Method for introducing sirna into cells by photochemical internalisation. RU patent 2510826.

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to a method for introducing an siRNA molecule into the cytosol of a cell, and can be used in medicine. The method involves contacting said cell with an siRNA molecule, a carrier and a photosensiting agent, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent. The carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, branched polyethyleneimine (PEI), a betacyclodextrin amine polymer, a PAMAM dendrimer molecule, and a cationic peptide such as polyarginine or L- or D-arginine copolymers. The method is used to inhibit the target gene expression, to obtain a cell or a cell population containing the siRNA molecules, as well as to treat or to prevent a disease where the inhibition of one or more genes may be effective, including to treat a malignant growth.

EFFECT: invention enables the PCI-mediated site-specific siRNA delivery into the cytosol of a cell.

27 cl, 15 dwg, 1 tbl, 13 ex

The present invention relates to a method of introduction of short interfering RNA (siRNA) in cells, preferably in the cytosol of cells, using a photosensitizing agents and media molecule and cell irradiation by light with a wavelength, effective for activation photosensitizing agents, and to the use of this method for changing the activity of genes, for example, to suppress genes in vitro or in vivo.

The process of RNA interference occurs in many organisms and in the process of double-stranded non-coding RNA excision with specificity to the sequence inhibits gene expression. In nature this phenomenon genome protects the body from foreign implemented nucleic acids, such as transposons, the transgenes and viral genes.

Introduction of double-stranded RNA (dsRNA) in the cell starts the process of suppression of RNA, and any mRNA in the cell with the sequence corresponding to the entered dsRNA, is degraded. Cascades suppression of RNA involve the transformation of dsRNA in short interfering RNA (siRNA), which send ribonuclease on the homology of mRNA targets. Enzyme Dicer convert dsRNA in siRNA, the length of which, as a rule, is 20-25 nucleotides. Then siRNA going in containing endoribonuclease complexes known as RNA-induced complexes suppression (RISC)that are directed to complementary RNA molecules, where they break down and destroy mRNA target. A small number of dsRNA can suppress large number of mRNA targets because of the rising component of suppression of RNA (discussed in Hannon and Rossi (2004), Nature 431, 371-378).

The knowledge that siRNA molecules are key components of the cascade, led to the testing of chemically synthesized molecules siRNA in length from about 20 to 22 base pairs corresponding to the RNA or DNA sequences target. It was shown that these molecules are violating the expression of a sequence of target proteins in mammalian cells (Elbashir S.M. et al., (2001) Nature 411, 494-498). As a rule, siRNA length 20 nucleotides is long enough for the induction of gene-specific suppression, but short enough to evade host response. Decrease in the expression of genes-targets can be significant with 90% suppression induced by several siRNA molecules.

Thus, the siRNA technology was developed as a common method that is specific to the sequence of the gene suppression. Suppression of genes has many uses, both in vitro and in vivo, both as a research tool and as a therapeutic strategy. High efficiency and specificity that can be observed when using siRNA technology, makes this technology especially attractive.

In all cases, the delivery of siRNA molecules in cells is a big problem, because for the purpose of suppressing gene, it is necessary that siRNA molecules entered the cell in sufficient concentrations to be relevant. On the strength of the overwhelming response and its duration is affected by the number of siRNA, which bring in a cage, and it was shown that, through the provision of siRNA in high enough concentrations, even relatively weak molecule siRNA molecule can suppress your target. However, by contrast to this is the fact that it is known that the introduction of large quantities of siRNA in the cell can lead to undesirable effects, such as unintended effects (i.e. unwanted changes in the levels of expression of protein) or activation of innate cascades of the immune system.

As usually deliver siRNA into cells using standard protocols transfection for nucleic acids, such as the protocols with the use of liposomes, cationic lipids, anionic lipids and microinjection. siRNA is a double-stranded molecule and, in fact, delivery and cell capture molecules are more complex than in the case of antisense molecule that binds to serum proteins to capture cells. Use strategy, and for this purpose there are commercially available kits. As stated above, efficient transfection is highly desirable, since the efficiency of suppression of gene are at least partially dependent on the concentration of siRNA in the cell, however, the introduction of cells in high concentrations can also cause unwanted side effects.

Introduction to high levels often requires high concentration of reagents for transfection, and this can have adverse effects on the cells, including reduced viability of cells and various other side effects as phenotypic and phenotypically. Moreover, when using high concentrations of reagents, do not reach specific delivery.

Targeted delivery of molecules of nucleic acids, such as siRNA, as a rule, is not sufficiently reliable. For this purpose you can use viruses, however, in the case of this approach is a security issue, and systemic viral delivery difficult to achieve.

siRNA acts in the cytosol of cells, and for the molecule acted, it is necessary that the molecule was reached zitozole. Taking into account the above considerations, it may be desirable to develop an improved method for delivery of siRNA in the cytosol cells. Desirable properties this improved method include (i) the ability to provide specific time and place of delivery of siRNA molecules in the place of their actions, (ii) avoiding the use of high concentrations of transfection reagent and/or siRNA and/or iii) strengthened siRNA-suppressing cell lines. In particular, these methods may reduce the total number of complexes siRNA:lipid required to achieve a certain level of gene suppression or its improvement. In such ways the ratio of siRNA:reagent for transfection can be changed while maintaining a certain degree of suppression of a gene or its improvement. Increasing the ratio of siRNA:lipid is useful because it can minimize the inhibitory effects that you see in the application of high concentrations of reagents for transfection.

In General, the goal of improved alternative method can be called a desire to balance the need for effective and controlled delivery of siRNA in the cytosol with the reduction of adverse side effects, the result of high concentrations of reagents for transfection, or non-specific effects, for example, in a particular cell types. As mentioned above, the decrease in the total number of complexes siRNA:lipid and/or increase the ratio of siRNA:lipid can lead to this goal.

To achieve these objectives, the authors of the invention combined use of media (reagent for transfection) with a way photochemical internalization (PCI). Specific selected media delivers molecule siRNA into the intracellular compartments of the cell, for example, indacetamine vesicles, such as endosome and/or lysosome cells. Alternative intracellular compartments that can be captured complex siRNA:lipid include the Golgi apparatus and endoplasmic network.

As a result of PCI way there is a release of siRNA molecules from intracellular vesicles. It depends on the impact to the cage photosensitizing connection and subsequent exposure, and can be observed that the release of siRNA molecules occurs only after irradiation cells and, in fact, it is released into the cytosol where mediated by its effects can be subjected to spatial or temporal control. Only cells that (i) contain siRNA in their intracellular vesicles, ii) subjected to photochemical internalydeluded substances and iii) subjected to irradiation, will release the siRNA molecules in the cell cytosol to act on mRNA in the cell.

As a rule, to optimize the delivery of siRNA in the cytosol reagents for transfection must be used in high concentrations. The authors of the invention suddenly discovered that using low concentrations of reagents for transfection (and photochemical internalydeluded substances), directions of siRNA in the intracellular vesicles, such as endosome where it is to launch its release through the application of irradiation, you can use the stage transfection. Thus, the method allows siRNA to achieve its scope, without the necessity to use high concentrations of reagents for transfection or siRNA. Moreover, using PCI way you can control the time and place of release siRNA molecules from intracellular vesicles, such as endosome.

Thus, in the first aspect, this invention relates to a method of introduction of siRNA molecules in the cell cytosol, including contacts the specified cells with siRNA molecules, the media and photosensitizing agent and the irradiation of the cells of light with a wavelength, effective for activation photosensitizing agents. After activation of intracellular compartments in the specified cell containing the specified photosensitizing agent, release siRNA contained in these compartments, in the cytosol.

PCI is a method that uses a photosensitizing agent in conjunction with the stage of irradiation for activation of this substance, and it provides the release of molecules, jointly entered into the cell, the cell cytosol. The method allows molecules that are captured in the organelles of the cell, such as endoscopy, after exposure to escape from these organelles in the cytosol.

These methods use the photochemical effect as a mechanism of introduction of molecules, otherwise not penetrating (or badly penetrates through the membrane into the cytosol cells, which, therefore, does not lead to the widespread destruction of cells or cell death, if the method is suitable adjusted to avoid excessive production of toxic particles, for example, by reducing lighting time or dose photosensitizing agents.

This method is particularly advantageous for the introduction of siRNA in the cells because it allows the use of lower concentrations of the carrier or transfection reagent and/or siRNA than necessary in the case of conventional siRNA transfection, reaching thus inhibiting gene. Moreover, the time and place of exposure to release the siRNA molecules can be controlled so that it would be released only at that time and in that place that is desirable to achieve the desired effects. Essentially, the effect on the cells of siRNA and media minimized, to minimize unwanted side effects. This is the opposite of the standard methods of the introduction of siRNA into the cells, where it is impossible to control the time and place of release siRNA and require high concentration of the reagent for transfection. By reducing the number of media (ratio siRNA:native) compared to the amount that is taken to apply, or by reducing the total number of complexes siRNA:media, which apply to cells, you can also minimize the release of siRNA of intracellular compartments before irradiation PCI.

Further, it was shown that using for the delivery of siRNA media, as defined herein, with PCI, you can achieve significant effects on the suppression of the gene causing simultaneously cytotoxicity. For example, when using PEI (25000 MM) in the amount of 1 mcg/ml to 100 nm siRNA and light doses up to 40 seconds, there were no cytotoxic effects (see FIGU). In these conditions have been identified significant effects on the suppression of the gene (see figure 10).

RNA is a polymer of ribonucleotides, each of which contains the sugar ribose together with phosphate group, and a nitrogenous base (as a rule, adenine, guanine, cytosine, or uracil). As in the case of DNA, RNA can form a complementary hydrogen bonds, and RNA can be a double-stranded (dsRNA), stranded (ssRNA) or double-stranded stranded protruding end. "Small interfering RNA" (siRNA) belong to the double-stranded RNA molecules in length from about 10 to about 30 nucleotides that are specific inhibit protein expression by binding to molecules of mRNA. Preferably, siRNA molecules have a length of 12-28 nucleotides, preferably 15-25 nucleotides, preferably 19-23 nucleotides and it is most preferable 21-23 nucleotides. Thus, the length of preferred siRNA molecules is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides.

The length of one chain defines the length of the siRNA molecules. For example, siRNA, which is described as siRNA length of 21 of ribonucleotides (21-Mer), can contain two opposite RNA chains which hybridize with education 19 continuous steam. The remaining two ribonucleotide on each chain form the protruding end. When siRNA contains two chains various length, the length of siRNA defines a longer chain. For example, dsRNA, which contains a single chain length of 21 nucleotides and the second circuit length 20 nucleotides, is 21-measures.

Are desirable siRNA, which contain the protruding end. Protruding end may be 5'and 3'-end of the chain. Preferably it is on the 3'-end of the RNA chain. The stickout length can vary, but preferably it ranges from about 1 to approximately 5 nucleotides, and more preferably length is approximately 2 nucleotide. Preferably, siRNA of the present invention will contain 3'-protruding end of about 2 to 4 nucleotides. Preferably, 3'-protruding end has a length of 2 ribonucleotide. Preferably, 2 ribonucleotide containing 3'-protruding end, have oraselului (U) the grounds.

siRNA design for interaction with ribonucleotidic sequence target, in other words they are complementary target sequence, so they contacted the sequence of the target, i.e. one chain siRNA complementary section of the target sequence.

The siRNA molecules that have modified frames to improve their time-life (for example, as described in the Chiu et al., (2003), RNA. 9(9), 1034-48 and Czauderna et al., (2003), Nucleic Acids Research 31, 2705-2716). Thus, the term "siRNA" also includes such modified molecules. Indication of siRNA, therefore, covers derivatives or options siRNA, which Express the same function, i.e. interaction with the mRNA sequence the target. Preferred options include options that use a modified frame (as above) or one or more of not occurring of the grounds.

The method can be used to make more than one type of siRNA molecules in the cell. In other words, while in the cell, you can enter the siRNA molecules with different sequence. If the introduction is subject to many siRNA molecules, it is possible to carry out simultaneous binding of more than one siRNA molecules with the media. Alternatively, each type of siRNA molecules can separately be associated with the media.

There are several ways of obtaining siRNA, such as chemical synthesis, transcription in vitro expressing siRNA vectors and expressing cassette PCR. Such methods are well known in this area. See, for example, Pon et al., (2005) Nucleosides Nucleotides Nucleic Acids. 24(5-7): 777-81, Du et al., (2006), Biochem. Biophys. Res. Commun. 345(1):99-105 and Katoh et al., (2003), Nucleic Acids Res Suppl. (3): 249-50.

Similarly, the methods of constructing siRNA molecules to achieve the required result is well documented. You must first select a region-target siRNA. This can be done using various methods (see, for example, Jagla et al., (2005), RNA. 11(6):864-72 and Takasaki et al., (2006), Comput. Biol. Chem. 30(3):169-78).

