enzyme, thereby activating the biological activity of the molecule now in the target cell and dissolved by the cellulose. This then binds to the specific mRNA of the target cell and thereby inhibits gene expression in a known manner in this particular cell.

In all cells other than the predetermined target cells into which these inactive molecular constructs can also enter, the drug molecules remain inactive because the covalent bonds between the siRNA and the cellulose persist due to the absence of the enzyme cellulase. Due to the further covalent binding to the cellulose, the siRNA does not bind with the mRNA of this cell, or no RISC is initiated.

Advantageously, the covalent bond between siRNA and oligosaccharide, in contrast to the binding of siRNA to peptides without required linkage linker, which would remain at least partially after binding break at the siRNA and which could affect the biological activity of the siRNA. As a result, the active ingredients with very high biological effectiveness come to the intended use.

Although, for example, in the treatment of fungal infections in plants, the molecule constructs according to the invention to be transfected in their inactive (bound) form not only reach fungal cells, but (which can hardly be avoided in practice) can also reach plant cells, become selectively exclusively activated in fungi cells with the cellulase enzyme expressed there, the said molecule in its biological activity and inhibits the drug-related expression of the target gene. This gene expression and thus the protein formation for the survival of the healthy cells remains unaffected by this active ingredient despite the non-affected in this (or not intended for the intended biological effect) cells, but permanently inactive molecular constructs.

Due to the proposed highly selective effect of the molecules due to target cell enzyme specific inactivity / activation in fungal cells, the be administered to transfecting biologically inactive molecular constructs with appropriate cellulose binding easily fungus-infected plants.

The molecular constructs can also be bound to other substances (for example nanoparticles as a carrier system) for better transport into or onto the target cells and for their stabilization.

Moreover, in order to improve the transfection rate, known mechanisms of action which increase the tissue or cell selectivity (for example ligand / antibody / antigen-labeled nanoparticles) can be used.

While this mis-transfection can not be prevented by use of the invention, the mis-transfected molecules in these other cells than the target cells, although still undesirable, are not biologically active. This status also remains unchanged despite inventive molecule activation in or on the target cells, so that the biological effect is selectively exclusively in the target cells and in contrast to the known mechanisms a highly cell-selective modulation of gene expression is achieved.

The invention is not limited to applications for the treatment of fungal infected cells. The molecular constructs of the invention can be applied to all cells which express enzymes such as cellulase and are in an environment of cells which do not express this enzyme.

It is advantageous in the application of the said molecules for the treatment of fungal infections in plants that the inactivated for example by cellulose siRNA molecules are destroyed some time after their application by the UV radiation of sunlight and thus do not enter the food chain. Even if these inactivated molecules should nevertheless enter the food chain, they are nevertheless ineffective and therefore safe, since, for example, in the human or animal body, no corresponding enzymes for activating these siRNA molecules occur.

Also advantageous is the use of the said molecules also for the treatment of fungal infestation in humans or animals or their respective tissue ex vivo, as these express no endogenous cellulases or other fungal-specific enzymes.

Furthermore, the application of said molecules is also for the treatment of fungal infestation of food, plant products (eg fibers, hay, seed cereals), animal products (eg Haute, leather), but also of buildings (mold Walls and wallpaper, wood sponges, especially dry rot). In the latter case, in particular, lignases are also enzymatic target molecules.

Similarly advantageous is the treatment of wood-damaging insects and their larvae, in particular the domestic horse, with the said molecules.

One advantage of all building and wood treatments is that there are no chemical residues that could harm users. The said molecules are activated only specifically and degraded after application in a short time.

The invention will be explained below with reference to exemplary embodiments illustrated in the drawing.

Show it:

1 : Section of the general structure of a known siRNA

2 : Section of the general structure of a known cellulose

3 A siRNA-oligosaccharide molecular construct according to the invention in which a cellulose molecule has been coupled by way of example to the sugar phosphate backbone of the siRNA via an interface of the molecular construct which can be disrupted by the enzyme cellulase

In 1 is the general structure of the known structure of siRNA 1 shown. It is always, as generally shown, an organic base to a Zuckerphospat 2 bound. An organic base and one sugar phosphate each 2 are called nucleotide; the connection of the individual sugar phosphates 2 form the basic structure of the siRNA 1 ,

In 2 is the structure of an already known cellulose 3 shown schematically. This consists of five glucose molecules shown 4 , which are each connected alternately by 180 degrees sterically to each other to a linear macromolecule. For other oligosaccharides, depending on the substance, these individual sugar molecules may not be twisted or branched (eg starch, chitin, etc.). In the case of the cellulose shown, cellulases are capable of producing these individual glucose molecules 4 to split off from the macromolecule.

In 3 is the structure according to the invention of a proposed siRNA-oligosaccharide molecule 5 with the binding between the siRNA 1 and the cellulose 3 shown. In this case, the cellulose is in the case shown 3 to the 3 'position of the siRNA 1 covalently bound (interface 6 ), thereby increasing the biological activity of the siRNA 1 is inhibited. Complete inhibition of siRNA 1 can be achieved by the coupling of very long cellulose molecular chains (polysaccharide chains).

The enzyme cellulase, which occurs in the target cells, breaks down the cellulose 3 (Cellulose oligosaccharide) by the breaking of the covalent bond (interface 6 ) in its individual components and the siRNA 1 regains its biological activity.

