Method of introduction of pna molecule conjugated with positive peptide, in cytosol and/or nucleus by photochemical internalisation (pci)
SUBSTANCE: method provides contact of said cell with a peptide nucleic acid (PNA) molecule and a photosensitising agent and cell exposure to light at wave length effective to activate the photosensitising agent where said PNA molecule is conjugated with positive peptide. Also, there are described compositions containing such conjugated PNA molecules, the cells produced with the use of the method, and application of the method.
EFFECT: effective cell capture of peptide nucleic acid molecules conjugated with positive peptide.
37 cl, 13 dwg, 9 ex
The present invention relates to a method of introduction of the molecules of peptide nucleic acid (NCP), conjugated with positively charged peptides into cells, preferably in the cytosol and/or nucleus of the cells, using a photosensitizing agent and irradiation of the cells with light of a wavelength effective to activate the photosensitizing agent, and to the use of this method for assessing or changing the activity of a gene, for example, antisense or antigenic technology, and for such further applications, such as in a high-performance screening system impact products down-regulation of genes.
NCP are synthetic analogs of DNA in which the normal fosfomifira the link in the main chain of DNA is replaced by the link 2-amino-ethyl-glycine. Nucleotide bases are associated with uncharged repeating units of the main chain through methylcarbamyl of linkers.
As a result of such binding NCP Natarajan. They are also chemically stable and resistant to hydrolytic cleavage and contact with complementary chains of nucleic acids (DNA or RNA) with a higher affinity than natural nucleic acids.
Although hybridization NCP to complementary DNA and RNA is accompanied by formation of hydrogen bonds in Watson-Crick, can gender is given as a parallel, and antiparallel double helix. In addition, their hybrid complexes exhibit excellent thermal stability and show the unique properties of ionic strength. Due to these advantages and the fact that NCP resistant to nucleases and proteases, NCP usedin vitroin antisense (preventing translation of mRNA) or antigen (preventing replication or transcription of a gene) applications. Double helix PNK-RNA are not substrates for RNase H and therefore may cause the antisense effects based on steric blocking or broadcast RNA, or processing. Triple helix is the result of concatenating the NCP and DNA, which may interfere with the replication or transcription, causing antigenic effects. Observed no evidence of any General toxicity NCP.
Thus, the binding molecules of target nucleic acids, PNK have significant influence on the processes of replication, transcription and translation. NCP used in antigenic or antisense techniques, as shown, prevents the activity of DNA - and RNA-polymerases, reverse transcriptase, telomerase and ribosomes.
For these impacts, which will be successfully mediated, it is necessary that the molecules NCP has penetrated into the cells and, for most methods, in the nucleus, which contains some of the NC and all DNA in addition to mitochondrial DNA. Cellular and nuclear capture, it is also very slow and does not occur spontaneously. The improvement of cellular and nuclear capture NCP therefore represents a major problem that must be addressed before you can receive any real prospect of its development as a therapeutic drug or treatment, or for its wide application.
One approach for the delivery of the NCP in the cell is using microinjection (reviewed in Ray and Norden, (2000), FASEB J.14, 1041-1060). Microinjection is also time consuming and requires a large investment of time. In addition, the injection must be individually performed in each cell, and it is, therefore, more suited for a small number of cells and not appropriate for many of the techniquesin vivo. Cell damage is also a problem.
Shipping is also carried out by electroporation (Shammas et al., (1999), Oncogene 18, 6191-6200), which also has disadvantages, for example, it is not suitable for usein vivo.
Also tested such methods that Deplete membrane as a temporary permeability using streptolysin About (Faruqi et al. (1997), P.N.A.S. USA 95, 1398-1403), the permeability of the cell membrane using isolectin (Boffa et al. (1996), J. Biol. Chem. 271, 13228-13233) or detergents like Tween (Norton et al. (1996), Nat. Biotech 14, 625-620). D is by the way also not suitable for use in vivoand can cause damage to the cells.
Even if you can effort to implement molecules NCP in the cell, nuclear capture may not happen. NCP force was implemented for absorption by cells at high concentrationsin vitrohowever , for this was very high concentration (10 to 20 µm) (Folini et al. (2003), Cancer Research 63, 3490-3494). Maybe that's why it was noticed that need better ways of introduction of PNK cells.
Cell NCP deliveryin vitroas also shown, occurs when the introduction of cationic lipid in the form of a complex. In this particular method molecules NCP associated with a functional peptide, were hybridized with overlapping oligonucleotides, and the complex was mixed with a cationic lipid. Then the cationic complex lipid-DNA NCP was internalservice, carrying the NCP as a passive load (Hamilton et al. (1999), Chem. Biol. 6, 343-351).
In PNK no polyanionic charges required for condensation and complexation with cationic liposomes by electrostatic interactions. Hybrids PNK-DNA, in addition, have distributed negative charge, which is provided by DNA. The condensed particles can be formed during the interaction of hybrids PNK-DNA with cationic lipids, such lipoplex quickly integrate into mammalian cells culture (Borgatti et al. (2003), Oncol. Res. 13(5), 279-287; Borgatti et al. (2002), Biochem. Pharmacol. 64(4), 609-616; Nastruzzi et al. (2000), J. Control Release 68(2), 237-249). NCP can also be carried into the cells by covalent binding to lipids (Muratovska et al. (2001), Nucleic Acids Res. 29(9), 1852-1863; Ljungström et al. (1999), Bioconjug. Chem. 10(6), 965-972; Filipovska et al. (2004), FEBS Lett. 556 (1-3), 180-186).
Efforts were also attempts to get the design of peptide-NCP, which can be absorbed by the cells more efficiently. Branden and Smith (2002, Methods in Enzymology 346, 106-124) used the so-called system Bioplex, by means of which the NCP used to associate a functional peptide with DNA in order to increase the delivery of DNA. You can also add polyethylenimine (PEI), to improve the condensation of nucleic acids.
This system is designed to deliver nucleic acids into the cell, and it takes advantage of the NCP as a tool for DNA binding, which must be delivered to the peptides, which are designed to improve the delivery of DNA.
Certain peptides known to mediate the delivery of molecules across the cell membrane. Also tried to bind the NCP with such cellular peptides-conveyors or penetrating into the cell peptides to try to improve the ability of the NCP to penetrate into the cell. We developed a variety of peptide transporters for transportation NCP in a cage./p>
NCP developed as a tool for anti-telomerase activity, conjugatively with the peptide for the internalization of HIV-tat (SEQ ID NO: 1 RKKRRQRRR) and penetrating into the cell Antennapedia peptide (SEQ ID NO: 2 RQIKIWFQNRRMKWKK), and she, as we have seen, had little effect as antisense molecules, reducing telomerase activity. These experiments also showed only a small decrease in telomerase activity; conjugated with tat NCP reduced telomerase activity only up to 73% of the control level at 48 hours, and conjugated with Antennapedia NCP was implemented 50% inhibition only at very high concentrations of >30 μm (Folini et al., 2003, see above).
Also described peptides, which are able to mediate the transport NCP in the core. For newly synthesized nuclear proteins, as shown, requires a specific amino acid sequence in order to reach the core and pass through the nuclear membrane. Such nuclear localization signals, when present in proteins that are not present endogenously in the nucleus, may also direct these proteins to the nucleus.
NCP also conjugatively with a nuclear localization signal (NLS) (SEQ ID NO: 3 PKKKRKV) in an attempt to direct the NCP in the cell nucleus. This NLS, as has been shown, mediates the transfer of large T-antigen SV40 through the nuclear membrane. Bogdanovi 10 μm PNK-NLS cells, his presence in the nucleus was shown after 24 hours. This effect, as has been shown, does not depend on the sequence of the NCP, but strongly depends on the sequence of the NLS; conducting a randomized sequence of the NLS (SEQ ID NO: 4 KKVKPKR), conjugated with the NCP, showed only a minimum number of NCP in the nucleus (Cutrona et al., (2000), Nature Biotechnology 18, 300-303). These results were compared to the functional study, where it was shown that PNK-NLS (wild type) (where NCP represented the antigen to myc) inhibited cell growth, whereas NCP conjugated with a randomized sequence of the NLS, were affected significantly more similar to the impacts of NCP control.
Branden et al. (1999, Nature Biotechnology 17, 784-787) similarly showed that although the conjugation NCP with peptides could increase nuclear transport NCP dependent on the sequence of the NLS way, no nuclear localization was not observed in the inversion of the NLS sequence.
Additional studies have also suggested that to obtain the NCP, which will be successfully transported into the nucleus, it is necessary to konjugierte molecule NCP as with the cell membrane peptide-Transporter, and NLS (Braun et al. (2002), J. MoI. Biol. 318, 237-243). Cell membrane peptide-Transporter believed to imports, the NCP, and NLS, as I believe, then further transmits the NCP in the core. In these experts who the cops NLS, as shown, essential for nuclear transport due to the structure containing penetrating into the cell the peptide with only one peptide sequence lysine-lysine, remained in the cytosol.
Interpretation of the above results is complicated by the fact that Richard et al. (J. Biol. Chem., (2003), 278(1), 585-590) demonstrated that fixation of cells, even under mild conditions of fixation, can cause artifacts in such experiments, and nuclear staining was observed in the absence of the NCP in the nucleus even when soft controlled conditions.
Thus, although it is shown that under certain conditions the NCP or NCP, conjugated with penetrating into the cell peptides can enter the cell, or, as recently shown, in endosome (Richard et al., 2003, above), in most cases, the biological effect mediated by the NCP, you must move the NCP in the kernel.
You may notice that there remains a need for such a reliable and reproducible method of introduction of the molecules of the NCP that is capture in a cage, such as the cytosol, preferably in the kernel, without the need to use high concentrations of NCP.
The inventors have surprisingly discovered that the molecules of the NCP, which is conjugated to a positively charged peptides that undergo endocytosis, and upon release from endosomes using m is using the technique of photochemical internalization (PCI) data molecules are transported into the nucleus.
Thus, according to the first aspect of the invention provides a method for introducing a molecule NCP in the cytosol, preferably in the nucleus of the cell, involving contacting the specified cell with the molecule NCP and photosensitizing agent and irradiation of the cells with light of a wavelength effective to activate the photosensitizing agent, in which the specified molecule NCP conjugated to a positively charged peptide.
PCI is a technique that uses a photosensitizing agent in combination with the stage of irradiation for activation of such agent and shall internalization of molecules simultaneously introduced into the cell. This approach allows the molecules that are captured by cell organelles such as endosome, to escape from these organelles in the cytoplasm after irradiation. The primary method of photochemical internalization (PCI) is described in WO 96/07432 and WO 00/54802, which are included here as a reference. As described above, the chimeric molecule (which is for use in the present invention is a conjugate PNK-peptide) and photosensitizing agent is brought into contact with the cell. Photosensitizing agent and chimeric molecule trapped in a cell membrane-bound by subcompartments inside the cell. When exposed to the cell with light of appropriate length in the wave activates the photosensitizing agent, which directly or indirectly causes the formation of toxic products that destroy intracellular membrane compartments. This enables the chimeric molecule to be released into the cytosol.