The method according to this invention provides moving molecules siRNA into the cytosol. However, it will be clear that the capture of each molecule in contact with the cell, unattainable. But is achievable significant and increased capture relative to the background when not in use PCI or media.

Preferably ways on this invention allows the capture of siRNA molecules to adequate levels, so that their effect is obvious expressed in the products of these cells. To achieve this, you can adjust the corresponding concentration of siRNA, subject to contact with a cell, for example, for achieve a reduction in the expression of the target genes by at least 10%, for example, reduced by at least 20, 30, 40, 50, 60, 70, 80 or 90% (for example, in the expression of one or more proteins encoded by the genome of target) after incubation with cells within, for example, 24, 48, 72 or 96 hour (for example, from 24 to 48 hours). Similarly, to achieve the reduction specified above, you can adjust the type and/or the concentration of media type and/or the concentration of photosensitizing agents and the exposure time.

It can be measured by determining the level of protein in the cell using standard methods, known in the field, such as Western blotting. The reduction of protein depends on the time-life is a protein, i.e. the previously existing protein will be removed in accordance with its half-life. Thus reach reduce the expression of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% relative expression in the same time without siRNA with the time half-life.

Alternative it can be measured from the point of view of effect of siRNA molecules on the amount of mRNA that is present in the cell, for example, the method can be performed to achieve a decrease in the levels of mRNA at least 10%, for example, reduced by at least 20, 30, 40, 50, 60, 70, 80 or 90% after incubation with cells within, for example, 24, 48, 72, or 96 hours, for example, from 24 to 48 hours, relative to the levels of mRNA target sequence at the same time without siRNA. It can also be measured using standard methods, known in the field, such as how hybridization or blotting and RT-PCR.

Because these methods require much fewer media or substances for transfection (and/or fewer siRNA, depending on whether the reduction in the total number of complexes, or subject to modification ratio siRNA:native or substance for transfection, or both of them)than in standard ways transfection of siRNA molecules, also improving transfection can be expressed using the method of this invention from the point of view of the number of media or substances for transfection that are required to achieve a certain degree of reduction in the expression of protein or mRNA levels. For example, the way in this invention preferably allows a certain decrease in the expression of the protein target or mRNA levels (e.g. at least 10%, for example, at least 20, 30, 40, 50, 60, 70, 80 or 90%as described above) using the concentrations of the carrier and/or concentration of siRNA, which, for example, at least 10, 20, 30, 40, 50, or 60% below the number of media that you want to achieve the same level of decrease in the expression of the protein target or mRNA levels without PCI.

Comparisons can also be between levels decrease the expression of protein or levels of mRNA that look at a certain concentration of siRNA and media, in the presence and absence of PCI. For example, the method according to this invention preferably allows a decrease in the expression of the protein target levels or mRNA at least 10%, for example, at least 20, 30, 40, 50, 60, 70, 80 or 90%, as described above, in comparison with the expression of a protein or mRNA levels provided by the conduct of the way in the absence stage of irradiation technology PCI.

As used herein, "communication" refers to the implementation of physical contact cells and photosensitizing agents and/or siRNA and media with each other in an environment suitable for the internalization of the cells, for example, preferably at 37 degrees C in appropriate culture medium, for example, when a 25-39ºC.

Photosensitizing substance is a substance that is activated when illuminated with the appropriate wavelength and intensity with the formation of active particles. Convenient to the substance could be a substance which is localized in the intracellular compartments, in particular, in endosomes or lysosomes. A number of such photosensitizing substances known in the field, and they are described in the literature, including in WO 96/07432, which is incorporated herein by reference. In this regard, mention can be made of di - and tetraculturology aluminum phthalocyanine (for example, AlPcS 2a ), from sulphonated tetraphenylporphine (TPPS n ), "blue Nile", derivatives of chlorin e 6 , uroporphyrin I, phylloerythrin, hematoporphyrin, methylene blue, which has been shown to be localized in endosomes and lysosomes cells in culture. In most cases this is a consequence of antozianov capture photosensitizing agents. Thus, photosensitizing substance preferably is a substance that is captured in the inner cell compartments, for example, complementary mechanism and/or endosome. Further appropriate photosensitizing agents for the application of the invention described in the WO 03/020309, which is also incorporated herein by reference, namely sulfatirovanne mesotetraphenylporthyrin, preferably TPCS 2a .

However, you can also use other photosensitizing agents, which are located in other intracellular compartments, for example, in the endoplasmic reticulum, or the Golgi apparatus. It is also convenient to be able to operate the mechanisms by which the effects of photochemical processing are other components of the cell (i.e. to components of other than restricted by a membrane, i.e. prisoners in the membrane compartments). Thus, for example, one possibility may be to photochemical processing destroyed molecules important for intracellular transport or vesicle fusion. Such molecules may not necessarily be located in limited membrane compartments, but photochemical damage such molecules, however, can cause photochemical internalization complexes media:siRNA, for example, through the mechanism, in which photochemical effects on such molecules lead to reduced transport molecules, be internalized (i.e. siRNA molecules)in a degrading vesicles, such as complementary mechanism, so that the molecule to be internalization can be selected in the cytosol to degradation.

Examples of molecules, not necessarily located in limited membrane compartments are few molecules of the transport system of microtubules, such as dyneins and components dynactin; and, for example, rab5, rab7 sensitive to N-ethylmaleimide factor (NSF), soluble protein accession NSF (SNAP), etc.

Classes are suitable photosensitizing substances that can be mentioned, thus, include porphyrins, phthalocyanines, purpurin, chlorines (in particular, derivatives of chlorin with porphyrins, described below), benzoporphyrin, lysosomotropic weak base, naphthalocyanine, cationic dyes and tetracyclines or their derivatives (Berg et al., (1997), J. Photochemistry and Photobiology, 65, 403-409). Other suitable photosensitizing agents include tekstilni, deformity, partizany, bacteriochlorin, kitoblarni, hematoporphyrin derivatives and their derivatives, endogenous photosensitizing agents induced by 5-aminolevulinic acid and their derivatives, dimmers or other conjugates between photosensitizing agents.

Preferred photosensitizing agents include TPPS 4 , TPPS 2A , AlPcS 2A , TPCS 2a and other affinnye photosensitizing agents. Other suitable photosensitizing agents include the connection of 5-aminolevulinic acid or esters of 5-aminolevulinova acids or pharmaceutically acceptable salts.

"Exposure" of the cell to activate photosensitizing agents refers to the use of light directly or indirectly, as described in this document hereinafter. Thus, the cells can be lit with the light source, for example, explicitly (for example, on a separate cells in vitro) or indirect, for example, in vivo, when cells are located under the surface of the skin or in the form of a layer of cells, not all of which are covered directly, i.e. without screen from other cells.

In this way, the molecule siRNA, subject to introduction into the cell, associate or associate or kongugiruut with one or more molecules of the medium or substances for transfection that act, facilitating or improving the capture photosensitizing agents or siRNA molecules in the cell. This affiliation, Association or conjugation can be performed before contacting siRNA molecules and its media cage or during a specified contact by contact of these molecules.

Molecule media can be associated, or related conjugated molecules siRNA or siRNA, and with photosensitizing agent. Thus, for example, siRNA can be attached to the media by the charge-charge interactions. As mentioned above, you can use more than one media, and the media can be associated, related or konjugierten more than one molecule siRNA, or more than one kind of siRNA molecules.

Preferably media contains the connection, preferably in liposomal the structure that contains two or more groups amines, i.e. is polyamines and which is cationic and preferably protoirey (i.e. can be protonated that it had one or more additional hydrogen atoms in a suitable reaction conditions) at different pH values. Different pH lead to different values protonirovannykh atoms in a separate molecules and/or in different molecules.

The term "protoirey" is used in this document to indicate that the group could accept the hydrogen atom, i.e. protonema the group is acceptyour hydrogen group. Obviously, the ability of the group to accept hydrogen depends not only on the structure of the group, but also from the pH value, which affects the group. Preferably specified protonema group contains nitrogen atom and he is an atom, which accepts the hydrogen atom.

As denoted in the present paper, "cation" means that total, or aggregate, the charge on the molecule is +1 or more. His preferably measured at physiological pH values, i.e. at a pH of 7.2. The molecule can have a higher charge, for example, +2 or greater, +3 or more, +4 or more, +5 or more, +6 or more, +7, +8, +or more than 9 +10 or more, +11 or more, +12 or more, +13 or more, +14 or more, +15, +20, +25 or more 50 or more, +75 or more, 100 or more, +150 or more, +200 or more, +250 or more, +300 or more, +400 or more, +500 or more, +750 or more, or +1000 or more.

Cationic poliaminy for use in accordance with the methods on this invention are as defined below, and they include

(a) lipophily in liposomal composition,

(b) polyethylenimine (PEI), with a value of M n 500-20000 by GPC,

(c) polymer metalldichtungen formula

where X is an integer from 1 to 100, inclusive, and n is an integer from 4 to 10, inclusive,

(d) dendrimer containing an amine group, and

(e) cationic peptide.

Preferably, polyamine, as denoted in the present document contains the group's primary or secondary amines, or a mixture (for example, at least two groups of primary amines). Preferably, the site polyamine has at least 2, 3, 4, 5 or 6 of the nitrogen atoms and charge at least +1, +2, +3, +4 or +5 (or at least+6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +20, +25, +50, +75, +100, +150, +200, +250, +300, +400, +500, +750 or +1000) at physiological pH values, for example, some or all of the amino groups are charged. Preferably at least one (for example, at least 2, 3, or 4) nitrogen group, for example, NH, is unloaded at physiological pH values. The pKa value at which the last Amin polyamine is protonated, for example, lipopoliamin, preferably is approximately 5.5, i.e. at lower pH, or the addition of acid compounds, last Amin, subject to the protonation, protonated at pH less than or equal to 5.5.

In one embodiment, the media contains lipophily in liposomal composition. Under leopolitano mean amphiphilic molecules containing at least one hydrophilic region Polyamin (i.e. which contains two or more groups amines) and lipophilic region. Lipophilic area may contain one or more lipophilic chains.

Region polyamine in lipopoliamin preferably has the formula (I)

where m is an integer greater than or equal to 2, and n is an integer greater than or equal to 1, it is possible to m varied between different carbon-based groups included between two amines, i.e. each group (CH) m-NH can have different values m, and m can be the same or can be different when it occurs in a specified formula. At each position R 1 is a hydrogen or connective group for lipo-part of lipopoliamin or very lipo-part, as described in this document forth, and it can be the same or can be different each carbon atom. R 2 is a hydrogen or connective group for lipo-part of lipopoliamin or very lipo-part, as described in this document hereinafter. Preferably, m is between 2 and 6, inclusive, more preferably 3 or 4, and n is between 1 and 5, inclusive, more preferably 3.

When R 1 or R 2 is a very lipo-part or a connection group, which is attached to lipo-part polyamine, formula (I) represents lipophily. Therefore formula (I) is the area polyamine only when R 1 or R 2 are not lipo-part or not connecting the group to which is attached lipo-part.

Preferably, the area polyamine corresponds to the following formula

where R 2 and from 1a R to R 1j are as R 1 defined above, and preferably R 1a is a connection to the group, and the rest of the group R 1 and R 2 are hydrogen.

Connection group contains links that are stable under normal conditions.

Preferably R 1 or R 2 represent a hydrogen atom or radical General formula II:

each R 3 and R 4 , which can be identical or may differ, is a saturated aliphatic radical C p H 2p+2 or unsaturated aliphatic radical C p H 2p or C p H 2p-2 , and p is an integer between 12 and 22, inclusive, and R 5 represents hydrogen atom or an alkyl radical, containing from 1 to 4 carbon atoms, optionally substituted phenyl radical.

Alternate each R 1 or R 2 can be a radical General formula III:

in which X is a group of methylene (-CH 2 -) or carbonyl group (CO-), and each and R R 6 7 that may be identical or may differ, is a saturated aliphatic radical C p' H 2p'+2 or unsaturated aliphatic radical C p' H 2p' or C p' H 2p'-2 , and p' is an integer number between 11 and 21, inclusive.

Regardless of the values of m and n, only one of the symbols R 1 and R 2 can be a radical General formula (II) or (III).

When n is between 2 and 5, the values of m in various fragments

may be identical or may vary.

In a preferred embodiment of the formula (I) n is 3, and the values of m in fragments

are identical or differ and be 3 or 4, or R-1 or R 2 is a: either radical General formula (II), in which each R 3 and R 4 represents an alkyl radical containing 12 to 22 carbon atoms, and R 5 represents hydrogen atom or R-1 or R 2 is a radical General formula (III)in which each R 6-X - R 7-X- is albanology radical containing 12 to 22 carbon atoms.