Advantageous is an application kit in which the biologically active molecules to be used are provided with the bound oligosaccharides having selected structures with respect to the particular enzymes of the target cells, or with which the oligosaccharides are coupled to the siRNA to achieve the desired inhibitory effect can. The application kit should be in ampoule form containing all necessary ingredients, conveniently also a selection of suitable transfection systems (such as nanoparticles, polyethyleneimines or lipids), other additives (such as antibodies, ligands and polyethylene glycol) and one or more probes or syringes with cannula for injecting the mixture from the contents of the ampoules to the medium containing the target cells. Some or all reagents may also be in dried form, in particular lyophilized. They are dissolved in water or buffer before use.

LIST OF REFERENCE NUMBERS

1

siRNA (schematically represented section of the chemical structure)

2

sugar phosphate

3

Cellulose (schematically represented section of the chemical structure)

4

glucose molecules

5

siRNA-oligosaccharide molecule

6

disruptible interface between siRNA 1 and cellulose 3

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 5898031 [0003]
• US7056704 [0003]
• US 6506559 [0005]
• US 6107094 [0006]
• DE 102007008596 A1 [0013]

Cited non-patent literature

• Elbashir SM et al .: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001 May 24, 411 (6836), 494-8 [0003]
• Liu Y et al .: Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 Feb 24, 43 (7), 1921-7 [0003]
• Zhang M et al .: downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells, RNA Biol. 2004 May, 1 (1), 74-7 [0005]
• Gilmore IR et al .: Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22, Curr Drug Deliv. 2006 Apr, 3 (2), 147-5 [0005]
• Morrissey et. al .: "Chemical Modifications of Synthetic siRNA", Pharmaceutical Discovery, May 1, 2005 [0007]
• Vernejoul et al .: Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models, Cancer Research 62, 2002, 6124-31 [0007]
• Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting through systemic application of polyethyleneimine (PEI) -complexed siRNA in vivo, Gene Ther 12 (5), 2005, 461-6. [0007]
• Ikeda et. al., "Ligand-Targeted Delivery of Therapeutic siRNA", Pharmaceutical Research, Vol. 8, August 2006 [0008]
• QN Nguyen et al .: Light controllable siRNAs regulate gene suppression and phenotypes in cells, Biochim Biophys acta, 2006 [0011]

Claims (11)

Biologically active molecules, based on siRNA, for biologically inactive transfection into a target cell to inhibit the expression of genes in this after biological activation by binding to mRNA and forming a "RISC" complex, characterized in that the biologically active Molecules ( 5 ) for the purpose of their biological inactivation outside the target cell, at least one covalent bond which can be disrupted only by the cell-specific enzyme (s) of this target cell ( 6 ) with at least one oligosaccharide ( 3 ), each of which has one or more selected for the or the target cell-specific enzymes and the breakable covalent bond ( 6 ) each having a significant chemical structure. Biologically active molecules according to claim 1, characterized in that the at least one oligosaccharide ( 3 ) for the purpose of breaking the bond ( 6 ) has the chemical structure of cellulose by the cell-specific enzyme cellulase. Biologically active molecules according to claim 1, characterized in that the at least one oligosaccharide ( 3 ) for the purpose of breaking the bond ( 6 ) has the chemical structure of starch by the cell-specific enzyme amylases. Biologically active molecules according to claim 1, characterized in that the at least one oligosaccharide ( 3 ) for the purpose of breaking the bond ( 6 ) has the chemical structure of chitin by the cell-specific enzyme chitinase. Biologically active molecules according to claim 1, characterized in that the at least one oligosaccharide ( 3 ) for the purpose of breaking the bond ( 6 ) has the chemical structure of pectin by the cell-specific enzyme pectinase. Biologically active molecules according to claim 1, characterized in that the at least one oligosaccharide ( 3 ) for the purpose of breaking the bond ( 6 ) has the chemical structure of proteoglycans by the cell-specific enzyme chondroitinase. Biologically. active molecules according to claim 1, characterized in that the at least one oligosaccharide ( 3 ) for the purpose of breaking the bond ( 6 ) has the chemical structure of hyaluronic acid by the cell-specific enzyme hyaluronidase. Biologically active molecules according to one or more of claims 1-5, characterized in that the at least one oligosaccharide ( 3 ) or the siRNA ( 1 ), other structures, such as peptides, polyethylene glycol, cholesterol, nanoparticles are covalently bonded. Biologically active molecules according to claim 1, characterized in that the siRNA ( 1 ) both a bond with the at least one oligosaccharide ( 3 ) as well as having a binding with at least one peptide. A method for cell-specific activation of siRNA, wherein this is preferably transfected biologically inactive into a target cell to inhibit there after their biological activation with binding to mRNA and by forming a "RISC" complex, the expression of genes, wherein the siRNA for the purpose their biological inactivation outside the target cell or before transfection is covalently bound to one or more oligosaccharides each having at least one selected to the one or more target cell-specific enzymes and significant for the breakable covalent bond chemical structure and that the covalent binding of the oligosaccharides or the For the purpose of their biological activation, siRNA is broken up in each case by the target cell-typical enzyme. Application kit for the preparation and administration of the biologically active molecules according to one or more of claims 1 to 8, consisting at least of - At least one ampoule (ampoule A), each containing a selected for the purpose of inactivating the siRNA oligosaccharide with a selected, the target cell enzyme entprechenden, chemical structure, in particular Celloluse, and / or at least one ampoule (ampoule B), each contains a siRNA (single-stranded or double-stranded) specific for binding the selected oligosaccharide and for a target gene, At least one further ampoule (ampoule C) with a transfection system, in particular nanoparticles, polyethylene or lipids, Dilution and reaction buffer for the contents of ampoules A to C, - One or more probes or syringes with cannula and other materials required for injection of the mixture of the ampoule contents in the medium containing the target cells and Instructions for administration with a comparison of the selectable oligosaccharides for respective enzymes of the target cell.

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