Such methods use a photochemical effect as a mechanism for the introduction of molecules, otherwise penetrating through the membrane, in the cytosol of cells in a way that does not lead to the total destruction of the cells or cell death, if the methodology accordingly adapted in order to avoid excessive formation of toxic products, for example, by reducing the time of exposure or dose of the photosensitizer.
Particularly surprisingly, when to release the NCP in a cage uses a method of PCI or certain penetrating into the cell sequence, any sequence of the NLS is not required to enter the NCP in the cell and for its subsequent transfer into the nucleus. Everything you need to NCP was associated with a peptide that has at least a single overall positive charge.
Thus, without wishing to be bound by theory, it appears that, when using PCI, the presence of a positively charged peptide may contribute to the capture molecules NCP in a cell in cellular compartments such as endosomal compartments, and additionally, after wisweb the Denia or the internalization of molecules NCP in the cytosol, charged peptide is then further mediates the directed movement of molecules NCP in the core. As a consequence, it requires only minimal modification of the molecule NCP for directional move it to the right place localization, and does not require conjugation of long amino acid sequences or multiple amino acid sequence with a molecule NCP.
Also surprising that only one peptide required to perform both of these functions, i.e. the directional capture molecules NCP in the cell, and also facilitate transfer to the nucleus, as soon as the molecule PNK-peptide is released or internalized in the cytosol.
Nuclear localization signals have been studied in some detail, and it has been shown that certain amino acid consensus sequences necessary to ensure an efficient directional nuclear transfer. In particular, the metabolic pathway importin identified as the method by which molecules can pass into the kernel. Such "classic" NLS with a high content of arginine/lysine as a sequence of large SV40T antigen that interacts with the protein-importname α+β. The complex moves through the Central channel of the nuclear pore complex and dissociates in the kernel. Stage of the Association and dissociation of I which are energy-dependent mechanisms (discussed by Cartier et al. (2002), Gene Therapy 9, 157-167). There are, as I believe, other metabolic pathways for nuclear import, although they are not as well characterized.
It is therefore surprising that when the PCI according to the invention does not require not only the classical NLS sequence, and moreover, any sequence with a total positive charge of one or more able to mediate nuclear localization. This is demonstrated by the fact that the sequence SEQ ID NO: 5 GHHHHHG, functioning also as SEQ ID NO: 3 PKKKRKV, and additionally that the Tripeptide only with a single positive charge (SEQ ID NO: 6 AKL) had the ability to send NCP, first in endosome and subsequently into the nucleus (see examples).
Next, a surprising observation is that the sequences that were originally identified by their ability to direct proteins in cell organelles such as peroxisomes and mitochondria by conjugation to molecules NCP, is also able to direct molecules PNK first endosome and subsequently into the nucleus (see examples).
The exact role of PCI in the method according to the invention is not known, but it certainly is the main implementation of the method, because without PCI molecules NCP, bearing positively charged peptide, do not penetrate into the cytosol or the nucleus in any material degree.
Efficiency is also apparently, not dependent on the total length conjuguemos peptide, and positively charged peptides with a length of 3 amino acids are equally function as well as peptides with a length of 29 amino acids. The effect also does not depend on the ratio of the charge to the length of the positively charged peptide and the specific charged amino acids, which is included in the sequence.
Used herein, the term "NCP" refers to a molecule, peptide nucleic acid, which acts as an analogue of the DNA and is based on pseudopeptides the skeleton is attached to nucleotide bases. NCP can be in free linear form or may be in the form of a double helix or self-closing, for example, bis-NCP.
Also considered derivatives of the standard form NCP, for example, in which one or more pseudopeptide monomers constituting the polymer, it is possible to modify or to get them derived, for example, to obtain the modified properties, such as lysine or other amino acid analogues. Similarly, one or more of the used grounds can be modified, if necessary, for example, using variants not found in nature. Thus, the NCP includes derivative of the standard form, provided that such derivatives retain the corresponding function is national property, that is capable of forming dependent on the sequence of the complex with DNA and/or RNA. In other words, the derived NCP matches in charge and structure, given the complementarity, sequencing of DNA or RNA.
Molecule NCP may be any sequence or to make any length. Preferably the molecule NCP contains the length less than 25, for example less than 20 grounds. Preferably the molecule NCP contains the length of more than 6 bases. For example, you can use molecules from 6 to 20 bases. The length of the oligomer NCP from 12-17 units optimal. The sequence length is primarily determined by the necessary specifics of the method used. Methods DNA, for which you must have more than 25 bases, can usually be performed with a much shorter sample NCP. Long oligomers NCP, depending on the sequence, are prone to aggregation and are difficult to purify and characterize. In addition, the shorter the sequence, the more it specific. Therefore, the impact of errors is greater than that for short sequences. In addition, oligomers NCP 20 units used without any problems associated with the aggregation.
Such molecules, their chemical properties and methods of synthesis known in the field (Ray and Norden, 2000, above), and they can get any item is chodashim ways.
Molecule NCP can be an antisense molecule NCP or molecule NCP, complementary gene (antigenic molecule), which can form the characteristic triple structure. Molecule NCP can also be a probe, that is, it can be associated with the sequence of the target nucleic acid, and it can therefore bear the label.
The method according to the invention transports the conjugate PNK-peptide in the cytosol, preferably in the core. In addition, will be fully understandable that capture every one of the molecules in contact with the cell, is not achievable. Significant and improved grip relative to the previous levels, which does not use any PCI, it is also achievable. Preferably, the methods according to the invention allow the capture molecules NCP at this level is sufficient that their effect on replication, transcription, or translation clearly expressed by the products of such cells. The corresponding concentration of conjugates PNK-peptide, which will be in contact with the cell, can be selected to achieve this goal, for example, to decrease expression of the target genes by more than 10%, for example, more than 20, 30, 40 or 50% decrease after incubation with the cells within, for example, 24, 48, 72 or 96 hours, for example, from 24 to 48 hours (see, for example, Fig.9). Level red eye reduction is of the amount of protein depends on the time half-life of the protein, that is, pre-existing protein will be deleted in accordance with its half-life. Thus, the decrease in expression of more than 10, 20, 30, 40 or 50% is relative to expression at the same time without NCP so that the half-life is taken into account.
The term "cell" is used here to cover all eukaryotic cells (including cells of insects and fungal cells). Typical examples of "cells" thus include all types of animal cells mammals and memleketim, plant cells, insect cells, fungal cells and protozoa. In addition, preferably, the cells are mammalian cells, such as cats, dogs, horses, donkeys, sheep, pigs, goats, cows, mice, rats, rabbits, Guinea pigs, but most preferably humans.
Used herein, the term "contacting" refers to bringing the cells and photosensitizing agent and/or conjugate PNK-peptide in physical contact with each other under conditions suitable for internalization into cells, for example, preferably at 37°C in a suitable nutrient medium.
Photosensitizing agent is an agent that is activated by light of the appropriate wavelength and intensity to form the activated products. Normally, such an agent can represent the manage an agent, which is localized in intracellular compartments, especially the endosomes or lysosomes. A number of these photosensitizing agents known in the field and described in the literature, including WO 96/07432, which is included here as a reference. In this respect, it is possible to mention di - and tetrasulfonated phthalocyanine aluminum (for example, AlPcS2a), from sulphonated tetraphenylporphine (TPPSn), Nile blue, chlorin derivatives e6, uroporphyrin I peloritan, hematoporphyrin and methylene blue, which has been shown to be localized in the endosomes and lysosomes of cells in culture. This occurs in most cases due to the capture of the photosensitizer by endocytosis. Thus, the photosensitizing agent is preferably an agent that is captured in the internal compartments of lysosomes or endosomes. Additionally appropriate photosensitizers for use according to the invention described in WO 03/020309, which is also included here as a reference, namely, from sulphonated meso-tetraphenylporphine, preferably TPCS2a.
In addition, other photosensitizing agents that are located in other intracellular compartments such as the endoplasmic reticulum or the Golgi apparatus, can also be used. Also presumably, is that there may be mechanisms when the action of which the impact of photochemical processing occur in other cell components (i.e. components other than the limited membrane compartments). Thus, for example, one possibility may be that photochemical processing destroys the molecules that are important for intracellular transport or vesicle fusion. Such molecules may not necessarily be limited membrane compartments, but photochemical damage of such molecules can also lead to photochemical internalization of molecules vectors, for example, by a mechanism in which photochemical effects on these molecules lead to reduced transport of the chimeric molecules (i.e. molecules NCP) in such destructive bubbles, as a complementary mechanism, so that the chimeric molecule could be released into the cytosol to degradation. Examples of molecules that are not necessarily located in the limited membrane compartments, are a few such molecules transport system of microtubules, as dynein and dynactin components; and, for example, rab5, rab7, a factor that is sensitive to N-ethylmaleimide (NSF), soluble protein accession NSF (SNAP), and so on.
Classes of suitable photosensitizing agents that may be mentioned, therefore, include porphyrins, phthalo is Yanina, the purpurin, chlorin, naphthalocyanine of benzoporphyrin, cationic dyes, tetracyclines and isometropia weak bases or their derivatives (Berg et al., J. Photochemistry and Photobiology, 1997, 65, 403-409). Other suitable photosensitizing agents include texaphyrin, pheophorbide, Partizani, bacteriochlorin, kitoblarini, hematoporphyrin derivatives and their derivatives, endogenous photosensitizers induced by 5-aminolevulinic acid and their derivatives, dimers or other conjugates of photosensitizers.
Preferred photosensitizing agents include TPPS4, TPPS2a, AlPcS2a, TPCS2aand other amphiphilic photosensitizers. Other suitable photosensitizing agents include the connection of 5-aminolevulinic acid or esters of 5-aminolevulinic acid or its pharmaceutically acceptable salt.
The term "irradiation" of the cell to activate the photosensitizing agent refers to the application of light directly or indirectly, as described below. Thus, cells can be lit by a light source, for example, directly (for example, isolated cells ofin vitroor indirectly, for example,in vivowhen cells are under the skin surface or in the form of a layer of cells, not all of which are covered directly, i.e. without shielding drugalert.
The term "peptide", as defined here, includes any molecule that contains any number of amino acids, that is, one or more amino acids. Preferably, furthermore, the peptide is a polymer of consecutive amino acids.
Preferably positively charged peptide consists of from 3 (or 4, 5 or 6) to 30 amino acids in length, more preferably from 3 (or 4, 5 or 6), 25, 3 (or 4, 5 or 6) to 20, or from 3 (or 4, 5 or 6) to 15 amino acids in length. In the most preferred implementation, the peptide consists of less than 10 amino acids in length, for example 3, 4, 5 or 6.
The peptides can be obtained by any convenient means, such as direct chemical synthesis or by recombinant methods, by the expression of nucleic acid molecules corresponding sequence in the cell.
Positively charged molecule capable of moving molecules NCP, with which it is conjugated in the cell and then into the cytosol and also, preferably, in the kernel.