Especially preferred are a 5-carboxypropylbetaine (DOGS) and 5-carboxylterminated of dipalmitoylphosphatidylcholine (DPPES).

The synthesis of the above mentioned leopolitano described in US 5476962.

The following examples of leopolitano for use in accordance with the invention, includes triptorelin 2,3-dialerace-N-[2-Springerville]ethyl-N,N-dimethyl-1-propanamine (DOSPA), 1,3-dialerace-2-(6-carboxypentyl) Propylamine (DOSPER) and RPR-120535 (Ahmed et al. (2005) Pharmaceutical Research 22 (6), 972-980). Patterns preferred leopolitano specified figure 7.

In DOSPA connection group is a

In DOSPER connection group is a

And the above patterns, thus representing a further suitable examples of connecting groups.

Lipophilic area can be as defined above for R 3 , R 4 R 5 or R 6 , or it can be any saturated or unsaturated hydrocarbon chain, cholesterol or other steroid, natural or synthetic lipid lipid able to form or lamellar hexagonal phase. The length of the hydrocarbon chain can be from 10 to 30 carbon atoms, for example, 12-28, 14-26, 16-24, from 18 to 22 carbon atoms.

Media preferably represents JetSI TM or JetSI-ENDO TM , both of which are available from Polyplus transfection. Alternatively, the media can be a Transfectam®available from Promega.

Under liposomal composition imply that cationic charged amphiphilic compound (i.e. lipophily) combined with a neutral auxiliary lipid such as DOPE (mileypartyintheusa), with the formation of liposomes. Essentially, liposomally composition of lipopoliamin represents containing lipophily composition, in which lipophily not represented in the form of the liposomes. In other words, such structures do not contain, in addition to leopolitano, any neutral auxiliary lipids. Examples of auxiliary lipids are neutral phospholipids, cholesterol, glycerophosphocholine and diacylglycerol. Preferably the composition of lipopoliamin contains only lipophily described in this document.

It is known that, as a rule, a combination of substances for transfection with auxiliary lipids, such as DOPE, will increase the efficiency transfection, and, therefore, surprising that improved and more selective degree of inhibition can be achieved by not including these compounds in the composition in the application of the methods on this invention.

Media preferably represents not lipofectamine 2000, lipofection, jet PEI, or media that has the composition of these commercially available reagents for transfection. The composition of lipofectamine 2000 is a 3:1 (Rev/about.) liposomal composition of poly lipid triptorelina 2,3-dialerace-N-[2(sprintersexual)ethyl]-N,N-dimethyl-1-propanamine (DOSPA) (name of Chemical Abstracts Registry: triptorelin N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxyphencyclimine)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenoic)-1-propanamine), and neutral lipid mileypartyintheusa (DOPE).

Composition lipofection is a mixture of 1:1 DOTMA (bromide 1,2-valeronitrile-3-trimethylammonium) and DOPE (mileypartyintheusa).

The media is also preferable is not siPORT or media that has this commercially available reagent for transfection.

In an alternate embodiment, the media represents a connection polyamine, which is cationic (and preferably protoirey) at physiological pH values, for example, polyethylenimine (PEI). PEI exists in many different structural options, but the most interesting are the options with a value of M n (srednecenovogo molecular weight) of 600 or more for GPC (gel permeation chromatography). For example, PEI may have a value of 500-700 M n, 500-750, 750-1000, 100-1250, 1000-1250, 1250-1500, 1000-20000, 1100-15000, 1200-12500, 1250-10000, 1500-7500, 1750-5000, 2000-4000 or 2500-3500.

Brednikova molecular weight is a way to determine the molecular weight of the polymer. Polymer molecules, even molecules of the same type have different sizes (length chains for linear polymers), so that the average molecular weight will depend on the method of averaging. Brednikova molecular weight represents the total, average, average molecular mass of individual polymers. It is determined by measuring the molecular weight of a polymer n molecules, the summation of the masses and dividing by n.

You can use a line of the form PEI or non-linear, for example, extensive PEI (which, for example, may have a low molecular weight values MM, described in this document).

Under extensive PEI mean PEI, which contains the group's tertiary amines, as well as the group's primary and secondary amines. The number of groups tertiary amines, relative to primary and/or secondary amines indicates the rate or degree of branching in the polymer. Usually ramified PEI contains the group's primary, secondary and tertiary amines in the ratio 0,5-1,5:1,5-2,5:0,5-1,5, for example, 1:2:1 (i.e. the ratio of 2:1 for groups of secondary and tertiary amines), but there are also extensive PEI with branching structure so that they contain relatively more or less the number of groups tertiary amines and can be used in the present invention. Examples of alternative ratios are from 1:1 to 3:1 (groups from secondary to tertiary amines), for example, between 1.2:1 to 2.8:1, 1.4:1 to 2.6:1, from 1.6:1-2.4:1, from 1.8:1 to 2.2:1.

Molecular mass PEI preferably less than 30 kDa or 25 kDa, for example, less than 15, 10, 5 or 2 kDa.

An example suitable PEI available from Sigma (408719 polyethylenimine (average MM ~800 Yes on LS, the average value Mn ~600 by GPC, low molecular weight, contains no water). Other commercially available reagents based PEI include Poly Sciences, Inc PEI (branched, MM 10000), US Biological Exgen 500, Polyplus transfection jetPEI TM , Sigma ESCORT TM Transfection reagent, and cool simple point pointers rubber TransIT TKO®.

As described above, in a preferred embodiment, as the molecules of the medium you can use one or more of polymers of metalldichtungen, i.e. the connection polyamine is a polymer of metalldichtungen. Suitable polymers of metalldichtungen and methods of synthesis of these molecules is described in Hwang et al., (2001) Bioconjugate Chem, 12, 280-90.

Suitable polymers of metalldichtungen and diagram showing their synthesis of the corresponding monomers are listed below:

As stated above, n can be an integer from 4 to 10, inclusive, preferably from 5 to 8, inclusive, or 6 to 7, inclusive. It is most preferable is a 4, 6 or 8. X can be any integer. X preferably constitutes from 1 to 100, from 10 to 50, from 15 to 25 from 1 to 20, for example, from 2 to 15, from 3 to 12, from 4 to 10, from 5 to 8, or 6 to 7, inclusive. It is most preferable X is a 4 or 5.

In the following preferred embodiment, as the molecules of the medium you can use one or more of dendrimers, the group containing amines (for example, polyamidoamine (PAMAM) dendrimer), i.e. the connection polyamine is a dendrimer containing an amine group. Dendrimers are a class with macromolecular structure, called "dense star"-polymers.

Unlike classical polymers, dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific characteristics of the size and shape and highly functionalized leaf surface. Thus, dendrimers are artificially obtained or synthesized molecule, which is derived from extensive units or monomers, with the formation of monodisperse, woody or generational patterns. Synthesis of monodisperse polymers requires a high level of control synthesis, which is reached by means of step-by-step reactions, form a single layer of monomers dendrimers, or "generation", at the same time. Each dendrimer consists of multifunctional Central molecules with woody wedge attached to each functional area. The Central molecule called "0 generation." Each successive repeating unit along all branches forming the next generation, the generation 1 and generation 2" and so on until the final generation.

The manufacturing process, thus, is a series of repetitive steps, starting with the Central initiating the core. Each stage of growth represents a new "generation" of the polymer with the larger diameter of molecules, double quantity of the reaction surface areas and approximately double molecular weight relative to the previous generation. For example, the generation of PAMAM dendrimer described in Esfand et al., (2001) Drug Discovery Today, 6(8), 427-36 and Kukowska-Latallo et al., (1996), 93(10), 4897-902.

Suitable dendrimers include all dendrimers, the group containing amines, for example; dendrimers with centers of triethanolamine, NH 3 or Ethylenediamine, which are attached amine containing monomers. Especially are preferred PAMAM dendrimers. Preferably, dendrimer consists of monomers polyamine, for example, having the General formula H 2 N(CH 2 ) m-NH-(CO) n -(CH 2 ) o , where m and o are the numbers from 1 to 10, preferably 1 or 2 and n represents 0 or 1.

PAMAM dendrimers shown Fig. Each "generation" is the addition of two new groups H 2 N-CH 2-CH 2-NH-CO-CH 2-CH 2 - each end of the amino groups of the previous generation; as also illustrated in the figure.

The table below shows the calculated properties of functional PAMAM dendrimers with surface amines for generations.

Generation

Molecular mass

The measured diameter (A)

Surface groups

Preferably PAMAM dendrimer has a molecular weight of 1000-235000 or 3000-117000, for example, 6000-60000 or 14000-30000 Yes.

Dendrimers can also be defined with account of their generation, and essentially dendrimer (for example, PAMAM dendrimer) preferably is a dendrimer 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 generations, in particular, 2-6 generation.

As mentioned above, the following alternative embodiment, as the molecules of the medium you can use one or more of cationic peptides, i.e. the connection polyamine is a cationic polypeptide. Various cationic peptides are known in this area.

As defined in this document, "peptide" includes any molecule that contains any number of amino acids, i.e. one or more amino acids. However preferably peptide is a polymer of consecutive amino acids.

Peptides can be obtained in any convenient way, for example, direct chemical synthesis or recombinant methods by the expression in the cell molecules of nucleic acids with the appropriate sequence.

As denoted in this document in respect of peptides, "cation" means that total, or aggregate, charge peptide is +1 or more at physiological pH values, i.e. pH of 7.2. Consider that the amino acid has a +1 if it is the prevailing type at physiological pH values is positively charged, when present in the sequence of the peptide. Each of these amino acids in the peptide provides additional positive charge in the calculation of the final charge peptide. Peptide can contain one or more negatively charged amino acid residues, and remains neutral, provided that the total charge peptide (calculated by summing the charges peculiar to each amino acid) is positive.

Preferably, cationic peptide contains the remains of L - and D-lysine, L - and D-arginine, L - or D-histidine and/or ornithine. More preferably peptide enriched by one or more of these residues, for example, it contains 10-100%, 20-80%, 30-70%, 40-60% or 50% of positively charged residues. Examples of these include peptides poly-L-lysine, poly-D-lysine, polyhistidine, histidinolovorans poliisin and poliorkitis or copolymers residues L or D lysine, L and D-arginine, L or D histidine and/or ornithine with other amino acids, for example, with one or more of alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, Proline, serine, threonine, tryptophan, tyrosine, and valine.

These cationic peptides can be determined from the point of view of their molecular weight. Essentially, they preferred to have a mass at least 1000 Yes, 1500 Yes 2000 Yes, 2500 Yes, 5000 Yes, 7500 Yes, 10000 Yes, 15000 Yes, 20000 Yes, 25000 Yes, 30000 Yes, 40000 Yes, 50000 Yes, 60000 Yes, 70000 Yes, 80000 Yes, 90000 Yes or 100000 Yes.

Alternative cationic peptides can be determined from the point of view of their length. Preferred cationic polypeptides have a width of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500 amino acids.

Highly preferred cationic peptide is poliklinik, in particular, polyorganic MM with at least 15000 kDa.

Without communication with theory, I believe that the Association of media molecule siRNA based on the interaction of a positively charged media negatively charged molecule siRNA, which leads to the formation of complex siRNA-media. Complexes siRNA-media interact with anionic es on the cell surface and captured by endocytosis.

As a rule, associations, linking or conjugation media molecule siRNA or siRNA, and with photosensitizing agent, reached by a simple mixing of the two components in appropriate conditions and concentrations and allowing components to interact. Thus, in a preferred embodiment, the method includes the additional step of contacting the specified siRNA with the specified media. The conditions under which spend this stage of the contact, and the corresponding concentration for each of the media and siRNA molecules can easily determine specialist in the field of conducting conventional testing. Examples of suitable conditions are as specified in the examples. For example, a molecule of siRNA and media molecule can be mixed, for example, shaking, and allowing to stand, for example, at room temperature. Then molecule siRNA and media molecule can be left approximately 10-20, or 10-30 20-40 minutes before contact with the cell.

For transfection, for example, in the hole of a standard 6-hole tablet use preferably 10 nm to 200 nm, for example, 15-150 nm, 20-100 nm, 20-100 nm, 30 to 90 nm, 40-80 nm or 50-70 nm siRNA, although you can test other concentration. Determination of the optimal concentration of siRNA for use in the way is a matter of practice.

The cells that cause siRNA and the media, received with the use of standard methods of cultivation of cells. If the cells are fixed cells, they preferred to have 50-70% or 25-50% with closing of monolayer.

Media molecule siRNA can be mixed in various proportions based on standard protocols. For example, as shown in examples 1 through 6, 8,4 MKL 2 mm media, made in aqueous solution, can mix with 2.8 mg siRNA in 2 ml environment for application in 6-well plate. Similarly, 4,2 MKL 2 mm media, made in aqueous solution, can be mixed with 1.4 mcg siRNA in 1 ml environment for application in 6-well plate. The media does not have to be at a concentration of 2 mm, suitable ranges include 0,5-5 mm, 1-3 mm, 1,5-2,5 mm (for example, produced in aqueous solution). For each ng siRNA, you can use the amount, equivalent to only about 6 nanomolar media (e.g., 1-10, 2-8) in solution with photosensitizing agent.

For PEI, preferred concentration is 1 mcg/ml and 10 mcg/ml (for example, 0.5-20 or 1-15 mg/ml) 100 nm siRNA, for example, in 1 ml environment for application to 6-well plate. It was shown that it is particularly effective for PEI with a molecular mass 1200-25000 Dalton (for example, 1300-2000 Dalton), as shown in example 9. The concentration used PEI can be modified to achieve the ratio of charges and/or the ratio N/P, which is achieved using the terms mentioned in example 9.

For polymers of metalldichtungen suitable concentration of 100 mg/ml (for example, 10-1000, 20-800, 30-600, 40-400, 50-200 mg/ml) with 50 nm siRNA, for example, in 1 ml environment for application in 6-well plate. It is based on the use of polymer as described in example 11. The values of n and X will influence the charge of the molecule and, as such, can be fit different concentrations. Used the concentration of polymer metalldichtungen can be modified to achieve the ratio of charges and/or the ratio N/P, which is achieved using the terms mentioned in example 11.

For a group amine containing dendrimers, suitable concentration of 100 mg/ml (for example, 10-1000, 20-800, 30-600, 40-400, 50-200 mg/ml) 100 nm siRNA, for example, in 1 ml environment for application in 6-well plate. The concentration used groups amine containing dendrimer, can be modified to achieve the ratio of charges and/or the ratio N/P, which is achieved using the terms mentioned in example 12.

For cationic peptides, suitable concentration is 0.35 mg/ml or 0.7 mcg/ml (for example, 0,1-20, 0,2-15, 0,3-10 mcg/ml) with 100 nm siRNA, for example, in 1 ml environment for application in 6-well plate. As outlined in example 13, these concentrations used in series with the media on the basis of Polyrhinia with molecular weight 15000-70000 and >70000. Molecular mass and the amino acid composition of peptide will influence the charge of the molecule, and, in fact, can be fit different concentrations. Used concentration of cationic peptide can be modified to achieve the ratio of charges and/or the ratio N/P, as achieving using the conditions specified in example 13.

The ratio of the two components can be expressed as the ratio of charges, and this must also be taken into account. Preferably, the charge ratio between the carrier and siRNA is at least 1+/- (i.e. 1 positive charge on the negative charge), 5+/-, 10+/-, 20+/-, 30+/-, 40+/-, 50+/-, 60+/-, 70+/-, 80+/-, 90+/-, 100+/-; 200+/-, 300+/-, 400+/- or 500+/-.

The ratio of charges depends on the charge of each component (i.e. from siRNA and media) and on the number of each component that is present.

Alternatively, the ratio of the two components can be expressed as the ratio N/P, i.e. the ratio of residue nitrogen to the phosphate oligonucleotide. Because not every atom of nitrogen media always is a cation, the ratio N/P is not the same as the ratio of charges. The ratio N/P depends on the chemical composition of each connection and the number of each connection, which is present. Suitable values for the ratio N/P include 1-500, for example, 2-450, 3-400, 4-350, 5 are 300, 6-250, 7-200, 8-150, 9-100, 10-80, 15-60, 20-50, 30-40, 1-25, 1-20, 1-15, 1-10, 1-5.

Preferably the media is such that the concentration you choose to use, does not release from intracellular compartments without the stage of irradiation, PCI, and the release of intracellular compartments see stage after exposure PCI. Suitable concentration and the ratio of siRNA and media mentioned above.

The media, as a rule, are made for transfection in aqueous solution (for example, in the water). Then, the original solution (for example, 20 mm in ethanol) can be diluted (for example, in the water) to the appropriate concentration for application. In US5476962 describes the composition of leopolitano.

As mentioned above, the media siRNA can also be used as a carrier for photosensitizing agents. However, alternative photosensitizing substance can be used without the media or with alternative media that are not used in accordance with this invention for siRNA. These alternative media are to be designated herein as the media for a photosensitizing agents, and they include polycation, such as poliisin (for example, poly-L-lysine or poly-D-lysine), polyethylenimine or dendrimers (for example, cationic dendrimers, such as SuperFect7); cationic lipids, such as DOTAP or lipofection, and peptides.

In order to target molecules siRNA and/or photosensitizing substances on specific cells (for example, malignant cells or tissue, siRNA molecules and/or photosensitizing agent and/or the media can be linked to or konjugierte with a specific target molecules, which will provide specific cellular capture siRNA molecules in the required cells or tissue.

You can use many different target molecules, for example, as described in Curiel (1999), Ann. New York Acad. Sci. 886, 158-171; Bilbao et al., (1998), in Gene Therapy of Cancer (Walden et al., eds., Plenum Press, New York); Peng and Russell (1999), Curr. Opin. Biotechnol. 10, 454-457; Wickham (2000), Gene Ther. 7, 110-114.

The target molecule can be associated, or related conjugated molecules siRNA, with the media, with photosensitizing substance or two (for example, siRNA and bearer or siRNA and photosensitizing the substance or the media and photosensitizing substance) or all three of these groups, and you can use the same or different target molecules. As mentioned above, you can use more than one target molecules simultaneously.

The method according to this invention can be applied in practice, as described below. The method according to this invention, siRNA molecules, together with its carrier and photosensitizing connection (not necessarily with the same media or media photosensitizing agents) applied simultaneously or consistently on the cells, and then photosensitizing connection, the media and siRNA molecules are endocytosis or otherwise move to endosome, complementary mechanism or other intracellular limited membrane compartments.

siRNA, media and photosensitizing connection can be applied to cells together or sequentially. As a rule, siRNA mixed with a carrier, as described above, so that the education of the complex, which is then injected into the cell simultaneously with photosensitizing connection. Alternative complex siRNA:media and photosensitizing connection, you can enter sequentially. The complex siRNA-media and photosensitizing connection can be captured by the cell in one or in several intracellular compartments (for example, they can work together to move).

Then siRNA is released under the effect on the cells of light with a wavelength suitable for firing photosensitizing connections, which in turn leads to the destruction of the membranes of intracellular compartments and subsequent release of siRNA, which can be in the same compartment that photosensitizing agent, in the cytosol. Thus, in these ways, final effects on the cells of light leads to the release of siRNA from the same intracellular compartment, which is a photosensitizing agent, and its penetration into the cytosol.

In WO 02/44396 (which is included in this document as links) described the way in which the order of stages can be changed so that, for example, photosensitizing substance in contact with the cells and activated exposure before molecules subject internalization (and the media), are in contact with the cells. This adapted method has the advantage of not having to molecule subject internalization, were present in the same cell subcompartment that photosensitizing substance during irradiation.

Thus, in a preferred embodiment, implementation specified photosensitizing agent, the specified media and specified siRNA molecules (such as a complex carrier:siRNA) is applied to the cell together, or specified photosensitizing substance is applied separately from the specified media and specified siRNA molecules. As a result, they can be captured by the cell in one of intracellular compartment and then can be specified irradiation. Photosensitizing agent, the media and the molecule siRNA can be an individual, or they can be made as dendrimer molecules (see, for example, Nishiyama N et al., (2005) Nat Mater. 4(12):934-41).

In an alternate embodiment, the method can occur through contacting the specified cells with photosensitizing agent, contact the specified cells media and siRNA molecules, subject to the introduction of, and exposure specified cells of light with a wavelength, effective for activation photosensitizing agents, where the specified irradiation is carried out to cellular capture the specified siRNA molecules and the media in the intracellular compartment that contains the specified photosensitizing agent, preferably before cell capture specified molecules and the media in any intracellular compartment.

The specified exposure can be carried out after the cellular capture molecules and molecules of the medium in the intracellular compartment, regardless of localized whether the specified molecule siRNA and photosensitizing agent in some intracellular compartments during exposure to light. However, in a preferred embodiment, irradiation is carried out to cellular capture molecules to be internalized.

As used herein, "internationalization" refers to the delivery of molecules in the cytosol. In this case, "internalization", thus, includes a step release of molecules of intracellular/membrane-bound compartments in the cell cytosol.

As used herein, "cellular capture" or "translocation" refers to one of the stages of internationalisation, in which molecules outside of the cell membrane, are trapped in a cage that they were on the inside of lying outside the cell membrane, for example through endocytosis or other mechanisms of capture, for example, in the intracellular limited membrane compartments, for example, endoplasmic network, Golgi body, complementary mechanism, endosome etc., or with them.

Stage contacting cells with photosensitizing agent and siRNA molecules and the media can be any convenient or desirable way. Thus, if the stage of contact should conduct in vitro cell convenient ways to support in the aquatic environment, such as, for example, an appropriate medium for cultivation of cells, and in the corresponding point of time photosensitizing agent and/or molecule siRNA and media, you can simply add to the environment in the relevant conditions, for example, depending on concentration and during the relevant time period. For example, cells can be subjected to community engagement siRNA molecules and the media in the presence of serum-free medium.

Photosensitizing substance exposed to contact with the cells in the corresponding concentration and within an appropriate period of time, which can easily identify qualified specialist with common methods and they will depend on such factors as the specific photosensitizing agent and the type and location of the target cells. The concentration of photosensitizing agents should be such that after the capture of the cell, for example, in one or more of its intracellular compartments, or in conjunction with, and activation through irradiation, one or more cellular structures were destroyed, for example, to one or more of intracellular compartments were lysis or destroyed. For example, photosensitizing agents, as described in this document, can be used in concentrations, for example, from 10 to 50 mcg/ml For use in vitro, the range can be much broader, for example, 0.05-500 g/ml For treatment of the person in vivo, photosensitizing substance can be used in the range of 0.05-20 mg/kg of body weight, with the systemic administration or 0.1-20% in solvent for local use. Animals have smaller concentration range may vary and can adjust accordingly.

Incubation of cells with photosensitizing substance (i.e. a "contact"can vary from several minutes to several hours, for example, even up to 48 hours or more, for example, from 12 to 20 or 24 hours. The incubation period should be such that photosensitizing substance was being captured by the relevant cells, for example, in the intracellular compartments of these cells.

After incubation of cells with photosensitizing substance may not necessarily follow the incubation period with not containing photosensitizing substance environment, before the cells exposed to light or add siRNA molecules and the media, for example, for 10 minutes to 8 hours, mainly, from 1 to 4 hours.

Molecule siRNA and the media (for example, as a pre-formed complex siRNA:the media) are in contact with the cells in the corresponding concentration and during the relevant time period.

Identification of suitable doses of siRNA molecules for use in the methods of the present invention is common practice for professionals in this field. For applications in vitro, illustrative dose of siRNA molecules will be approximately 1-100 nm siRNA, and for applications in vivo she will be approximately 10 -6 1 g siRNA for injection in humans. For example, siRNA molecules can be entered at levels less than 500 nm, for example, less than 300 nm, especially preferably less than 100 nm and 50 nm, for example, from 1 to 100 nm, or from 5 to 50 nm, where this concentration reflects the levels in contact with the cell.

As mentioned above, it was found that the contacts you can start even a few hours after adding photosensitizing agents and conduct of exposure.

The corresponding concentration can be defined depending on the efficiency of the capture of interest siRNA molecules in interest cells and final concentration that it is desirable to achieve in the cells. Thus "time transfection" or "time of taking cells" i.e. time for which the molecules are in contact with the cells can range from a few minutes up to several hours, for example, you can use the time transfection from 10 minutes to 24 hours, for example, 30 to 10 hours or, for example, from 30 up to 2 hours or 6 hours. You can also use a longer incubation period, for example, from 24 to 96 hours or more, for example, 5-10 days.

Increased time transfection, as a rule, leads to increased capture of interest molecules. However, the shorter the incubation period, for example, 30 minutes to 1 hour, can also lead to higher specificity capture molecules. Thus, the choice of time transfection for any way, you must find the appropriate balance between getting enough capture molecules, while maintaining sufficient specificity processing PCI.

In vivo, the appropriate way and time of incubation, through which the molecule siRNA, media and photosensitizing agents subject to contact with target cells will depend on factors such as the route of administration and the type of siRNA molecules, media and photosensitizing agents. For example, if the molecule siRNA and the media is injected into the tumor, tissue or body being treated, the cells near the point of injection will be in contact and, therefore, have a tendency to capture siRNA molecules more quickly than cells, located at greater distances from the point of injection, which, probably will be in contact with siRNA molecules at a later time and in a lower concentration.