Mentioned here the phrase "positively charged" means that the overall, or total, the charge of the peptide is +1 or more at physiological pH, i.e. at a pH of 7.2. The amino acid is considered +1, if the predominant type amino acids at physiological pH values positively charged, in cases when it is stood in the e peptide. Each amino acid in the peptide introduces an additional positive charge when calculating the final charge of the peptide. The peptide may contain one or more negatively charged amino acid ostatu, and neutral residues, provided that the total charge of the peptide (calculated by adding together the charge made by each amino acid) is positive. Molecule NCP nezarazeno and, thus, does not contribute to the total charge of the molecule. In addition, you must understand that this charge represents the charge part of the peptide, which is important and which is examined when determining the presence of a positively charged peptide.
The charge of the peptide, therefore, depends on its amino acid composition. Some amino acids are charged at normal physiological pH values. Positively charged amino acids are lysine (K), arginine (R) and histidine (H) and is considered to be +1 on the above scale. Aspartic acid (D) and glutamic acid (E) carry a negative charge at most physiological pH values and are treated as -1 on the above scale. Other naturally occurring amino acids are believed to have no charge. Can have any number of positively charged or negatively charged amine is acid, provided that the overall charge of the peptide is a +1 or more.
Used in peptides amino acids for use according to the invention does not have to be a naturally occurring amino acid. One of the more amino acids in the peptide can be replaced is not found in nature, for example, derivatizing amino acid. Such amino acids are similarly assessed based on their contribution to the charge of the peptide. Thus, like naturally occurring amino acids, if the predominant type of amino acids are positively charged at physiological pH, it is immaterial whether or not such charge from derivateservlet group (for example, the introduced amino group) or the group is also present in naturally occurring amino acid, provided that the total charge is +1 or more.
The peptide may be present as the site of the hybrid molecules, for example, associated with such non-protein molecule, as an organic polymer, which can, for example, be used as a cross-linking group. The peptide can also join separate component, which can be protein in nature, but which effectively does not depend on the peptide, for example, Natarajan or is a separate structural configuration. In such cases, the peptide presented the yawl would be an exposed, preferably the peripheral area, and would determine the charge of such area as appropriate peptide.
Positively charged peptide can be konjugierte or N-end or C-end of the molecule, the NCP, and can join with or without a cross-linking group, as 8-amino-3,6-dioxanonane acid, 2-aminoethoxy-2-toxicsuse acid (AEEA) or disulfide linkers. Preferably, moreover, the peptide kongugiruut directly covalent binding. Particularly preferably, the conjugate is not present any other components, in addition to the NCP and peptide.
Previous studies have shown that only a classical nuclear localization signals transported conjugated molecules in the core. However, as mentioned above, surprisingly, it is shown that the capacity for nuclear localization peptide depends only on the charge and not on the sequence, when implementing the method of internalization using PCI. Peptides with a net charge of +5 showed the highest degree of absorption, and it is, as found, did not depend on the sequence, providing a boost. The charge of the peptide is >1, preferably from +1 to +10, for example, from +2 to +8, such as from +3 to +6, for example, +4 or +5.
Preferably, the peptides to join NCP rich residues K, R and/or H. Especially predpochtite who correctly use a series of sequential charged residues. Preferably, other residues used in the peptide, neutral. Thus, for example, the peptide may possess or contain the sequence: Xn(Y)m-Xoin which X represents a neutral residues and Y is a positively charged residue, which may be the same or different in each position in which they appear, and n, m and o are integers ≥1, for example, in the range from 1 to 10, and n and o are preferably represents 1 or 2, and m preferably represents from 2 to 5. Particularly preferably, Y represents the same remainder in each position and is K, R or H.
Particularly preferred peptides are SEQ ID NO: 7 MSVLTPLLLRGLTGSARRLPVPRAKIHSL, SEQ ID NO: 6 AKL and SEQ ID NO: 5 GHHHHHG. SEQ ID NO: 7 MSVLTPLLLRGLTGSARRLPVPRAKIHSL and SEQ ID NO: 6 AKL are sequences for directed transport in mitochondria and peroxisomes, respectively, and yet they were able to directed migration into the kernel using the method described here PCI. This unexpected finding illustrates the dependence of the charge, but not on the sequence of peptides that are applicable according to the invention.
Positively charged peptide preferably is a NLS as SEQ ID NO: 3 PKKKRKV or randomized NLS SEQ ID NO: 4 KKVKPKR or reverse LS SEQ ID NO: 8 VKRKKKP, or this classic penetrating into the cell a peptide as HIV Tat SEQ ID NO:1 RKKRRQRRR or penetrating into the cell Antennapedia peptide SEQ ID NO: 2 RQIKIWFQNRRMKWKK. It is possible to estimate, for example, determining the extent of nuclear transfer or penetration into the cell without PCI. Peptides capable of significant nuclear migration or penetration into the cells, in such circumstances, would be considered NLS or penetrating into the cell peptides. The peptide also preferably does not represent polylysin. Additionally, the NCP, or the peptide may contain such additional modifications as fluorescent labels at the ends.
Conjugates PNK-peptide, as described herein, constitute additional aspects of the invention.
Referred to herein, the term "algae" refers to the binding together of the peptide and molecules NCP for the formation of a single structural unit under physiological conditions. The NCP and the peptide is preferably linked covalent bond.
Molecule NCP and the peptide can be synthesized or cleaned separately and then combine, for example, using this molecule is a spacer, as Fmoc-NC603H11-OH (Branden et al., 1999, see above), or they can be chemically synthesized as a single molecule, for example, using the strategy (Btoc). In this way the monomers NCP oligomers are synthesized in a length of 20 bases, using protocols standard peptide synthesis. For the suits PNK use protection fluorenylmethoxycarbonyl (Fmoc) N-terminal amino group of the monomer and benzylaminocarbonyl (Bhoc) to protect A, C and G ekzoticheskih amine groups. Group Bhoc associated with XAL-label for synthesis, which allows the quick removal of protection and cleavage of the oligomer from NCP resin. Typical outputs of the associated product is >95%. Synthesis ends with the removal of oligomer TFMSA from resin. The oligomer is purified by using HPLC with reversed phase (Viirre et al. (2003), J. Org. Chem. 68(4), 1630-1632; Neuner et al. (2002), Bioconjug. Chem. 13 (3), 676-678).
Thus, the positively charged peptide, apparently, responsible for the absorption of the NCP in the cage, and as soon as it is released from intracellular compartment for its nuclear capture.
More than one type of molecule NCP, i.e. molecules NCP different sequences, you can enter or make at the same time. Similarly, you can enter or make molecules NCP carrying more than one type of the positively charged peptide.
Optionally, one or the other, or both of the photosensitizing agent and a conjugate molecule NCP, which must be entered in the cells, you can attach or link, or konjugierte with one or more molecules of the carrier or with molecules for the directional migration, which may impact on the provision or increase of the capture of the photosensitizing agent or conjugated molecule, the NCP, or may have an impact on healthy lifestyles is to cancel the transfer or delivery of data objects to a specific cell type, tissue or intracellular compartment. In the case of conjugated molecules NCP directional transfer in the core can also be achieved by the component peptide conjugated according to the invention.
The examples of carriers include polylysine or other polycation, dextran sulfate, various cationic lipids, liposomes reconstituted LDL-particles or sterically stable liposomes. Such systems media can, in General, to improve the pharmacokinetics and increase cell capture conjugated molecules NCP and/or photosensitizing agent, and may also direct the molecule NCP and/or photosensitizing agent in the intracellular compartments, which are particularly favorable for the implementation of photochemical internalization, but they, in General, do not possess the ability to direct the molecule NCP and/or photosensitizing agent to specific cells (e.g. cancer cells) or tissue. In addition, in order to achieve a specific or selective directional migration, media molecule, the molecule NCP and/or the photosensitizer can be linked to or konjugierte with certain molecules for the directional migration, which will provide a specific cell capture molecules NCP in the desired cell or tissue. Such molecules for the directional migration may also direct the molecule in utricularia compartments, which is particularly favorable for the implementation of photochemical internalization.
You can apply many different molecules for the directional migration, for example, as described in Curiel (1999), Ann. New York Acad. Sci. 886, 158-171; Bilbao et al., (1998)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 media molecule and/or a molecule for directional migration can be associated, linked or konjugierte molecule with the NCP, photosensitizing agent, or with both, and you can use the same or a different media or molecules for directional migration. As mentioned above, you can simultaneously use more than one carrier and/or molecules for directional migration.
The preferred carrier for use in the present invention includes a polycation such as polylysine (for example, poly-L-lysine or poly-D-lysine), polyethylenimine or dendrimers (such cationic dendrimers, as SuperFect®); such cationic lipids, as DOTAP or lipofectin, and peptides.
The method according to the invention can be performed as described below. In the method according to the invention chimeric molecule and photosensitizing compound is applied simultaneously or sequentially in the cells, after which the photosensitizing compound and the molecule undergoes endocytosis or friend the mi methods are moved to endosome, complementary mechanism or other intracellular limited membrane compartments.
Conjugate PNK-peptide and photosensitizing compound can be applied to the cells together or sequentially. They can be absorbed by the cell in the same or different intracellular compartments (for example, they can be moved together). Conjugate PNK-peptide is then released when exposed to cells with light of suitable wavelength to activate the photosensitizing compound, which in turn leads to the destruction of intracellular membrane compartments and the subsequent release of a molecule, which can be located in the same compartment, as photosensitizing agent into the cytosol. Thus, in such methods the final stage of the cells by light leads to the release of molecules of interest from the same intracellular compartment that photosensitizing agent, and it is in the cytosol.
More recently, WO 02/44396 (which is incorporated here by reference) describes the way in which the order of the stages can be modified so that, for example, a photosensitizing agent interacts with cells and is activated by irradiation before the molecule, which will internalizing thus be delivered into the cell, interacts with cells. This atop the new method takes advantage of this fact, that chimeric molecules need not be present in the same cell subcompartment that photosensitizing agent.
Thus, in a preferred implementation of the specified photosensitizing agent and the specified molecule NCP is used in the cell together or sequentially. As a consequence, they can be absorbed by the cell in the same intracellular compartment, and you can then carry out the specified irradiation.
In an alternative implementation of this method can be done by contacting the specified cells with a photosensitizing agent, contacting the specified cell with the molecule NCP, which you must enter, and irradiation of the specified cell with light of a wavelength effective to activate the photosensitizing agent, where the specified irradiation is carried out until the cell specified capture molecules NCP in the intracellular compartment containing the specified photosensitizing agent, preferably before cell capture specified molecule into any intracellular compartment.
The specified irradiation can be performed after cell capture molecules in the intracellular compartment, localized whether or not the specified molecule NCP and photosensitizing agent in the same intracellular compartments during the attack by the Oia light. In one preferred implementation of the radiation, in addition, is carried out until the cell capture chimeric molecule.
Used herein, the term "internalization" refers to cytosolic delivery of molecules. In this case, the "internalization" also includes the stage of release of the molecules from the intracellular/membrane-bound compartments in the cytosol of the cell.
Used herein, the terms "cell capture" or "moving" refers to one of the stages of internalization, in which molecules external to the cell membrane, are absorbed by the cell so that they discover inside located outside of the cell membrane, for example, when endocytosis or other mechanisms to capture, for example, in the intracellular limited by the membrane compartment or related, for example, the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes, and so on.