However, despite the fact that the situation in vivo is more complex than in vitro, the fundamental concept of the present invention is nevertheless the same, i.e. the time after which the molecules are in contact with target cells must be such that to exposure appropriate number of photosensitizing agents were captured by the target cells and either: (i) before or during irradiation molecule siRNA was or captured, or is subject to seizure after sufficient contact with the target cells, in the same or in different intracellular compartments, or (ii) after irradiation molecule siRNA were in contact with the cells over a period of time, sufficient to ensure its capture in cells. Given molecule siRNA is captured in the intracellular compartments, invalid activation photosensitizing agents (for example, the compartments, which contain the substance), the molecule siRNA can be captured before or after exposure.

Stage of irradiation with light to activate photosensitizing agents may occur in accordance with the methods and processes that are well known in this area. For example, the wavelength and intensity of the light can be selected in accordance with the photosensitizing agent. Suitable light sources are well known in this area.

The time during which the cell is exposed to light in the methods of the present invention, may vary. The effectiveness of internalization of siRNA molecules in the cytosol increases with the exposure of light to the maximum, after which increases damage cells and, thus, cell death.

Preferred stage duration of exposure depends on factors such as target, photosensitizing the substance, quantity photosensitizing agents that have accumulated in the target cells or in the target tissue, and the overlap between the absorption spectrum of the photosensitizing agents and the spectrum of emitted light source. As a rule, the length of time for the stage of irradiation is of the order from several minutes to several hours, for example, preferably up to 60 minutes, for example, from 0.5 or 1 to 30 minutes, for example, from 0.5 to 3 minutes, or 1 to 5 minutes, or 1 to 10 minutes, for example, from 3 to 7 minutes, and preferably approximately 3 minutes, for example, from 2.5 to 3.5 minutes. You can also use a shorter exposure times, for example, from 1 to 60 seconds, for example, 10-50, 20-40 or 25-35 seconds.

The appropriate dose of light can be selected by an expert in the field, and they will also depend on photosensitizing agents and the number of photosensitizing agents gained in the target cells or target tissues. For example, the dose of light, usually used for photodynamic treatment of malignant tumors with photosensitizing substance with Photofrin and the predecessor of protoporphyrin 5-aminolevulinic acid, is in the range of 50-150 j/cm 2 with a range of flux density of less than 200 mW/cm 2 in order to avoid hyperthermia. Doses of light, as a rule, are lower when using a photosensitizing agents with higher extinction coefficients in the red region of the visible spectrum. However, for the treatment of non-malignant tissues with less accumulated photosensitizing agents, the total required amount of light can be essentially higher than in the case of treatment of malignant tumors. Furthermore, if you want to preserve the viability of the cells, you need to avoid excessive levels of toxic particles and the settings you can adjust the suitable way.

How this invention can lead inevitably to the destruction of a certain number of cells by means of photochemical processing, i.e. by means of formation of toxic particles when activated photosensitizing agents. Depending on the alleged use this cell death may be of no consequence and, in fact, it may be advantageous for some applications (for example, in the treatment of malignant tumors). However preferably cell death avoid, and, as noted in this document, the method can be conducted so as to cause a strict inhibition of expression (i.e. strict effect siRNA) in the absence of cell toxicity. Is a highly preferential achieving strict inhibition of expression in the absence of a common cellular toxicity or effects on cell viability. How this invention can be modified in such a way that part or proportion of surviving cells were regulated by selecting doses of light depending on the concentration of photosensitizing agents. Also, such methods are known in this area.

In applications in which a viable cells are desirable, essentially all of cells, or a large majority (for example, at least 50%, more preferably at least 60, 70, 80 or 90% of the cells) are not destroyed. Cell viability after treatment by means of PCI, you can define the standard methods, known in the field, such as the test MTS (see examples).

Cytotoxic effects can be achieved by using, for example, gene therapy, in which a molecule siRNA internalized in tumor cell method on this invention, for example, to suppress gene.

How this invention can be applied in vitro or in vivo, for example, or for processing in situ or ex vivo treatment with the subsequent introduction of the treated cells in the body, for a variety of purposes, including inhibition of the expression of certain genes, for example, in the methods of gene therapy, and the creation of screening assays.

Thus, real the invention relates to a method of inhibiting the expression of the target genes through the introduction of siRNA molecules in the cell that contains the specified gene target, method, as described herein above, where this molecule specific siRNA inhibiting the expression specified of the target genes.

"Specific inhibition" refers to a dependent sequence from inhibition of the target genes. Expression of genes that contain the sequence that is quite identical level of nucleic acids used molecule siRNA, be violated by siRNA molecules. As noted above, have been developed that allow qualified to design siRNA molecules with the appropriate sequence to invoke a specific sequence inhibition of expression.

"Gene target" refers to the gene whose expression to suppress and which is targeted research or manipulation.

These methods can be used to change the expression profile of cells, for example, for the study of cell circuits, or to determine the effect of the expression of a specific gene, or for therapeutic purposes.

How this invention can also be used to treat any disease, which is useful suppression, reparation or mutation in one or more genes. For example, genes that sverkhekspressiya in malignant tumors, can be suppressed by the introduction of appropriate siRNA molecules (Lage (2005) Future Oncol 1(1):103-13). Alternative diseases that can be treated include neurodegenerative diseases such as Huntington's disease and Alzheimer's disease and viral infections, such as hepatitis (for example, B and C and HIV.

Thus, the next aspect of this invention relates to a composition containing a molecule siRNA, media molecule (preferably as a set with the specified molecule siRNA) and optional separately also photosensitizing agent, as described in this document. In the next aspect of this invention relates to a specified compositions for use in therapy.

Alternative present invention relates to a set containing molecule siRNA, molecule carrier and optionally also photosensitizing agent, as described in this document. Preferably specified set (or product) is designed for simultaneous, separate or sequential use in medical therapies.

Alternative described, the present invention relates to the use of siRNA molecules and media as described in this document, to obtain medicinal products for the treatment or prevention of disease, disorders or infection by changes in the expression of one or more genes to target the specific patient. Optional indicated the drug can only contain one of the specified siRNA molecules or media and it can be used in the ways in which this molecule siRNA or media not present in the specified medicinal product, intended for introduction to the specified patient in the treatment or prevention of specified diseases, disorders or infection. Optional specified medicinal product may contain photosensitizing agent. Preferably indicated the drug is intended for gene therapy, i.e. treatment for diseases or disorders characterized by abnormal gene expression or which is useful suppression of one or more genes. This change includes the suppression of the specified expression.

In accordance with the variants of implementation, different from the above mentioned photosensitizing agent and specified siRNA molecules and the media is subjected to community engagement cells or tissues of a patient at the same time or consistently and these cells irradiated by light with a wavelength, effective for activation photosensitizing agents, and irradiation is carried out before, during or after the cellular capture the specified siRNA molecules and media in the intracellular compartment that contains the specified photosensitizing agent, preferably before cellular capture the specified floating molecules in any intracellular compartment.

As defined in this document, the "treatment" refers to reduce, mitigate or eliminate one or more of the symptoms of diseases, disorders or infections that are subjected to treatment, relatively symptoms before treatment. "Prevention" refers to the slowing or preventing the onset of symptoms, breach or infection.

The compositions of the present invention can also contain the cell containing the molecule siRNA, which internalisaton in the cytosol of the specified cells way on this invention. This invention further extends to such compositions for use in therapy, in particular, in the treatment of malignant tumors or in gene therapy.

Thus, another aspect of this invention refers to the cell or cell population containing molecule siRNA, which internalisaton in the cytosol of the specified cell, and the cell is produced by method of the present invention.

The next aspect of this invention relates to the use of such cells or cell population to get the song or the medicinal product for use in therapy, as described herein above, preferably in the treatment of malignant tumors or in gene therapy.

In addition, it the invention relates to a method of treatment of the patient, including the introduction of a specified patient cells or compositions of the present invention, i.e. the way, including the introduction phase of siRNA molecules in the cell, as described herein above, and the introduction of the specified cells obtained in this by the way, indicated patient. Preferably these methods used for the treatment of malignant tumors and in gene therapy.

In vivo, you can use any route of administration, common or standard in this area, for example, an injection infusion, local introduction, both on internal, and on external the surface of the body etc. For use in vivo, this invention can be applied to any tissue that contains cells, which places photosensitizing agent and siRNA molecules, including the area of the body fluids and solid tissues. You can treat all fabric, provided that photosensitizing substance is captured by the target cells, and the light can be properly delivered.

Thus, the songs on this invention can be made in any convenient way, in accordance with the technologies and processes, known in the pharmaceutical field, for example, using one or more pharmaceutically acceptable carriers or excipients. As denoted in the present paper, "pharmaceutically acceptable" refers to the ingredients that are compatible with other ingredients compositions and are physiologically acceptable to the recipient. The type and composition of media or materials of excipients, dosage, etc. you can choose a conventional manner in accordance with the selected and required by way of introduction, the aim of treatment etc. Dosage similarly, you can define an accepted way, and they can vary depending on the type of molecule, treatment goals, patient's age, route of administration, etc. In respect of photosensitizing agents, you should also consider the effectiveness/the ability to destroy the membrane under irradiation.

Ways described above, an alternative you can use to create a screening tool for high-performance methods of screening, in particular, to analyze the effects of suppression of a specific gene. siRNA aimed at one or more specific genes can be created and used in the way according to this invention, as described above. Thus, siRNA can be used to reduce the expression of a gene in a population of cells. Then, the resulting population of cells can be used as a screening tool to identify follow-up effects of suppression of the gene with standard methods.

Earlier attempts to reduce the expression of genes with normal and chemically modified oligonucleotide antimuslim were limited to issues related to the degradation of the nucleases antisense oligonucleotides, the emergence of non-specific effects and/or lack of affinity to the target. Using the method of this invention for the introduction of siRNA, these problems can be overcome.

The result of this change in gene expression can influence the expression of other genes. Thus, through the impact on normal test gene expression, you can determine the change of the pattern of expression of other genes. The identification of these genes and the impact test gene expression has on them allows the researcher to draw conclusions about the functions of the gene, for example, about its future functions. Genes that are affected by the change in normal test gene expression, you can activate or repress, but the total change of expression pattern indicates the role of the gene in normal cell functioning and consequences of violations of its regulation.

Using standard methods, well-known in this field, you can explore the effect of suppression or elimination of expression of interest gene. For example, this can occur through the search functional changes in cells (or cell population), such as changes in cell adhesion, protein secretion, or morphological changes. Alternative gene expression profile can be investigated directly by the analysis of patterns of mRNA and/or expression of protein, also with use of standard methods that are well known in this area.

Under the inhibition or decrease gene expression should be understood that the expression of interest gene reduced in comparison with the cell, which was not subjected way, i.e. with the wild-type cage or with normal cell. Change in the level of gene expression can be defined by standard methods, known in this area.

May occur complete inhibition of expression, so there is no measurable detection of gene expression, i.e. no measurable detection of mRNA or protein, or may occur partial inhibition of expression, i.e. a decrease by which the value of gene expression is lower than in the cell wild-type or in a normal cell. This can be assessed and monitored by comparing the effect of siRNA with a specific sequence with the effect of siRNA with a randomized sequence, i.e. with the same composition of nucleotides, but with a different order of sequence. It is preferable to make this method was suitable, the decrease in the expression is less than or 80% of the reference levels, e.g., <50%, preferably <20, 10 or 5% of the reference levels. Used cell and preferably will be a cell population, individual cells are genetically identical. Cells can be any of the cells, as discussed above.

The cell or cell population received in accordance with the methods on this invention can be used to obtain a library that forms the next aspect of this invention.

This invention will be described further on in more detail in the following non-limiting examples with reference to the following drawings in which:

Figure 1 presents the results of the experiment on the suppression of genes with the use of cell lines of OHS with various reagents for transfection with (black bars) and without (white columns) processing PCI using siRNA9. The graph below shows the levels of protein S100A4, from left to right; 1) lipid siPORT, 2) FuGene 6, 3) lipofectamine 2000, 4) lipofection, 5) jetSI and 6) jetSI-ENDO. The results are presented as a percentage of untreated control cells. Columns are averages for the three separate experiments. Error bars indicate standard error of the mean (SEM).

Figure 2 presents the results of the experiment suppress gene, where cells of various types were transpirirovat siRNA using jetSI-ENDO, processing through PCI and without it. The results in (A) show levels of the protein S100A4 in four cell lines in the processing of siRNA. The grey bars show conducting a randomized control siRNA with PCI, black columns shown effector siRNA without PCI and white columns shown effector siRNA with PCI. Cell lines from left to right: HCT-116, SW620, OHS and RMS. The columns represent the average of three separate experiments. Error bars indicate standard error of the mean (SEM). The results in (B) shows a Western blot, showing the different cell lines from top to bottom, HCT-116, SW620, OHS and RMS. On the top panel shows the load control with alpha-tubulin and the bottom panel shows the levels S100A4. In each of the bottom panel, tracks 1-3 show the levels of protein without processing PCI: untreated control (C), a randomized control (siRNA11) and effector (siRNA9). On the tracks 4-6 shows the levels of protein processing PCI: untreated control (C), a randomized control (siRNA11) and effector (siRNA9).