Stage contacting cells with a photosensitizing agent and a conjugate PNK-peptide can be accomplished in any suitable or desired manner. Thus, if the stage of interaction you want to spendin vitrothe cells can appropriately support in these aquatic environments, such as an appropriate environment for cell culture, and at the appropriate time photosensibility the ith agent and/or conjugate PNK-peptide can simply be added to the medium under appropriate conditions, for example, at an appropriate concentration and for an appropriate period of time.
Photosensitizing agent is brought into contact with the cells at an appropriate concentration and for an appropriate period of time, which can easily identify the person skilled in the art using conventional methods, and will depend on such factors as specific used photosensitizing agent and the type of target cells and the location. The concentration of the photosensitizing agent should be such that when the absorption in the cell, for example, in one or more of its intracellular compartments or associated with one or more of its intracellular compartments and when activated by irradiation, one or more cellular structures are destroyed, for example, one or more intracellular compartments lyse or destroy. For example, photosensitizing agents used in the examples can be used at a concentration of, for example, from 10 to 50 µg/ml For usein vitrothe range can be much wider, for example, 0.05 to 500 µg/ml For the treatment of manin vivophotosensitizing agent 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 introduction. For smaller animals the concentration range of the can is different and may be selected accordingly.
The time of incubation of the cells with a photosensitizing agent (i.e. time "contacting"can vary from several minutes to several hours, for example, even up to 48 hours or more, for example, from 12 to 20 hours. The incubation time should be such that the photosensitizing agent zahvatila appropriate cells, for example, in intracellular compartments in these cells.
Incubation of cells with photosensitizing agent may not necessarily be accompanied by a period of incubation with not containing the photosensitizer medium before the cells are subjected to exposure to light or as add molecule NCP, for example, in a period of from 10 minutes to 8 hours, especially from 1 to 4 hours.
Molecule NCP is brought into contact with the cells at an appropriate concentration and for an appropriate period of time.
Identify appropriate doses of molecules NCP for use in the methods of the present invention is a common practice for professionals in this field. For applicationsin vitroapproximate dose molecules NCP would be approximately 0.1-500 µg NCP in ml and for applicationsin vivoapproximately 10-6-1 g PNK per injection for human. For example, the conjugate PNK-peptide can be entered at levels of less than 50 μm, for example less than 30 μm, particularly preferably less than 10 μm, for example, from 0.1 to l micron, or about the 5 to 30 μm, where indicated concentration reflects the levels in contact with the cell.
As mentioned above, it was found that communication can begin, even a few hours after he added a photosensitizing agent and radiation.
A suitable concentration can be determined depending on the efficiency of capture molecules of interest NCP in cells of interest and the final concentration, which should be achieved in the cells. Thus, the "time of transfection" or "time of cell capture", i.e. the time during which the molecules are in contact with cells, can vary from several minutes to several hours, for example, you can use the time of transfection from 10 minutes to 24 hours, for example, from 30 minutes to 10 hours, or, for example, from 30 minutes to 2 hours or 6 hours. You can also use a longer incubation time, for example, from 24 to 96 hours or more, for example, 5 to 10 days.
Increased the transfection usually leads to increased capture molecules of interest. In addition, a shorter incubation time, for example, from 30 minutes to 1 hour, apparently, leads to improved specificity of the capture molecules. Thus, when choosing the time of transfection for any way an appropriate balance must be observed between getting enough : the ATA molecules, maintaining sufficient specificity processing PCI.
In vivothe appropriate way and time of incubation, at which the molecule NCP and photosensitizing agents come into contact with target cells, will depend on such factors as the route of administration and types of molecules NCP and photosensitizing agents. For example, if the molecule NCP is introduced into the tumor, tissue or organ to be treated, the cells around the injection site prokontaktirovat molecule with the NCP and, therefore, will have a tendency to capture the molecule NCP more rapidly than the cells located at greater distances from the site of injection, which probably prokontaktirovat molecule with NCP over a longer period of time and at lower concentrations.
Additionally, for molecules NCP, intravenous injection, it may take some time to achieve the target cells, and this may require a longer post-introduction, for example, several days, to ensure that sufficient or optimal number of molecules NCP has accumulated in the target cell or tissue. The same reasoning, of course, belong to the time of reference, necessary to capture the photosensitizing agent into the cells. Time required for individual cellsin vivoso, probably, will change depending on these and other the parameters.
However, although the situationin vivomore complex thanin vitrothe main concept of the present invention is still the same, i.e. the time during which the molecules come in contact with the target cells must be such that before there irradiation, the appropriate amount of photosensitizing agent is absorbed by the target cells and also: (i) before or during irradiation molecule NCP or was acquired or to be acquired after sufficient contact with the cell target in the same or a different intracellular compartment; or (ii) after irradiation molecule NCP is in contact with the cells for a period of time sufficient for the implementation of its capture in cells. In that case, if the molecule NCP is absorbed into the intracellular compartments exposed to when you activate the photosensitizing agent (for example, the compartments in which the agent), the molecule NCP can be absorbed before or after irradiation.
Stage irradiation with light to activate a photosensitizing agent may occur according to the methods and procedures well known in the field. For example, the wavelength and intensity of light can be selected in accordance with the photosensitizing agent. In this area known suitable light sources.
Time, those who tell which cells are exposed to light, in the methods of the present invention, can be modified. The efficiency of internalization of molecules NCP in the cytosol increases with increased exposure to light to maximum, after which increases the damage of cells and, therefore, the death of cells.
The preferred period of time for the stage of irradiation depends on such factors as the target, the photosensitizer, the amount of photosensitizer accumulated in the target cells or tissues, and the overlap between the absorption spectrum of the photosensitizer and the spectrum of emission of the light source. In General, the period of time for the stage of exposure is of the order of 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 from 1 to 5 minutes, or from 1 to 10 minutes, for example, from 3 to 7 minutes and, preferably, about 3 minutes, for example, from 2.5 to 3.5 minutes.
The appropriate dose of light may choose a specialist, and they will once again depend on the photosensitizer and the amount of the photosensitizer accumulated in the target cells or tissues. For example, the dose of light, usually used for photodynamic treatment of cancer by the photosensitizer Photofrin and 5-aminolevulinic acid, a precursor of protoporphyrin, are in the range of 50-150 j/cm2when the band tightly the t energy of less than 200 mW/cm 2to avoid overheating. The dose of light is generally lower when using the photosensitizer with higher extinction coefficients in the red region of the visible spectrum. In addition, for the treatment of non-cancer tissues with fewer accumulated photosensitizer total number of required light can be substantially higher than for the treatment of cancers. Moreover, if you want to maintain the viability of the cells, it is necessary to avoid the formation of excessive levels of toxic products and can accordingly choose the appropriate options.
The methods according to the invention can inevitably cause the death of some cells with photochemical processing, i.e. the formation of toxic products when you activate the photosensitizing agent. Depending on the intended use of this cell death may not have consequences and may, in fact, be advantageous for some applications (e.g., cancer treatment). Preferably, in addition, cell death avoid. The methods according to the invention can be modified so that the fraction, or the ratio of surviving cells regulate, choosing the dose of light on the concentration of the photosensitive agent. Again, such methods are known in this field.
In applications that require viable cells, with the society in all cells do not die, or essentially not kill most of the cells (e.g., at least 50%, more preferably at least 60, 70, 80 or 90% of the cells).
Regardless of the degree of cell death induced by activation of the photosensitizer, for the impact of NCP on the cells it is important that the dose of light is regulated so that some of the individual cells in which the effect of PCI, were killed when one of photochemical processing (although they may subsequently die from molecules introduced into the cells, if such molecules possess cytotoxic activity).
Cytotoxic effects can be achieved using, for example, gene therapy, in which the antisense molecule NCP internalized in the nucleus of tumor cells by the method according to the invention, for example, down-regulation of the gene.
The methods according to the invention can be usedin vitroorin vivofor example, either to processin situor to processex vivoaccompanied by the introduction of the treated cells in the body for a variety of purposes, including: (i) inhibition of expression of certain gene products, linking mRNA or intermediate products of splicing; (ii) the effect on the transcription of certain genes, directly affecting gene (e.g., by inhibiting transcription factors); (iii) use as probes forin situhybridization; (iv) and the use of studies-screening and (v) to perform site-directed mutagenesis or repair of defective genes in the target cell.
Thus, the present invention proposes a method of inhibiting the transcription or expression of the target genes by the introduction of the molecules of the NCP in the cell that contains the specified gene target in the manner described above, where this molecule NCP specifically binds to a specified gene target or by-product of its replication or transcription. Thus, for example, the specified NCP can be associated with DNA and/or RNA.
The term "specific binding" refers to dependent sequences linking the NCP with the molecule-target, which can be RNA or DNA. The term "gene target" means a gene or its fragment, which is able to contact the NCP and which is the target of research.
The present invention also proposes a method of identification or analysis of the level of the target genes or product its replication or transcription, where the method includes the introduction of the molecules of the NCP in the cell that contains the specified gene target, or the product of its replication or transcription, as described above, where this molecule NCP specifically binds to a specified gene target or by-product of its replication or transcription, and analysis levels associated NCP, to determine whether the level of a specified gene target, or the product of its replication or transcription. The easy way for this method, the molecule NCP can carry molecule with reporter group that can be identified in the analysis, for example, a radioactive label or means of signal generation. Analysis may be qualitative and/or quantitative.
The present invention also proposes a method of carrying out site-directed mutagenesis or repair of the target genes, preferably, the defective gene in the cell, where the method includes the introduction of the molecules of the NCP and the molecules of the oligonucleotide containing the desired sequence in the cell that contains the specified gene target, the method described above, where this molecule NCP specifically binds to a specified gene target, to form a fastening NCP. Such deformation of the normal nucleic acid, forming a triple helix, occurs at the site of the target and provides repair or recombination at this particular site. Donor nucleotide which can be associated with NCP or simply type in conjunction with the NCP contains the required nucleotide sequence. NCP, therefore, acts as a promoter repair/recombination.
These methods can be used for research, for example, diagnostic purposes, or to change the expression profile of cells, for example, to get Elenovo product selection or therapeutic purposes.
The methods according to the invention can thus be used for diagnostic purposes where the presence of a particular gene product or its replication or transcription informative due to the presence, stage or prognosis of the disease, condition or violation. Thus, the present invention further provides a method for the diagnosis of diseases, conditions or disorders, providing for the introduction of molecules NCP in the cell (which can be donein vitro,in vivoorex vivo) the method described above, where this molecule NCP specifically binds to a gene target or by-product of its replication or transcription, which indicates the presence of specified disease, condition or violation, and the analysis of the level associated NCP to determine the presence, stage or prognosis of developing this disease, condition or violation.
The methods according to the invention can also be used in the treatment of any disease in which favorable downward regulation, repair or mutation of one or several genes. For example, for genes that are highly expressed in cancer, you can lower the regulation of the introduction of the corresponding NCP-molecule.
NCP, inhibitory expression of mutant that causes the disease gene, could also shall be entered in combination with the replacement gene (i.e., in gene therapy, which includes therapeutic gene transfer or modification of existing genes in the cells of the patient), for example, in the treatment of cystic fibrosis, cancer, cardiovascular diseases, viral infections and diabetes. Other diseases, the treatment of which would have the benefit of decreasing regulation of one or more genes include leukemia and pancreatic carcinoma (Cogoi et al. (2003) Nucleosides Nucleotides Nucleic Acids 22(5-8), 1615-1618), amyotrophic lateral sclerosis (AMS) (Turner et al. (2003), Neurochem. 87(3), 752-763), Huntington's disease (Lee et al. (2002) J. Nucl. Med. 43(7), 948-956) and Alzheimer's disease (McMahon et al. (2002) J. MoI. Neurosci. 19(1-2), 71-76).
As described above, the NCP can also be used to modify an existing gene, thus, can be used to repair the defective gene, for example, to treat diseases that are causally associated with the expression of this defective gene or lack of expression of the normal form of this gene (Rogers et al. (2002), PNAS U.S.A. 99(26), 16695-16700; Faruqi et al. (1998), PNAS U.S.A. (5(4), 1398-1403).
Thus, in a further aspect of the invention features a composition comprising a molecule NCP and also not necessarily separately photosensitizing agent, as described herein, where this molecule NCP conjugated to a positively charged peptide. In a further aspect of the invention features specified and composition for the use in therapy.
An alternative is described that the present invention provides the use of molecules NCP, as described here, in preparations medicinal products for the treatment or prevention of diseases, disorders or infections by modifying the expression of one or more of target genes in the specified patient. Preferably specified drug used for gene therapy, i.e. for the treatment of diseases or disorders for which typical abnormal gene expression. This change may include lower regulation specified expression or increase regulation of a modified form of the specified gene.
According to various implementations, the above specified photosensitizing agent and the specified molecule NCP contact with the cells or tissues of the patient simultaneously or sequentially, and these cells irradiated with light of a wavelength effective to activate the photosensitizing agent, and the irradiation carried out before, during or after cell capture of the specified molecule NCP in the intracellular compartment containing the specified photosensitizing agent, preferably, to the cell specified capture molecules into the intracellular compartment. Thus, in an alternative aspect of the invention proposes a method of treatment or prophylaxis of time the development of the disease, breach or infection in the patient, providing for the introduction of molecules NCP in one or more cellsin vitro,in vivoorex vivoaccording to the methods described above, and where necessary (i.e. when the transfer isin vitroorex vivo) introduction these cells indicated patient, where this molecule NCP conjugated to a positively charged peptide.
As defined here, the term "treatment" refers to reducing, alleviating, or eliminating one or more symptoms of the diseases, disorders or infections, which need to cure, regarding symptoms before treatment.
The term "prevention" refers to the delay or prevention of symptoms, diseases, disorders or infections.
The compositions of the present invention may also contain a cell containing the molecule NCP, which was internalities in the cytosol or the nucleus of the specified cell by the method according to the invention, where this molecule NCP conjugated to a positively charged peptide. The invention additionally relates to such compositions for use in therapy, especially in the treatment of cancer or gene therapy.
Thus, in yet another additional aspect of the invention features a cell or population of cells containing molecule NCP, which was internalities in the cytosol or poison what about the specified cells in which the cell can be obtained by the method according to the present invention, where this molecule NCP conjugated to a positively charged peptide.
In yet another additional aspect of the invention provides the use of such cells or population of cells for the preparation of the composition or the medicinal product for use in therapy, as described above, preferably cancer therapy or gene therapy, where this molecule NCP conjugated to a positively charged peptide.
The invention further provides a method of treatment of a patient, introducing a specified patient cells or compositions of the present invention, i.e. a method comprising a stage of introduction of molecules into the cell, as described above, and introducing the thus obtained of the specified cells of the indicated patient. Preferably these methods are used to treat cancer or in gene therapy.
You can use any method of administrationin vivo, normal, or standard in this area, for example, injection, injection, local injection and internal and external surfaces of the body and so on. To apply ain vivothe invention can be used in respect of any tissue that contains cells in which localized photosensitizing agent and the molecule NCP, including localize the Oia in liquid media of the body as well as in solid tissues. All fabrics can be processed provided that the photosensitizer is captured target cells, and the light can be delivered properly.
Thus, the composition of the invention can be prepared in any convenient manner, in accordance with the procedures and methods known in the pharmaceutical field, for example, using one or more pharmaceutically acceptable carriers or excipients. Referred to herein, the term "pharmaceutically acceptable" refers to ingredients that are compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient. The nature and composition of media or substances, excipients, dosage and so on, you can choose the usual manner in accordance with the choice and necessary way of introduction, the goal of treatment, and so forth. Dosing similarly, you can define in the usual way, and they can depend on the nature of the molecule, the goals of treatment, age of the patient, the route of administration and so forth. In relation to the photosensitizing agent, the potential/ability to destroy the membrane by irradiation should also be taken into account.
The methods described above, can alternatively be used to create tools for screening for high-performance methods of screening, particularly for the analysis of the eff is mswb suppression of certain genes. NCP aimed at one or more specific genes, can be obtained and used according to the method of the invention, as described above. NCP can thus be used to reduce gene expression in a population of cells. The resulting cell population can then be used as a tool for screening to identify the effects of gene suppression in the forward direction along the chain sequence using standard methods. Thus it is possible to identify, for example, genes that are also affected by the suppression of the target genes. Optional to such identified genes can be sent NCP for further stages of screening, for example, to identify molecules involved in a particular event signal.
Thus, the invention also relates to a method of screening cells with altered pattern of gene expression, providing: (a) analysis of expression of the target genes or one or more genes of a cell or population of cells, which was obtained by introduction of molecules NCP according to the method according to the invention where the specified NCP specifically binds to a specified gene target or by-product of its replication or transcription and alters the expression of the specified one or more genes; and (b) comparing the expression specified Misha is and/or one or more additional genes with expression of these genes in the cells of the comparison, preferably the cells of the wild type.
The pattern of expression may be determined using any suitable method known in this field, for example, using a microarray carrying the probes that bind with molecules of mRNA (or cDNA) and which can be used to estimate the amount of each transcript. Cells comparison (control cells) denote any cell that is compared to the expression. Preferably, these cells represent the control cells, which did not enter the NCP. Particularly preferably, these cells represent a control wild-type cells, such as cells that were not exposed to such genetic manipulation, as when using NCP.
Previous attempts to reduce gene expression by normal and chemically modified antisense oligonucleotides were limited to nuclease degradation of antisense oligonucleotides, the occurrence of non-specific effects and/or lack of affinitiy to the target. When using the method according to the invention for the introduction of NCP can be overcome such problems.
Thus, in a further aspect the invention provides a method of changing the pattern of gene expression in the cell (for example, a population of cells to prepare a cell (or cell population) to use the education as a tool for screening (for example, for high-throughput screening), involving contacting the molecules of the NCP, is able to inhibit or decrease the expression of a gene, and a photosensitizing agent with a cell (for example, a population of cells and light cells (for example, a population of cells) light of a wavelength effective to activate fotosensibiliziruyuschimi agent, where this molecule NCP conjugated to a positively charged peptide. The invention additionally relates to such cells and method of screening of these cells, where certain properties of such cells, for example, levels expressii mRNA such cells studied, for example, on the microarray.
The phrase "altered pattern of gene expression" means that as a consequence of the presence of the indicated molecules NCP in the cell nucleus influences the transcription or translation of the gene to which it is directed.
As a consequence of such changes in gene expression can influence the expression of other genes. Thus, affecting the normal expression of the studied gene, it is possible to determine changes in the expression pattern of other genes. Identification of such genes and the impact of the expression of the studied gene, allows the researcher to draw conclusions about the functions of a gene, for example, about their function in the forward direction of the sequence. Genes that are affected by the ISM is out of the normal expression of the studied gene, can be subjected to increasing regulation or down-regulation, but the total change in the expression pattern indicates the role of the gene in a normal cell functions and consequences of the violation of its regulation.
Using standard methods, well known in this field, it is possible to study the effect of reduced regulation or loss of expression of the gene of interest. This can, for example, be accomplished through search such functional changes in the cells (or cell population), as changes in cell adhesion, secretion of the protein or morphological changes. Alternatively, the expression profile of genes can be directly studied by analyzing the patterns of mRNA and/or protein expression, again using standard methods that are well known in this field.
Inhibiting or reducing the expression of a gene, it should be understood that the expression of the gene of interest is reduced when compared to cells that were not subjected to the method, i.e. the cell wild-type or normal cell. The change in the level of gene expression can be determined by standard methods known in this field.
This full inhibition of the expression that there is no detectable gene expression, i.e. not detectable mRNA or protein, or perhaps a partial inhibition of expression, the EU is ü reduction, whereby the amount of gene expression is lower than in the cell of a wild type or normal cell. This can be evaluated and adjusted by comparing the effect of NCP with a certain sequence with the effect of NCP with a randomized sequence, i.e. with the same nucleotide composition but in different order. It is preferable for the applicable methods of reducing the expression to less than 80% of control levels, for example, <50%, preferably <20, 10 or 5% of control levels. Used cell (cell) preferably represents a population of cells, individual cells which are genetically identical. Cells can be any cells, as discussed above.
Prior to the development of this new technique of delivery, the NCP was impossible to use the NCP for such a system. The ability to use the NCP in this system has several advantages. Such known methods for introducing molecules into the cell, as the use of transfection agents, often bring variations in cell research, used in large-scale screening systems so that it is hardly possible to establish what the effects caused by the violation of gene expression, and which are caused by the transfection method. Indirect PCI shipping has few such effects, and this is also possible when the IP is the use of appropriate controls.
Some of the other substances used for delivery of molecules into the cell, can also cause non-specific effects on the study at screening. For example, short interfering RNA (siPHK), which is used for methods of suppression of the gene was reported to affect gene expression of interferon (Sledz et al. (2003), Natl. Cell Biol. 5(9), 834-839). Stability NCP high, and as such impact that it has on gene expression lasting even after a single injection.
The effectiveness of the NCP does not depend on systems of certain enzymes because of its inhibitory effect depends on the chemical interactions with the nucleotide molecules. The degree of ingibirovaniya constant in different types of cells. This is not the case for siPHK, for example, which depends on certain enzymes.
It was found that, surprisingly, the technique PCI is not of the expected problems of occurrence of nonspecific effects on gene expression.
The cell or cell population obtained according to the methods of the invention can be used to create a library that is an additional aspect of the invention.
Now the invention will be described in more detail with the aid of the following non-limiting examples with reference to the following drawings, in which:
Figure 1 shows the effect of the charge on Jn is eralization PNK, using the analysis using flow cytometry with FITC-PNK capture in OHS cells, HeLa and FEMXIII. Cells were incubated with various FITC-PNK at a concentration of 1000 nm for 24 hours at 37°C and analyzed using flow cytometry as described in experimental protocols. "-1" PINK, "0" PNK, "+1" PINK, "+5" PNC. The results are shown as mean values of fluorescence intensity relative to the total charge of different molecules NCP. The columns show three separate experiments, with 6 Parallels each. Error bars show the standard deviation from the mean.