Figure 3 shows the results of the experiment suppression of the gene in cells OHS (a) a dose-dependent inhibition (1-5 nm siRNA) through 96 h after irradiation, b) time-dependent inhibition (24, 48 and 96 h) with siRNA9. The results are presented as a percentage of untreated control cells. The columns represent the average of three separate experiments. Straps errors shows the standard error of the mean (SEM).

Figure 4 shows the results of suppressing gene by siRNA, transpirirovat with jetSI, processing PCI through 96 h after irradiation and without it. The results show the level of protein S100A4 (A) and RNA levels (B) after treatment cell lines OHS 100 nm siRNA, as indicated below. Black columns are the samples without PCI, and white columns are the samples subjected to PCI. Untreated control without processing PCI used as a control for all samples (not represented). Samples: 1 and 4) in a randomized control (siRNA11), 2 and 5) the effector (siRNA9), 3) untreated control. The columns represent the average of three separate experiments. Straps errors shows the standard error of the mean (SEM).

Figure 5 shows the distribution of fluorescently labeled siRNA in OHS after transfection (200 nm) with jetSI-ENDO, processing PCI and without it: (a) delivery of siRNA without processing PCI, left to right: phase contrast, fluorescence and the corresponding absorption, b) delivery of siRNA processing PCI: from left to right: phase contrast, fluorescence and the corresponding absorption, c) fluorescent patterns without processing PCI showing left to right: fluorescence, encased in endosomes (left box), and the fluorescence yield of endosomes (right box), zoom the image in the left rectangle, and the magnification of the image in the rectangle.

Figure 6 shows the results of suppressing gene using jetSI after processing OHS through 100 nm siRNA and jetSI at 50% of the recommended level, compared to a standard Protocol. The columns are: 1) randomised control siRNA without PCI, 2) effector siRNA without PCI, 3) untreated control with PCI, 4) randomized control siRNA with PCI, 5) effector control siRNA with PCI. The columns represent the average of three separate experiments.

Figure 7 presents the structure of your preferred leopolitano. (See also Ahmed et al., above and Behr et al. (1989) PNAS, 86, 6982-6).

On Fig shows the results of gene suppression using PEI as a carrier. Western blot displays load control (alpha-tubulin) in the upper panel and protein levels S100A4 in the lower panel. Track 1 = raw control (without PEI), 2 = 1 mm PEI + effector siRNA, 3 = 10 ul of PEI + effector siRNA, 4 = raw control (without PEI), 5 = 1 mm PEI + effector siRNA, 6 = 10 ul of PEI + effector siRNA. Tracks 1-3 presents without PCI, and tracks 4-6 presented with PCI.

Figure 9 presents the results of suppressing gene using PEI mass of 25 kDa as a carrier. Used samples indicated on the figure. A. The level of protein S100A4, quantitatively defined by scanning Western blots (using siRNA S100A4 with or without PCI, the average value of 3 separate experiments, where error bars represent SEM). B. the Example of the Western blot.

Figure 10 shows the effect on PCI activity siRNA media PEI used in various concentrations. The levels of protein S100A4 quantitatively determined by scanning Western blots. Black columns correspond transfection without PCI, while white columns correspond transfection with PCI. The results are the average of 3 separate experiments.

Figure 11 shows the results of experiments to determine the toxicity PEI separately and in combination with PCI and siRNA. Specify the number of PEI and the dose of the light used in experiments with PCI. A. Toxicity PEI without PCI. Specified MM various (extensive) test media PEI (average for 5 separate experiments). B. Toxicity of the combination of PCI, PEI (1 mg) and siRNA at different doses of light. Shows the tested samples and doses of light. Control - = raw control (without PEI but without PCI), Randomized, - = randomized control siRNA (PEI but without PCI), siRNA - = siRNA S100A4 (PEI but without PCI). Control +, Randomized + and siRNA + are the same as Control -, Randomized and siRNA-but with PCI (average for 5 separate experiments).

On Fig shown induced PCI delivery of siRNA molecules using a beta of ciclodextrina as a carrier. Tracks 1 and 4 = control without siRNA, tracks 2 and 5 = control conducting a randomized, siRNA, tracks 3 and 6 = siRNA S100A4. Bandwidth control tubulin and band S100A4 are as shown.

On Fig shown induced PCI delivery of siRNA molecules using polyamidoamine (PAMAM) dendrimers (G2-7) with the center of Ethylenediamine. (A) the Western blot, in which the upper band are a load control (alpha-tubulin), lower bands are levels S100A4. Samples in different tracks are: 1. PAMAM G6 with PCI. 2. PAMAM G1 with PCI. 3. Control with PCI. 4. PAMAM G6 without PCI. 5. PAMAM G1 without PCI. 6. Control without PCI. (B). The level of protein S100A4, quantitatively defined by scanning Western blots. Black columns are transfection without PCI, while white columns represent the transfection with PCI. The results are the average of 3 separate experiments. The figure indicates the various forms used PAMAM.

On Fig shows the structural formula GO, G1 and G2 PAMAM dendrimers.

On Fig shows the effect on PCI mediated by POLIKLINIKA delivery of siRNA. The level of protein S100A4 analyzed by Western blotting. Top track meets load control (alpha-tubulin), the bottom track is a levels S100A4. Samples gel were as follows: C+ = control with PCI, S+ = control conducting a randomized siRNA with PCI, R+ = siRNA S100A4 with PCI, C = control without PCI, S = Supervisory conducting a randomized siRNA without PCI, R = siRNA S100A4 without PCI. 1 = poliklinik MM 15000-70000 in the amount of 0.35 mg, 2 = poliklinik MM in 15000-70000 the amount of 0.7 mcg, 3 = poliklinik MM >70000 in the amount of 0.35 mg, 4 = poliklinik MM >70000 in the amount of 0.7 micrograms.

EXAMPLES

Materials and methods

Cell lines and cultivation conditions

HCT-116 (colorectal adenocarcinoma) and SW620 (colorectal adenocarcinoma) were obtained from the American type culture collection (Manassas, VA, USA). OHS (osteosarcoma) and cell lines RMS were obtained in the Norwegian Radium Hospital. All cell lines cultivated with the use of RPMI-1640 medium (Bio Whittaker, Verviers, Belgium or GibcoBRL, Paisley, UK), no antibiotics, but supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Linz, Austria) and 2 mm L-glutamine (Bio Whittaker, Verviers, Belgium). The cells were grown and incubated at 37 degrees C in a humidified atmosphere containing 5% CO2 . All cell lines tested, and found that they are negative against Mycoplasma infection before the experiments.

The light source and substance photosensitizing

Lumisource ® (PCI Biotech AS, Oslo, Norway) was used as a light source. Lumisource ® is a series of four fluorescent tubes designed to ensure homogeneous illumination of the treated area, emitting mainly blue light peaking at 420 nm. Photosensitizing agent, desulfuromonas tetraphenylporphine (TPPS 2a ) acquired from Porphyrin Products (Logan, UT, USA). TPPS 2a first was dissolved in 0,1M NaOH, and then diluted in phosphate-buffered saline (PBS), pH 7.5, to a concentration of 5 mg/ml and up to a final concentration of 0,002M NaOH. Photosensitizing substance protected from light and stored at -20 to use.

SiRNA transfection without PCI

Various reagents for transfection to deliver siRNA was estimated using: Lipofectin TM Reagent from Life Technologies Inc. (Gaithersburg, MD, USA), Lipofectamine TM 2000 from Invitrogen (Carlsbad, CA, USA), FuGene 6 from Roche Diagnostics (Mannheim, Germany), siPORT TM Lipid Transfection Agent from Ambion (Austin, TX, USA), jetSI TM and jetSI TM-ENDO from Polyplus transfection (Illkirch, France). All reagents for transfection processed in accordance with the descriptions of the manufacturer. All cell lines cultivated, as described in "Cell lines and conditions of cultivation", and were cultured for 24 h in 6-hole tablets to 50-70% of closing the monolayer before transfection for 24, 48 or 96 hours Reagent for transfection separately inflicted on the cells as untreated control, in addition to a randomized siRNA with reagents for transfection.

The Protocol used for jetSI/jetSI-ENDO, was as follows for the standard 6-hole tablet:

- Stage 1: For each hole, dilute 4,2 (8,4) MKL solution jetSI/jetSI-ENDO 100 ul of environment. Shake vigorously (important: do not pipette until mixture) and wait for 10 minutes (important: not to exceed 30 minutes).

- Stage 2: For each hole, dilute 1,4 mcg (100 nm) duplex siRNA and 100 ul of the environment. Gently shake.

- Add 100 ul of the solution environment jetSI 100 ul of solution siRNA and at once to mix the solution (important: do not mix the solutions in the reverse order).

- Immediately shake-mix solution for 10 seconds.

- Incubate for 30 minutes at room temperature to provide education systems (important: not to exceed 1 hour).

- In the process of formation of the complex, delete environment for the growth of tablets and add 0.8 ml containing fresh serum environment (and photosensitizing agent, if it is used), pre-heated at 37 degrees C.

- Add 200 ul of the solution jetSI/siRNA into each well and homogenize the mixture careful rotation of the tablet.

- Incubate tablet in required conditions of cultivation of cells within 18 hours, then rinse tablet three times a fresh environment and re-incubated with 2-4 ml environment.

SiRNA transfection with PCI

The granulosa cells and transpirirovat as in the case of "siRNA transfection without PCI", with a few modifications. To the environment when transfection added photosensitizing substance TPPS 2a (0.5 mg/ml). After 18 hours of incubation cells were washed 3 times with fresh medium and incubated within 4 hours before processing light. After 4 h, the cells were exposed to the effect of blue light (7 mV/cm 2 ) during the various periods of time (60-90 s), depending on the cell line, and re-incubated for 24, 48 and 96 hours, and then collected. To measure the effect PCI effector siRNA, randomised siRNA and transfection reagent for separately put in a different hole of the same tablet, with photosensitizing substance or without it, and perform the same processing. The cells were protected from light aluminum foil in the course of these experiments.

PCR (reverse transcriptase with detection in real time to S100A4

Total cellular RNA was isolated using a set GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Steinheim, GER) and for reverse transcription used the kit cDNA synthesis iScript (BioRad, Hercules, CA). Both sets used in accordance with the guidelines of the manufacturer. All PCR was performed in parallel detection in real time spent using SYBR Green I. For each PCR, 10 ul of cDNA, MCL 30 iQ SYBRGreen Supermix (BioRad), 300 nm each primer and not containing nucleases water was added to a final volume of 60 mm. Then, on 25 MKL each sample was applied on the tablet for PCR. This method ensures that the Parallels were true Parallels, and that for all occurrences of a mix PCR was sufficient. The design of primers was performed using software Primer Express from Applied Biosystems (Applied Biosystems, Foster City, CA). Used set of primers (direct primer 5'-AAGTTCAAGCTCAACAAGTCAGAAC-3' and reverse primer 5'-CATCTGTCCTTTTCCCCAAGA-3') amplificare segment of the 79 digested 2 and 3 of the exon sequence S100A4.

Reaction to the detection in real-time spent on iCycler (Bio-Rad) with the following Protocol amplification: the initial denaturation for 3 min at 95 º C, 50 cycles of denaturation for 10 s at 95ºC and annealing/extend over 35 with at 60ºC, one keeping 95ºC within 20, with subsequent keeping within 1 min at 55ºC, and the evaluation of the curve annealing of 80 stages, each 10 C, elevation 0,5ºC to the final temperature 95ºC. The quality of RNA samples confirmed by gene amplification in the household, TBP (direct primer 5'-GCCCGAAACGCCGAATAT-3 and reverse primer 5'-CGTGGCTCTCTTATCCTCATGA-3') and RPLPO (direct primer 5'-CGCTGCTGAACATGCTCAAC-3' and reverse primer 5'-TCGAACACCTGCTGGATGAC-3'). For the quantitative calculation used Gene Expression Macro, version 1.1 (Biorad). The program carries out calculations on the basis of the method ∆∆ CT, which allows comparison of the threshold cycle, obtained using different sets of primers on one set of samples.