Figure 2 shows relocalization FITC-PNK-NLS from intracellular vesicles into the kernel when using processing PCI using NCP 200 in OHS cells: (A) before and after processing, PCI (3 hours), (B) to the processing PCI: (i) when fitokontrol microscopy, (ii) by staining with FITC-PNK, (iii) if the LysoTracker staining, (iv) at Hoechst staining and (v) showing the combined staining; and (C) after treatment with PCI at colouring, as for (B).
Figure 3 shows nuclear localization in different cell types after PCI with the use of different molecules NCP, using fluorescent microscopy. Cells were incubated with various FITC-PNK at a concentration of 1000 nm for 24 hours and analyzed using fluorescence microscopy, as described in the experts is the mental protocols. (A) OHS-PNK-NLS (PNC), (B) OHS-PNK-MITO (PNC), (C) OHS-PNK-GHHHHHG (PNG), (D) HeLa-PNK-NLS (PNC), (E) FEMXIII-PNK-NLS (PNC)resulting from, left to right, fasovannogo image, staining with FITC-NCP, Hoechst, LysoTracker.
Figure 4 shows that the delivery of the NCP in the core does not depend on the language used by the fluorophore. The OHS cells were incubated with the NCP with FITC in the presence of the C - or N-Terminus (1000 nm) for 24 hours and analyzed using fluorescence microscopy as described in experimental protocols. The results, from left to right, show FITC associated with either C-or N-end.
Figure 5 shows the capture NCP 200 in the nucleus of various cells after PCI: (A) prior PCI, (B) after PCI, determining the dye FITC-PNK; cells FEMX1, FEMX5, HeLa, OHS, SW620, HCT116, WiDr, 293 and SaOs, respectively.
Figure 6 shows that the grip NCP depends on temperature; the OHS cells were subjected to 1000 nm NCP 200 when: (A) 4°C for 5 hours, and (B) 37°C for 5 hours. The results are shown from left to right, vasovegetative image, FITC/NCP, combined image; magnification is 10× the upper image 32× on the bottom of the image.
7 shows that the delivery of the NCP in the core does not depend on the type of fluorophore. Cells were incubated with PNC (1000 nm)conjugated with rhodamine, within 24 hours and analyzed using fluorescence microscopy as described in ex is elementarnykh protocols. The results, shown from left to right, represent vasovegetative image with rhodamine and combined image.
On Fig shows the influence of the differently charged molecules NCP on nuclear import after PCI. The OHS cells were incubated with NCP (1000 nm), as described in the experimental protocols. (A) 383, (B) 385, (C) 456, (D) 384, (E) 381, (F) 455. The results, shown from left to right, represent vasovegetative image, FITC image, the combined image.
Figure 9 shows the inhibition of S100A4 expression in cells OHS different NCP (1000 nm), as assessed using Western blotting: (A) dose-dependent inhibition PNC, (B) time-dependent inhibition of various NCP. The results are presented as percentage of control cells, and the columns represent the average of 3 separate experiments. Error bars show the standard deviation from the mean. Typical examples of Western blott for experiments shown in the image from (C) to (E); (C) and (D) test load (α-tubulin), (E) inhibition after 96 hours is left-to-right control, randomised NCP (PNC), PNK, PNK, (F) dose-dependent inhibition with PNC after 96 hours is left-to-right control, 100 nm, 500 nm, 1000 nm, 2000 nm.
Figure 10 shows the results of TS after processing PCI OHS cells. The results show that only the NCP non-toxic.
Figure 11 shows the results of the analysis of Western-blot showing that any impact on the levels of proteins in cells OHS is not observed in the directional migration NCP to codereuse region of S100A4 (PNC): (A) the upper strips are α-tubulin as a control boot, the bottom strips are S100A4, track 1 is the control without photosensitizer and without processing light, track 2 is the sample with a photosensitizer, but without processing the light, track 3 is the sample without photosensitizer, but with the processing light, track 4 is the sample with a photosensitizer and the processing light; (B) the upper strips are α-tubulin as a control boot, the bottom strips are S100A4, lane 1 - control, track 2 - conducting a randomized, NCP (PNC), lane 3 - (PNC) 1000 nm, lane 4 - (PNC) 2000 nm.
On Fig shows the relative expression of S100A4 mRNA in the treated PNK/PCI OHS cells compared with control. A) total RNA was isolated from cells OHS after processing PNK/PCI using PNK-AUG, PNK-5'UTR and PNK-conducting a randomized. Processed PCI OHS cells were isolated as control in addition to a randomized NCP. All samples were subjected to the action of reverse transcriptase and solutions cDNA, for example, the Ali to the analysis of real-time PCR, using SYBRGreen I as a reagent for detection. CTvalues obtained for the different samples showed small differences in gene expression. B) Analysis of the melting curve, showing only one interested in the product.
On Fig shows the levels of protein TYR 72 hours, using Western Western blot turns. Lanes were loaded as follows: 1. Control (without NCP), 2. Conducting a randomized NCP TYR (1 mm), 3. Conducting a randomized NCP TYR (10 mm), 4. NCP TYR UTR (1 mm), 5. NCP TYR UTR (10 mm), 6. NCP TYR AUG (1 mm), 7. NCP TYR AUG (10 mm). Alpha-tubulin is shown as a control of loading.
Cell line and culturing conditions
Cell lines human HeLa (cervical adenocarcinoma), WiDr (carcinoma of the rectum) and 293 (embryonic kidney) were obtained from the American type culture collection (Manassas, VA, USA). Human OHS (osteosarcoma) and FEMXIII (melanoma) were collected in the Norwegian Radium Hospital (Fodstad et al, (1986), Int. J. Cancer 38(1), 33-40; Fodstad et al., (1988), Cancer Res. 48(15), 4382-8). All cell lines were cultured in medium RPMI-1640 (Bio Whittaker, Verviers, Belgium), except for the cell line 293, which were cultured in DMEM (Bio Whittaker, Verviers, Belgium). Both have been used without antibiotics, but with the addition of 10% serum embryo calf (FCS; PAA Laboratories, Linz, Austria) and 2 mm L-glutamine (Bio Whittaker, Verviers, Belgium). Cell cultures were grown and incubated at 37 the C in humidified atmosphere, containing 5% CO2. All cell lines were tested and found that they were negative against infectionMycoplasma.
NCP-specific gene S100A4, including randomized NCP received from Oswell DNA Service (Southampton, UK). Modifications performed on one or both ends (see table 1). Target the 5'UTR (GeneBank access NM_002961, 2-15), the starting region AUG (63-82) and codereuse region in the second exon (98-118) was chosen based on previous experiments on the inhibition of NCP (Doyle et al., (2001), Biochem. 40, 53-64; Mologni et al., (1999), Biochem. Biophys. Res. Comm. 264, 537-543) and suppression of the S100A4 gene by ribozymes (Hovig et al., (2001), Antisense Nucleic Acid Drug Dev. Apr 11(2), 67-75). Sequences were aligned against a database of the human genome using BLAST search to eliminate sequences with significant homology to other genes. Royal solutions (1 mm) were prepared by dissolving NCP 10% triperoxonane acid and before use was heated to 50°C in order to ensure that the NCP was dissolved completely. Before using NCP was further diluted to working solutions (10 μm) in sterile water and stored at -20°C.
Based on the rules proposed by Elbashir et al. (Elbashir et al, (2001), Genes Dev. 15, 188-200), two targets were chosen against the coding region of the gene S100A4. The first target was a target against the sequence AA(N)19 (GeneBank access NM_002961, 343-361) and the second target was represented by a sequence AA(N19)TT (264-282). In addition, received a randomised control siPHK and fluorescently-labeled siPHK. All siPHK was ordered from Eurogentec (Seraing, Belgium). Tagging was performed on both strands using FITC at the 5'end of the antisense strand and rhodamine at the 3'-end of the sense strands. The GC content of the double helix was maintained in the range of 40-70% and all siPHK synthesized with dTdT sticky ends at their 3'-ends for optimum stability of the double helix siPHK. Two target sequences were also aligned against a database of the human genome using BLAST search to eliminate sequences with significant homology to other genes. Dried oligonucleotides siPHK resuspendable 100 μm DEPC treated water and stored at -20°C. the Annealing siPHK performed by to obtain separate aliquot and the cultivation of each oligo RNA concentrations up to 50 μm. Then 30 µl of each solution oligo RNA and 15 μl of 5× buffer for annealing were mixed at a final concentration of 50 mm Tris, pH 7.5, 100 mm NaCl in DEPC-treated water. Then the solution was incubated for 2 minutes in a water bath at 95°C, followed by a gradual cooling for 45 minutes on your desktop. Successful annealing was confirmed by nudemature the existing polyacrylamide gel electrophoresis.
TransfectionsiPHKand electroporation PNK
The OHS cells were cultured as described above and were cultured for 24 hours in 6-hole tablets 30 to 60% confluence before transfection. Tranfection conducted in nestorgames serum medium OPTI-MEM I (Invitrogen Corp., Paisley, UK) with different concentrations siPHK using the reagent lipofectin from Life Technologies Inc. (Gaithersburg, MD, USA), the reagent lipofectamine from Invitrogen (Catlsbad, CA, USA), (N-(1-(2,3-dialectics)propyl)-N,N,N,-trimethylammonium-methyl-sulfate (DOTAP) from Boehringer Mannheim (Mannheim, Germany), FuGene from Roche Diagnostics (Mannheim, Germany), the means of transfection lipids siPORT from Ambion (Austin, TX, USA) and the hydrobromide poly-L-lysine (MW 15000-30000) from Sigma (St. Louis, MO, USA), according to manufacturer's instructions. When electroporation of cultured cells OHS collected and resuspendable in fresh medium. Approximately 4×106cells were mixed with NCP (1-10 μm) in 300 μl of medium and incubated on ice for 10 minutes. The cells were electroporative in a cell with a size of 0.4 cm to the settings, 950 μf/250 V (ECM399, BTX, A Division Of Genetronics, CA). After electroporation the cells were incubated on ice for 30 minutes, diluted in T25 flasks and incubated at 37°C with 5% CO2within 24 hours, and analyzed by fluorescence microscopy.
PCI technology and processing
The sensitizer desulfosporosinus tetraphenylporphin (TPPS2a) was purchased from Porphyrin Products (Logan, U, USA). TPPS2afirst, was dissolved in 0.1 M NaOH and subsequently bred in zbuffering phosphate isotonic saline solution (PBS), pH 7.5, to a concentration of 5 mg/ml and the final concentration of 0.002 M NaOH. The photosensitizer was protected from light and kept at -20°C until use. When irradiated cells treated TPPS2awere subjected to blue light using LumiSource prototype (PCI Biotech AS, Oslo, Norway), containing a series of four fluorescent tubes (Osram 18W/67) with the highest flow rate of approximately 420 nm.
Before use, cells were cultured for 24 hours in 6-hole tablets at 37°C under 5% CO2. Then cells were incubated with various NCP and the sensitizer TPPS2a(1 μg/ml) for 18 hours. After capturing the cells were washed 3 times with fresh medium, and incubated in medium without sensitizer for 4 hours. Finally, the cells were subjected to blue light for 30 seconds and re-incubated for 24, 48 and 96 hours. During the experiment, the cells were protected from light with aluminum foil.
Cells were analyzed using an inverted microscope Zeiss Axiovert 200 equipped with filters for FITC (450-490 nm excitation filter BP, 510 nm beam splitter FT and 515-565 nm emission filter LP), rudamina (546/12 nm excitation filter BP, 580 nm light is elitel FT and 590 nm emission filter LP) and DAPI (365/12 nm excitation filter BP, 395 nm beam splitter FT and 397 nm emission filter LP). Images were obtained using a Carl Zeiss AxioCam HR, Version 5.05.10, and software AxioVision 126.96.36.199. Specific organelle markers were used to confirm the subcellular localization of the NCP. Localization of lysosomes was determined using a fluorescent microscope and LysoTracker Red DND-99 (Molecular Probes, Eugene OR). Nuclear localization was determined using Hoechst H33342 (Molecular Probes, Eugene OR).
Analysis of flow cytometry
Cells hydrolyzed by trypsin, centrifuged, resuspendable in 400 μl of medium for the cultivation and filtered through a nylon filter with cells at 50 μm before you can analyze on a flow cytometer FACS-Calibur (Becton Dickinson). For each sample collected 10,000 cases. Labeled with FITC NCP was measured with the filter 510-530 nm after excitation argon laser (15 mW, 488 nm). Dead cells were separated from a single viable cells by selection of forward scattering against lateral dispersion. Data were analyzed using CELLQuest software (Becton Dickinson).
The protein lysates were prepared in 50 mm Tris-HCl (pH 7.5)containing 150 mm NaCl and 0.1% NP-40 with pepstatin at a concentration of 2 g/ml Aprotinin (Sigma Chemical company, St Louis, MO) and leupeptin (Roche Diagnostics, Mannheim, Germany). Total lysate proteins (30 μg) from each sample, readelementstring in 12% SDS-polyacrylamide gel and transferred to membrane Immobilon-P (Millipore, Bedford, MA) according to manufacturer's instructions. As a control of loading and transfer, membranes were stained with 0.1% lidocainum. Membranes are then incubated in 20 mm Tris-HCl (pH 7.5)containing 0.5 M NaCl and 0.25% Tween 20 (TBST), with 10% dry milk (blocking solution) before incubation with rabbit polyclonal antibodies against S100A4 (diluted 1:300, DAKO, Glostrup, Denmark) and mouse monoclonal antibodies against alpha-tubulin (diluted 1:250, Amersham Life Science, Buckinghamshire, England) in TBST containing 5% milk powder. After washing, the immunoreactive proteins were visualized using secondary antibodies conjugated to horseradish peroxidase (diluted 1:5000 DAKO, Glostrup, Denmark), and systems enhanced chemoluminescence (Amersham Pharmacia Biotech, Buckinghamshire, England). The levels of protein S100A4 was represented as percentage of control samples, and alpha-tubulin was used as a control boot.
Measurement of cell viability using the MTS analysis
100 μl of cells were placed in 96-well plates and were grown for 24 hours at 37°C. Also included a negative control containing no cells. 20 μl of MTS reagent (tetragonia salt) was added 100 μl to each well and incubated for 2 to 4 hours at 37°C in the dark. Then read the absorbance at 490 nm.
PCR with reverse transcriptase real-time
Tile and cultivated and processed, as explained above. After photochemical treatment cells were incubated with various NCP within 96 hours and then harvested for RNA extraction. Total cellular RNA was isolated using the kit GenElute Mammalian Total RNA Miniprep (Sigma-Aldrich, Steinheim, GER) according to the manufacturer's instructions. For cDNA synthesis primers mix containing 50 pmol oligo-dT, 3 μg total RNA and dH2O up to 12 ál, were prepared for each sample. The mixture was denaturiruet for 5 minutes at 65°C and then quickly cooled on ice and mixed with 18 μl of the reaction mixture to a final concentration of 1× buffer First Strand buffer (Invitrogen), 10 mm DTT, 0.3 mm dNTP, and 6.5 ng/µl yeast tRNA and 200 U (6,6 U/μl) of the enzyme Superscript II. Synthesis of cDNA was carried out at 42°C for 50 minutes, followed by inactivation for 15 minutes at 72°C.
For PCR analysis was prepared 3 ten-fold dilution of each sample cDNA and led all three reactions, receiving a total of 9 tubes for PCR on the cDNA sample. PCR was performed in a total volume of 25 µl, using a final concentration of 3 mm MgCl2, 200 μm dNTP, 1× buffer for PCR, and 0.5 U platinum Taq, 2 μl of cDNA and 300 nm of primers, specific for S100A4 gene (forward primer 5'-AAGTTCAAGCTCAACAAGTCAGAAC-3' (SEQ ID NO: 9) and reverse primer 5'-CATCTGTCCTTTTCCCCAAGA-3' (SEQ ID NO: 10)). In addition, all reactions were labeled with 1 nm fluorescein, as required for iCycler. Results real-time PCR were obtained using the end-times is Denia 1:100000, SYBR Green I (Molecular Probes, Eugene OR) as the detecting agent. Cycles of amplification consisted of the following: 5 minutes initial denaturation at 95°C, followed by 40 cycles of 15 seconds at 95°C/30 seconds at 60°C for amplification product. The definition of products real-time PCR was performed using optical 96-well plates and detecting system of the iCycler iQ, manufactured by Bio-Rad Laboratories, CA. For each sample, the procedure was repeated three times. To determine false amplification products or primer dimers (which are equally marked by the introduction of SYBRGreen and can, thus, affect the reading fluorescence) melting curve, i.e. the loss of fluorescence during denaturation, included at the end of the Protocol PCR amplification. The melting profiles for each sample were compared with the profiles obtained for standard samples.
The cDNA chips
The microarray used in this application was performed in laboratory conditions, using a robotic printer Micro Grid II (Bio-Robotics, Cambridge, UK). These chips 15k cDNA person printed on slides, covered with aminosilane (CMT GAPS Corning Life Sciences, Corning, NY). For a detailed description of the contents of the chip, the inventors refer to: http://www.med.uio.no/dnr/microarray/index.html.
Clearing and staining RNA
Total RNA from cultured cells treated with PCI and different and PNK, was isolated as described above. For the analysis of possible effects in the forward direction of the sequence as a consequence of the suppression of the S100A4 gene, each individual chip hybridized with cDNA from cells treated with the active NCP (PNC or 453) and a randomized or control PNK (PINK (control) or 454 (conducting a randomized)). cDNA was obtained from 50 µg total RNA from each of the data cell cultures and differential were labeled Cy3 - or Cy5-dCTP (Amersham Pharmacia Biotech AB) during reverse transcription. The reaction mixture contained 20 anchored-dimensional primers oligo-dT (4 μg), 40 U Mcsina (Promega, Madison, WI), 1 standard buffer, 0.01 M DTT, 0.5 mm dATP, dCTP, dGTP, 0.2 mm dTTP. The mixture is incubated at 65°C in a water bath for 5 minutes. Then the test tube was transferred to a heating block at a temperature of 42°C was added 4 μl (4 nmol) of one of the two fluorophores in the corresponding tubes in addition to 400 U Superscript II (Invitrogen, Groningen, The Netherlands). After 60 minutes the reaction iactiveaware 5 ál of 0.5 M EDTA (pH 8.0). For hydrolysis of residual RNA was added 10 μl of 1 M NaOH and incubated the tube at 65°C for 60 minutes. Added 25 μl of 1 M Tris-HCl (pH 7.5) to neutralize the mixture. Labeled with Cy3 - and Cy5-cDNA was diluted to 0.5 × TE-buffer (pH 7.5) before you remove unbound dye and the concentration of the samples using columns Microcon YM (Ambion, Millipore Corporation, Bedford, MA).
Preliminary hybrid is the nation's slides
The cross slides were made UV at 150 kJ within 60 seconds. Directly before use, the slides are pre-hybridized to inactivate reactive groups on the surface of the slide and then washed of unbound DNA. A small slide holder, filled with a solution for pre-hybridization, pre-heated at 50°C for 30 minutes. Hybridization solution contained 1% (wt./about.) fraction V bovine serum albumin (BSA) (Sigma-Aldrich), a 3.5 × SSC and 0.1% SDS. Slides were incubated in a pre-heated solution at 50°C for 25 minutes, directly after incubation the slides were transferred to a clean stand for slides and washed twice with shaking in ultrapure water at room temperature. To denature the DNA to form single-stranded chain slides was dissolved in freshly boiling water for 2 minutes and then quickly immersed in propan-2-ol and shook for 30 seconds. The slides were dried by centrifugation.
Hybridization and scanning
Hybridization mixture in an amount of 45 μl consisted of 15 μl of each of the labeled probes, 16 μg poly A (Amersham Pharmacia Biotech AB), 4 g yeast t-RNA, 1,25 × solution of Denhart, 5 μg BSA, a 3.5 × SSC (pH 7.5) and 0.3% SDS. The final mixture was heated for 2 minutes at 100°C and centrifuged for 10 minutes at 13 before nasenyana microchip under a LifterSlip (Erie Scientific Company, Portsmouth, NH). Then the slide was placed in a hybridization chamber ArrayIT (Telechem, Sunnyvale, CA) and incubated overnight in a water bath at 65°C. Before scanning the cover glass was removed in a solution of 0.5 × SSC and 0.1% SDS. Then the slide was twice washed in the same solution for 5 minutes at room temperature, followed by washing 2 times for 5 minutes in wash solution is 0.06 × SSC. Slide finally dried by centrifugation. Scanning was performed using a scanner ScanARRAY 4000 (Packard Biosciences, Biochip Technologies LLC, Meriden, CT) and obtained data from the images using the software GenePix Pro 4.0 (Axon Instruments Inc., Union City, CA). The data is stored, analyzed and processed using the BASE (Lao H et al. BioArray Software Environment: A Platform for Comprehensive Management and Analysis of Microarray Data, Genome Biology 3(8): software 0003.1-0003.6 (2002).), and calculated corrected for background intensity for each spot by subtracting the average value of the pixels in the local area of the background of the mean of the pixels in the spot.
Example 1: Cell capture molecules PNK
The first series of experiments related to the issue of cell capture, that is, if the penetration NCP associated with short peptides with different total charges across the cell membrane in various cancer cell lines are human or not. For detection of compounds within the cell NCP noted FIT or N-, or C-Termini. Table 1 shows in detail the NCP used in the experiment, including their target sequence, chemical modification and charge.
Cell capture was measured by flow cytometry. NCP-related peptide with a total negative charge (PNC), did not penetrate into the cells OHS (figure 1, a total charge of -1). Since the capture of negatively charged molecules was practically absent even after 24 hours, the inventors investigated the possibility of tranfection into cells by electroporation. In this case, the grip was very weak. In contrast to the negatively charged NCP neutral NCP without charge and without any of the associated peptide (PNC) and NCP-related peptide with neutral net charge (PNC), both were internalizable in low-grade (figure 1, a total charge of 0 for PNC, PNC not shown).
Further, the inventors investigated the positively charged NCP. Capture explicitly observed when the NCP was associated with the peptide with a total charge of +1 (PNK) (Fig 1, the total charge of +1). However, when the inventors increased the total charge to +5 (PNC), the inventors observed a nearly 5-fold increase of cell capture, compared to NCP with charge +1 (figure 1, a total charge of +5).