Microscopic research

Cells were incubated with complexes siRNA/jetSI-ENDO as described (siRNA transfection with PCI and without it) and analyzed with the processing of PCI or without it in 48 hours using the inverted microscope Zeiss Axiovert 200, equipped with filters to FITC (excitation filter BP 450-490 nm, the beam splitter FT 510 nm and emissionnye filter LP 515-565 nm), and rhodamine (excitation filter BP 546/12 nm, the beam splitter FT 580 nm and an emission filter LP 590 nm). The painting was received with use of Carl Zeiss AxioCam HR, Version 5.05.10 and software AxioVision 3.1.2.1. The images were obtained using Adobe Photoshop 7.0 (Adobe, San Jose, CA) and the Zeiss LSM Image Browser (Version 3).

Western Western blot turns

Protein lysate received in 50 mm Tris-HCl (pH 7.5), containing 150 mm NaCl and 0.1% NP-40 with 2 g/ml pepstatin, Aprotinin (Sigma Chemical Company, St. Louis, MO) and lapatina (Roche Diagnostics, Mannheim, Germany). Total protein lysate (30 micrograms) of each sample were divided by 12% SDS-polyacrylamide gel electrophoresis and carried on membranes Immobilon-P (Millipore, Bedford, MA) in accordance with the manufacturer's guidance. As load monitoring and control of movement, membranes were stained 0,1% ahmedovym black. After this membrane incubated 20 mm Tris-HCl (pH 7.5), containing 0,5M NaCl and 0.25% Tween 20 (TBST) with 10% of dry milk (blocking solution) before incubation with rabbit polyclonal antibody against S100A4 (diluted 1:300, DAKO, Glostrup, Denmark) and murine monoclonal antibody against the alpha-tubulin (diluted 1:250, Amersham Life Science, Buckinghamshire, England) in TBST containing 5% dry milk. After washing, immunoreactive proteins were visualized using conjugated to horseradish peroxidase secondary antibodies (diluted 1:5000 DAKO, Glostrup, Denmark), and enhanced chemiluminescent system (Amersham Pharmacia Biotech, Buckinghamshire, England). The levels of protein S100A4 recorded as a percentage of the control sample and as load control used alpha-tubulin.

Example 1

Suppression of gene expression in cell lines of OHS

Cells OHS was transpirirovat designed molecule to target on the expression of the protein S100A4 using different systems for transfection. In each case used a standard Protocol transfection, in accordance with the manufacturer's instructions. When used photosensitizing agent, it was a TPPS 2a (0.5 mg in volume transfection 1 ml, excluding jetSI, which used the amount transfection 2 ml). The time of exposure in each case amounted to 60 seconds.

The original 20 microns solution siRNA mixed with each of the various reagents for transfection and transpirirovat 6-hole tablets with a final volume of 1 ml (except jetSI and jetSI-ENDO, with the final volume was USD 2 ml).

Lipid siPORT = tested 2 and 4 MCL, in combination with 1.4 mcg siRNA in 1000 MKL

FuGene 6 = tested 4,2 MKL and 8.4 MCL, in combination with 1.4 mcg siRNA in 1000 MKL

Lipofectamine 2000 = tested 4.2 and 7 MCL, in combination with 1.4 mcg siRNA in 1000 MKL

Lipofectin = tested 4.2 and 7 MCL, in combination with 1.4 mcg siRNA in 1000 MKL

jetSI = 8,4 MCL, in combination with 2.8 mg siRNA in 2000 MKL

jetSI-ENDO = 4,2 MCL, in combination with 1.4 mcg siRNA in 2000 mm.

Used molecule siRNA was a molecule against mRNA sequence S100A4 (registration number GenBank NM_002961). Specific siRNA with a sequence

5'-UGAGCAAGUUCAAUAAAGA-3'

3'-ACUCGUUCAAGUUAUUUCU-5'

Dried oligonucleotides siRNA resuspendiruetsa in the amount of 100 microns in processed DEPC water and stored at -20. Annealing was performed through separation into individual aliquots and dilution of each of the oligonucleotide RNA concentrations up to 50 microns. Then 30 MKL each solution of the oligonucleotide RNA and 15 ul 5X buffer for annealing were United to the final concentration 50 mm Tris, pH 7.5, 100 mm NaCl in the treated DEPC water. Then the solution is incubated for 3 minutes on a water bath at 95 º C, followed by a gradual cooling within 45 minutes on the desktop. After annealing confirmed by gel electrophoresis 4% agarose gel NuSieve (data not shown).

In figure 1 you can see that the dependent PCI suppression of the gene can be achieved through the application of tools for transfection jetSI and jetSI-ENDO. When these two tools for transfection use in the absence of PCI (i.e. exposure, but in the absence of a photosensitizing agents), gene expression approximately 75% of control by measuring levels of a protein using the Western blot, but when, in addition, use PCI, gene expression is reduced approximately up to 15% of the control.

In contrast, the use of lipid SIPORT or Fugene 6 as a means for transfection not achieved any significant changes in gene expression, and the use of lipofectamine 2000 and lipofection led to inhibition of gene expression, regardless did also PCI.

Example 2

Suppression of genes in different cell lines

Four different cell lines, cells HCT 116, SW620, OHS and RMS was transpirirovat 50 nm siRNA (jetSI-ENDO = 4,2 MKL, together with 1.4 mcg siRNA), designed to suppress the expression of S100A4 (see example 1), using the standard Protocol jetSI-ENDO, in the presence and absence of photosensitizing agents (0.5 mg/ml TPPS 2a ). The cells were subjected to irradiation and protein levels S100A4 determined through Western blots 96 hours after exposure (exposure were the following, OHS = 60 s, SW620 = 80 C HCT116 = 90 C, RMS = 70).

Examples of the results are presented on FIGU, and the results are presented graphically on figa. Each type of cells impact on transfetsirovannyh cells through processing PCI led to high levels of gene suppression, while the cells that were transfuziology siRNA S100A4, but which were not subjected processed PCI showed significantly less suppression of the gene. It was shown that the effect is specific, because conducting a randomized siRNA had no effect on gene expression, both in the presence and in the absence of PCI. No significant differences in the effect of suppressing gene between different cell lines that were tested.

Example 3

The effect of concentration of siRNA and time

Molecule siRNA, designed to suppress the expression of protein S100A4, was transpirirovat in the cells of OHS in concentrations of 1 to 5 nm, using 4,2 MKL jetSI-ENDO, and subjected to processing PCI, as described above.

The levels of protein in the cell lysates were measured using the Western blot and they are shown as a percentage of protein levels in untreated control cells on figa.

The effect of suppressing gene has increased with the concentration of siRNA, the effects of which were subjected cells, although there is a slight difference between the effects of suppression identified in the case of 4 nm 5 nm siRNA.

Randomised molecule siRNA also used in all experiments, and it was shown that it does not affect gene expression. Essentially, the effect of inhibiting gene is specific.

On FIGU shows the effect of time on the suppression of gene by changing the period of time between the exposure of cells and collection of cell lysates for analysis. You can see that on the longer is the time of the cells left after exposure and before you collect, the more inhibits the expression of the gene see.

Example 4

The suppression of the gene in cells OHS after processing siRNA PCI

Cells OHS was transpirirovat 100 nm or siRNA randomized siRNA as described above, and determined the effect of processing on PCI levels of the protein and mRNA through 96 h after irradiation by Western blotting and RT-PCR and compared with the levels of the protein and mRNA in the untreated controls.

The results, shown in figure 4, show that in relation to the levels of protein and mRNA, large values of suppressing gene for gene S100A4 see when siRNA that are specific to this gene, transferout with jetSI ENDO and cells processed PCI (track 5 figa and B). A small decrease in the levels of protein and RNA detected without processing PCI (track 2 figa and B), but it is greatly enhanced by processing PCI.

Example 5

Cytosolic delivery labeled siRNA

Cells OHS was transpirirovat 200 nm FITC-labeled siRNA using jetSI ENDO ((jetSI-ENDO = 8,4 MKL, together with 2.8 mg siRNA in the amount of transfection 1000 MKL = 200 nm solution siRNA), with photosensitizing agent and without it. Then transfetsirovannyh cells exposed to radiation.

Cells OHS was transpirirovat 100 nm siRNA using jetSI. In contrast to the above experiments, jetSI used in lower concentrations, i.e. in 50% of concentration, as recommended in the standard Protocol. Standard Protocol for 6-hole tablet is: 2.8 mg siRNA + 8,4 MKL jetSI in 2000 MKL environment, with getting 100 nm siRNA in each well.

In contrast, for a 6-hole tablet, 1,4 mcg siRNA mixed with 4.2 MKL jetSI in 1000 ml of environment, receiving 100 nm siRNA in each well. The total number of complexes, thus, was reduced by 50%.

After transfection of cells or subjected to processing PCI (dose of light for this experiment was 30 C, with 0.5 mg/ml TPPS 2a ) or left unprocessed, and suppression of gene was determined using RT-PCR. Effector siRNA was able to reduce the expression of a gene S100A4 to less than 20% from untreated control, while without PCI, and using a randomized siRNA (or to the processing PCI, or without it) showed a small decline.

This demonstrates that the combined use of media PCI not only provides the advantage of selective release siRNA molecules, but with lower concentrations of funds for transfection you can achieve high levels of suppression of genes in combination with PCI. By comparing 5 tracks figure 6 5 track figure 1 you can see that even with only 50% of the funds for transfection, which are used in example 1, when also using PCI, the degree of inhibition of the gene is significantly higher.

Example 7

Transfection using PEI as a carrier

siRNA-target chose against mRNA sequence S100A4 (registration number Gene Bank NM_002961). siRNA 481-499 used as an effector for the sequence, see example 1).

Polyethylenimine (PEI) was evaluated in relation to the induced PCI delivery. Cell lines cultivated (RPMI-1640 supplemented with 10% FBS, 10 ml L-glutamate, 10 ml Hepes) in the 6-hole tablets to 50-80% of closing the monolayer before transfection. As the standard concentration used 100 nm siRNA.

Used PEI was from Sigma and it was diluted in sterile water and received the original solution containing 1000 MKL PEI and 9000 MKL sterile water. From the original solution of 1 and 10ul used for transfection of cells 1,4 mcg siRNA (100 nm in each well).

Used PEI from Sigma (408719 polyethylenimine (average MM ~800 LS, Mn average ~600 through the GPC, extensive, low-molecular, anhydrous)).

For transfection, received two solutions, the solution A: siRNA diluted in 100 ul of serum free (OPTI-MEM (I) environment. Solution B: PEI was diluted in 100 ul serum-free medium. Solutions A and B are mixed through careful hashing and incubated at room temperature for 30 minutes Then mixed mortar added to cells (1 ml 100 nm siRNA).

Processing PCI was performed, as described above for jetSI. Dose of light to experiment PEI was 40 C. levels of the protein were measured by Western blotting through 96 h after irradiation.

From pig you can see that the expression of protein S100A4 decreases in the samples shown on tracks 5 and 6, in the samples that were subjected transfection with PEI and processing PCI.

Example 8

The effect on PCI activity siRNA using media PEI mass of 25 kDa

SiRNA transfection. All cell lines cultivated, as described in "Cell lines and conditions of cultivation", and were sown in a 6-hole tablets at 25-50% closing monolayer before transfection. Conducted education complex siRNA and media careful hashing and incubated for 30 min before adding to the cells. The cells were transpirirovat siRNA, the media and or photosensitizing substance (TPPS 2a = 0.5 mg/ml)or without it, and incubated for 18 h, then washed 3 times with fresh environment and before processing the light again incubated within 4 hours After 4 h, the cells were exposed to the effect of blue light (7 mV/cm 2 ) during the various periods of time (0-60 C), depending on the experiment, and before collecting re-incubated within 96 hours To measure the effect of PCI at suppressing gene-specific siRNA, randomised siRNA and transfection reagent for separately put in a different hole one plate, with photosensitizing substance or without it, and have the exact same treatment. During the experiments, the cells were protected from light aluminum foil.

Mediated siRNA S100A4 suppression of gene was measured on the protein level, with the processing of PCI and without a branching polyethylenimine (PEI) weight 25 kDa (1 mg/ml) and siRNA aimed at gene S100A4, at a concentration of 100 nm. Followed the above Protocol, using doses of light 30s, as with the formation of complexes and transfection in containing serum environment. On figa shown that the application of PCI, S100A4 siRNA, as a rule, reduced the level of S100A4 up to 5-15% of the level of untreated controls (processed with PEI, but without siRNA). In contrast, S100A4 siRNA without PCI was only able to reduce the level S100A4 to 100-80% of control. The levels of untreated controls were compared with controls, but instead are specific to S100A4 siRNA used siRNA (data not shown). On FIGU experiment is shown by Western blots. Top bands correspond load control (alpha-tubulin), and the lower band corresponds to the levels S100A4, as indicated in the figure. As can be seen from these blots, siRNA significantly suppresses S100A4 when using PCI, than would be the case for cells, which were introduced S100A4 siRNA, which have not been processed PCI, where it was impossible to identify significant suppression of genes.

Example 9

The effect on PCI activity siRNA media PEI, used in various concentrations

Effects on PCI activity S100A4 siRNA investigated for use different formulations of branched PEI in various concentrations (0.1 mg/ml 1 mg/ml, 10 mg/ml and 100 g/ml, was using 1 ml of medium per well). The tested samples PEI (all branched) were as follows: PEI MM (Yeah)

6. 25000

Followed the Protocol described in the section "Materials and methods", using doses of light 30s, as with the formation of complexes and transfection, containing serum environment. siRNA used at a concentration of 100 nm. As you can see from figure 10, the use of PCI can significantly increase the effect of suppressing gene through S100A4 siRNA media PEI. This effect is particularly pronounced at lower levels PEI (1 and 10 mg/ml), where the effect without PCI was very low, except PEI 4 (1800 MM) at a concentration of 10 mcg/ml. When these two concentrations PEI PCI greatly amplified the suppression of the gene for all tested samples PEI, except PEI 1 (800 MM).

Without PCI degree of suppression of gene increases with increasing concentrations of PEI, PCI, this effect is not as pronounced, except that you cannot see any suppress gene (either PCI or without it) from 0.1 mcg/ml PEI. One possible explanation for this is that the number of PEI is not high enough for the formation of the complex with all siRNA, which leads to negatively charged complexes, which are not captured by the cells. Without PCI, apparently, there is a tendency towards increased suppression of the gene with increasing molecular mass media PEI; PCI this effect is not so obvious, also excluding PEI 1 (800 MM). The effect PEI without PCI MM at higher and higher amounts is probably a consequence of the described antisovetskih property PEI, existing only at high concentrations PEI.

This shows that PCI can replace this effect, giving the opportunity to implement low quantities and low MM polyethylenimine, this property is highly beneficial to avoid toxicity and other problems with the media PEI.

Example 10

Toxicity studies

First assessed the toxicity of various compositions PEI (MM 800-25000) separately (without processing PCI). In this analysis, the cells OHS sown in 96-well plates and allowed them to attach themselves during the night in containing serum environment. Then Wednesday deleted and cells were incubated with the environment and different formulations PEI in various concentrations within 20 PM Then contains PEI environment was removed, and each hole solution was added MTS (dilution 1:6, 100 ul/well) (Promega, Madison, WI, USA), and tablets re-incubated for 4 more hours Measured the absorption at 490 nm.

As you can see from figa, toxicity is increased with increasing molecular weight composition PEI (for example, 25000 PEI against 800 PEI) and with increasing quantities of PEI. It is important that the composition of PEI in the amount of 1 mg/ml (not shown) and 10 mcg/ml does not indicate significant toxicity. For these samples, significant biological effect siRNA can be achieved with PCI, while the effect without PCI was very low (see example 9).

Investigated the effect on PCI mediated beta-tsiklodekstrina delivery of siRNA. In these experiments used a dose of 60 light with and followed the Protocol described in the section "Materials and methods". Beta cyclodextrines, as described above, with n=6 and X=4 diluted in sterile water and spent the formation of complexes and transfection in serum-free medium. As you can see from the Western blot (Fig), beta-cyclodextrine at a concentration of 100 mg/ml (used 1 ml per well), which formed complexes with 50 nm (0,7 mcg) siRNA, was effective for induced PCI delivery of siRNA (3 track), while in these conditions delivery without PCI was ineffective (track 6). Beta cyclodextrines used in this study consists of molecules beta-cyclodextrin, conjugated through the bridge Amin responsible for binding to siRNA, and described in Hwang SJ. et al. (2001, Bioconjugate Chem. 12, 280-290).

Example 12

Induced PCI delivery of siRNA molecules using polyamidoamine (PAMAM) dendrimers (G2-7) with the center of Ethylenediamine

Polyamidoamine (PAMAM) dendrimers were assessed in relation to the induced PCI delivery of siRNA. PAMAM diluted in sterile water and transfection conducted in containing serum environment. Different types of PAMAM dendrimers (G2-7) at a concentration of 100 mg/ml (used 1 ml per well) were subjected to the formation of complexes with 100 nm (1,4 g) siRNA and transfection was in accordance with the methods described above. In these experiments used a dose of 30 seconds of light As you can see from the Western blot, (Figo) PCI can significantly enhance the activity of siRNA (tracks 1 and 2) in circumstances where siRNA/PAMAM separately was ineffective against suppressing gene (tracks 4 and 5). As you can see in FIGU, this effect was also evident for several other types of PAMAM dendrimers, indicating that can PCI mainly to strengthen the delivery of siRNA through dendrimers based polyamine.

Different types of PAMAM used in the study, containing various the number of surface groups amines, are:

G2 = Molecular formula: [NH 2 (CH 2 ) 2 NH 2 ]:(G=2);dendri PAMAM(NH 2 ) 16

G3 = Molecular formula: [NH 2 (CH 2 ) 2 NH 2 ]:(G=3);dendri PAMAM(NH 2 ) 32

G4 = Molecular formula: [NH 2 (CH 2 ) 2 NH 2 ]:(G==4);dendri PAMAM(NH 2 ) 64

G5 = Molecular formula: [NH 2 (CH 2 ) 2 NH 2 ]:(G=5);dendri PAMAM(NH 2 ) 128

G6 = Molecular formula: [NH 2 (CH 2 ) 2 NH 2 ]:(G=6);dendri PAMAM(NH 2 ) 256

G7 = Molecular formula: [NH 2 (CH 2 ) 2 NH 2 ]:(G=7);dendri PAMAM(NH 2 ) 512

Generation

The measured diameter (nm)

Surface amino

Example 13

Induced PCI delivery of siRNA using hydrochloride poly-L-arginine as a carrier

Poliklinik diluted in sterile water and held transfection in serum-free medium. Tested two types of carriers on the basis of Polyrhinia (molecular weight 15000-70000 and >70000). Poliklinik in the amount of 0.35 or 0.7 mcg/ml (used 1 ml per well) were subjected to the formation of complexes with 100 nm (1,4 g) siRNA and followed the Protocol described above, evaluating the expression S1004A by Western blotting after a dose of 30 seconds of light As you can see from Fig, there is a significant difference in the efficiency of suppression of gene between treated and untreated by PCI samples. Thus, although all processed through PCI samples (R+samples on Fig) show significant suppression of the gene, it was impossible to observe the effect of suppression in the respective samples, not processed PCI (R-samples on Fig). Thus, PCI is effective in suppressing gene with both test different carriers on the basis of Polyrhinia and used both concentrations, indicating that the PCI can significantly enhance the delivery of siRNA through media-based cationic peptide.

1. Way the introduction of siRNA molecules in the cell cytosol where this method includes: i) contacting the specified cells with siRNA molecules, the media and photosensitizing agent, and (ii) the exposure of the cells of light with a wavelength, effective for activation photosensitizing agents, where specified the media contains cationic polyamine selected from (a) of lipopoliamin in liposomal part where the specified lipophily is a 5-carboxypropylbetaine (DOGS), JetSI™ or JetSI-ENDO™, (b) extensive polyethylenimine (PEI) with molecular weight from 1,2 kDa to 25 kDa (c) polymer of metalldichtungen formula

where X is an integer between 1 and 20, inclusive, and n is an integer from 4 to 10, inclusive, (d) molecule of the dendrimer FRAMES, and (e) cationic peptide selected from POLIKLINIKA or copolymer L or D arginine.

2. The method according to claim 1, where molecular weight PEI is less than or equal to 25 kDa.

3. The method according to claim 1, where molecular weight PEI is less than 25 kDa.

4. The method according to claim 1, where specified PEI has a value M n 500-20000 by the GPC.

5. The method according to claim 4, where specified PEI has a value M n 500-1500 by the GPC.

6. The method according to claim 1, where this media molecule is a molecule dendrimers FRAMES generation 2-6.

7. The method according to claim 1, where the length of siRNA molecules is 12-28 nucleotides.

8. The method according to claim 1, where this cell is a mammal cells.

13. The method according to claim 1, where for transfection using 10 nm to 200 nm siRNA.

14. The method according to claim 1, where, in addition, there is the media photosensitizing agents selected from polycation, polyethylenimine, dendrimers, cationic lipid and peptide.

15. The method according to claim 1, where siRNA mixed with a carrier so that was formed complex, which is then injected into the cell simultaneously or sequentially with photosensitizing agent.

16. The method according to claim 1, where the conduct by contacting the specified cells with photosensitizing substance, contact the specified cells media and siRNA molecules, subject to the introduction of, and exposure specified cells of light with a wavelength, effective for activation photosensitizing agents, where the specified irradiation is carried out before cell seizure specified siRNA molecules and the specified media in the intracellular compartment that contains the specified photosensitizing agent.

17. The method according to article 16, where the specified irradiation is carried out before cell seizure specified siRNA molecules and the media in any intracellular compartment.

18. Method of inhibiting the expression of the target genes through the introduction of siRNA molecules in the cell that contains the specified gene target, by the way, as defined in any one of claims 1 to 17, where this molecule specific siRNA inhibiting the expression specified of the target genes.

19. Composition containing molecule and siRNA molecule media, and also photosensitizing agent, not necessarily present separately, for use for the treatment or prevention of disease, in case of which may be useful suppression of one or more genes by in vivo introduction siRNA molecules and media in one or more cells, as defined in the method according to any one of claims 1 to 17, where the specified media contains cationic polyamine selected from (a) of lipopoliamin in liposomal part where the specified lipophily is a 5-carboxypropylbetaine (DOGS), JetSI™ or JetSI-ENDO™, (b) extensive polyethylenimine (PEI) with molecular weight from 1,2 kDa to 25 kDa (c) of polymer metalldichtungen formula

where X is an integer between 1 and 20, inclusive, and n is an integer from 4 to 10, inclusive, (d) molecule of the dendrimer FRAMES, and (e) cationic peptide selected from POLIKLINIKA or copolymer L or D arginine.

20. The method of obtaining the cells or cell population containing siRNA molecules in the cytosol of each cell, where this method includes the implementation of the method according to any one of claims 1 to 17 in the cell or cell population.

21. Composition for treatment of disease or impairment, which is characterized by an abnormal gene expression or which is useful suppression of one or more genes that contain the cell or cell population containing molecule siRNA, which internalisaton in the cytosol of the specified cell, and the cell obtained by the method according to claim 20.

22. Set containing molecule siRNA, media molecule and photosensitizing agent to use for the treatment or prevention of disease, in case of which may be useful suppression of one or more genes by in vivo introduction siRNA molecules and media one or more cells, as defined in the method according to any one of claims 1 to 17, where the specified media contains cationic polyamine selected from (a) of lipopoliamin in liposomal part where the specified lipophily is a 5-carboxypropylbetaine (DOGS), JetSI™ or JetSI-ENDO™, (b) extensive polyethylenimine (PEI) with molecular weight from 1,2 kDa to 25 kDa (c) of polymer metalldichtungen formula

where X is an integer between 1 and 20, inclusive, and n is an integer from 4 to 10, inclusive, (d) molecule of the dendrimer FRAMES, and (e) cationic peptide selected from POLIKLINIKA or copolymer L or D arginine.

23. The use of siRNA molecules, media and optional photosensitizing agents to obtain drugs for the treatment or prevention of disease, in case of which may be useful suppression one or more genes, through changes in the expression of one or more of target genes in the specified patient way in which the specified siRNA molecules are introduced into the cell containing one or more of target genes, the manner specified in any one of claims 1 to 17, where this molecule siRNA specific changes the expression specified one or more of target genes, and where the specified media contains cationic polyamine selected from (a) of lipopoliamin in liposomal part where the specified lipophily is a 5-carboxypropylbetaine (DOGS), JetSI™ or JetSI-ENDO™, (b) extensive polyethylenimine (PEI) with molecular weight from 1,2 kDa to 25 kDa (C) of polymer metalldichtungen formula

where X is an integer between 1 and 20, inclusive, and n is an integer from 4 to 10, inclusive, (d) molecule of the dendrimer FRAMES, and (e) cationic peptide selected from POLIKLINIKA or copolymer L or D arginine.

24. The use of cells or cell population containing molecule siRNA, which internalisaton in the cytosol of the specified cell, and the cell is obtained by the method according to claim 20, to obtain drugs for the treatment or prevention of disease, in case of which can be useful suppression of one or more genes, through changes in the expression of one or more genes to target the specific patient.

25. The application of paragraph 24, where the specified drug is intended to treat malignant tumors.

26. The method of treatment or prevention of disease, disorders or infection, in which case it may be useful suppression of one or several genes in the patient, including the introduction of siRNA molecules and media in one or more cells in vivo in accordance with the methods on any one of claims 1 to 17.

27. The method according to p, which is used for the treatment of malignant tumors.