The results from the above experiments conducted to glue the framework of OHS, FEMXII and Hela, are summarized in table 2.
To identify the localization of molecules NCP used staining. Fluorescence labels attached to the molecule NCP, were used to identify the NCP within the cell. The Hoechst staining and staining with LysoTracker used to identify nuclear and lysosomal compartments, respectively. On figa shown that after PCI molecules NCP distributed inside the cell. On figv and C shows that the molecules of the NCP are distributed in the nuclei after PCI (i.e., their distribution coincides with Hoechst staining).
In order to investigate whether the cell capture from the conformation of the peptide NLS or only charge from the authors of the invention linked NCP with a 29-amino acid signal mitochondrial import with a total charge of +5 (PNK) (pigv). As a second control to PNC (figa) the authors of this invention have replaced the original amino acid NLS sequence PKKKRKV (SEQ ID NO: 3) alternative sequence GHHHHHG (+5) (SEQ ID NO:5, PNK) (figs). The relative levels of cell capture for three different designs NCP presented the same levels that were detected using microscopy. In all cases, the NCP was localized in the nuclei after PCI. This also happened when I carried out the procedure on HeLa cells and FEMXIII (fig.3D and E).
In order to investigate any who is one of the differences in cell capture, which could relate to the orientation of the NLS, the inventors have linked peptides and N- (PNC), and C-ends (PNC). No differences in the levels of capture were not observed (figure 4). The inventors also tested various NCP and their cell capture in different cell lines (HeLa, WiDr, 293, OHS, FEMX5, SW620, HCT116, SaOs), but did not observe significant changes (figure 5).
Data of the inventors show that the chimeric capture NCP strongly depends on the total charge of the molecule peptide and does not depend on the conformation of amino acids. Here the inventors have shown that the capture molecules NCP increases when net positive charge on kongugirovannom peptide becomes larger.
Example 2: the Mechanism and localization of seizure PNK
In order to assess the capture process for modified NCP, the inventors have firstly investigated their capture at different temperatures. The results of the inventors showed no capture cell at 4°C, although the seizure was observed at 37°C (Fig.6). Moreover, the inventors under fluorescent microscopy showed the grain-like fluorescent spots in certain areas in the immediate vicinity of the nuclear envelope (pigv). Finally, the inventors observed a good overlap between the intracellular localization of structures NCP and marker for endosomes/lysosomes (ysoTracker Red DND) (data not shown).
The results of the inventors show that involved endocytosis. This can be caused by internalization through bordered bubbles. In the experiments of the inventors capture NCP authors of the invention were blocked at 4°C.
The results of the inventors confirmed discoveries Kuismanen and Saraste (Kuismanen E et al. (1989) Methods. Cell. Biol. 32, 257-274), which showed that endocytosis can be blocked at low temperatures. The temperature dependence, in addition, it was confirmed overlapping localization NCP and LysoTracker.
Endocytosis can be divided into several basic types: caused by receptor-dependent claritin, independent of claritin and phagocytosis. However, to clarify certain type of endocytosis further research is needed. The assumption is that molecules NCP authors of the invention are captured by independent claritin endocytosis, because there is no evidence due to receptor-dependent claritin endocytosis. NCP was associated with various peptide signals with the same charge; the results were the same, indicating that the capture does not depend on a particular receptor. The conclusion is that the positively charged molecule NCP, most likely, has a close relationship with cell membrane than negatively charged the traveler, which in turn will increase endocytotic grip.
Example 3: Effect of processing PCI
Data on microscopy authors of the invention have shown that the NCP with neutral/positive net charge localized in the endosomes/lysosomes. Data on microscopy authors of the invention have clearly shown that the design NCP moved from endosomes/lysosomes in the nucleus after treatment with PCI (figure 2 and 3). In order to confirm relocalization NCP, the inventors have also used the staining of the nucleus Hoechst as described above, see figure 2 and 3.
Example 4: Nuclear import of molecules PNK
For nuclear directional transfer NCP must overcome major barriers: the most important are the cell membrane, intracellular membrane and nuclear envelope. After the release of NCP from endosomes/lysosomes, the inventors wanted to explore the ability to localization of NLS peptide was whether the orientation of the NLS peptide is important for nuclear localization. To resolve these issues, the authors of the invention linked peptide NLS with NCP as with N-and C-end. Data on microscopy authors of the invention have shown that the NCP associated with the NLS peptide or via the N-or C-end, move into the nucleus (figure 4). Signal amplification fluorescence was observed, increasing the exposure time and concentration design FITC/NCP. In order clause is to kanalizirovat any possible incompatibility between different types of fluorophores, the inventors changed the fluorophores from NCP to FITC rhodamine (Rho). It is, moreover, made no visible change in localization (7). The inventors have also linked FITC either N-or C-ends of the NCP, again no changes in the localization or efficiency (figure 4).
Next, the inventors investigated whether a capacity for nuclear localization PKKKRKV (SEQ ID NO: 3) is simply a consequence of changes in the charge in the charge NCP or the result of a specific amino acid sequence. In order to control the capacity for nuclear localization of the NLS peptide, the inventors tested the NCP alternative peptide having the same net charge (+5), but with substituted amino acids (PNK, figs). Also, the inventors have studied nuclear import neutral NCP (PNK and 385, figs and B, respectively), and also with NCP-related peptide signals for importing directed to the mitochondria and peroxisomes (PNK, PNK, figv and 8D, respectively). Surprisingly, the inventors have demonstrated that all neutral and positively charged investigated NCP moved into the nucleus after treatment with PCI.
In conclusion, the results of the inventors demonstrated that the NCP with neutral/positive net charge not only spontaneously move from among the s in complementary mechanism at a high level, but also move from cytosol in the core after photochemical treatment. Efficiency between the neutral and positively charged NCP changes, probably as a direct consequence of changes in cell capture, not nuclear capture.
Example 5: Inhibition of the expression of S100A4 by using NCP/PCI
In order to assess the functional ability NCP as an inhibitor, the inventors synthesized the NCP for the directional transfer to three different target areas of the gene S100A4. The inventors chose chimeric homoparental PNK 14 i.e. for directional migration by the end of the 5'-UTR (PNC) and two NCP with mixed bases from 20 n to the starting codon (PNC) and coding region in the second exon (PNC). The inventors wanted to explore whether it is possible to realize lower regulation of S100A4 dependent on dose. The inventors have therefore affected cells OHS different concentrations (100-2000 nm) PNC within 96 hours and checked for viability, and the presence of protein S100A4 Western blotting (figa and F). Data of the authors of the invention clearly demonstrate a dose-dependent inhibition activity S100A4, with a decrease in the transmission of signals, starting with a concentration of 100 nm using PNC. Typical examples of suitable controls is shown in fig.9D. Moreover, financial p the tats authors of the invention shown, what PNC at a concentration of 1000 nm caused maximum inhibition of the expression of S100A4. On the basis of MTS data that measure mitochondrial integrity as a measure of the viability of the cells (see experimental protocols for a detailed description), there is no appreciable toxicity using NCP concentrations less than 2000 nm (figure 10, lines 5 and 6). Thus, the concentration of 1000 nm, the NCP was chosen for all subsequent experiments.
To evaluate, there was a downward regulation of protein levels Sl00A4 dependent on time by the way, the inventors incubated the cells with NCP for 24, 48 and 96 hours (pigv). After 24 hours the level of protein S100A4 was decreased by 45% (PNC) and 35% (PNC). With a longer exposure time (48 hours) expression was decreased to 25% (PNC) and 35% (PNC), compared with the control level, respectively. Finally, expression of S100A4 was decreased to 10% (PNC) and 20% (PNC), compared with control cells incubated with the NCP within 96 hours after PCI (FIGU, C and E). Data of the authors of the invention have indicated that the NCP, and directed to the starting area AUG (PNC), and by the end of the 5'-UTR (PNC), inhibit the expression of S100A4, and that the NCP directed to the starting area AUG, was the most effective inhibitor. On the contrary, the inventors have not found any Engibarov the Oia expression of S100A4 by using NCP, directed to the second exon (PNC), as determined by Western blotting (11).
Also examined the relative expression of S100A4 mRNA. Total RNA was isolated from cells OHS after processing PNK/PCI using PNK-AUG, PNK-5'-UTR and PNK-randomized. Processed PCI OHS cells were chosen as a control in addition to a randomized NCP. All samples were subjected to the action of reverse transcriptase and solutions cDNA were subjected to analysis of real-time PCR using SYBRGreen I as a reagent for detection. CTvalues obtained for the same solutions, showed small differences in gene expression. The results are shown in table 3.
Example 6: Inhibition of the expression of S100A4 by using siPHK
The inventors compared the ability of the NCP to inhibit the expression of S100A4 with the ability siPHK to do the same. To analyze the efficiency of transfection siPHK and distribution, the inventors have noted one of the four siPHK the rhodamine and FITC. The inventors then tested different transfection reagents and concentrations. Data microscopy authors of the invention showed no capture using either FuGene, Lipofectamin, siPORT or Lipofectin (data not shown). However, the capture showed how using DOTAP, and poly-L-lysine and poly-L-lysine as the most effective agent. The authors izopet the deposits we used poly-L-lysine in all subsequent experiments.
In the experiments of the inventors siPHK designed according to Elbashir et al. ((2001), Genes Dev. 15, 188-200). In addition to siPHK designed to the selected gene target, designed control siPHK getting randomised siPHK, a BLAST search was carried out in GenBank, to remove false hybridization. The OHS cells were incubated with siPHK during different time periods and with different concentrations, with subsequent measurement of the level of protein S100A4 performed using Western blott. In addition, the inventors did not observe any down-regulation of S100A4 expression after 24, 48 and 96 hours, using 20, 50, 100 nm siPHK directed to two regions of the gene S100A4 (data not shown).
NCP may act by blocking transcriptional processes through their ability to form stable patterns triple helices, complex, built in the thread or with a displaced strand with DNA. Such complexes can create structural barriers to sustainable blocking the function of RNA polymerase and may thus be able to act as antigenic funds. At the level of the broadcast effect of antisense NCP based on steric blocking or RNA processing, transport to the cytoplasm, or broadcast. The failure of the NCP to activate RNase H eliminates the possibility of unintentional degradation of non-target mRNA. Updat the Executive, the lack of negatively charged main circuit prevents the binding of NCP with many proteins inside and outside of cells, usually associated with negatively charged macromolecules. Inhibitory effect PNC is in agreement with Doyle et al. (2001, see above), which demonstrated that the NCP towards the end of the 5'-UTR, were effective inhibitors of luciferase mRNA. Moreover, experiments on broadcast, performed on cell-free extracts showed that the NCP blocked the broadcast dependent on dose when was aimed close to the start codon AUG RNA (Knudsen & Nielsen (1996), Nucleic Acids Res. 24, 494-500). No effect was observed when the NCP was sent to sequences in the coding region. These results confirm the results obtained by the authors of the invention with PNC and PNC directed to the starting area and AUG second exon S100A4, respectively (figures 9 and 11). The patterns of expression of S100A4 in cells exposed to a randomized PNK/202, and the control cells without NCP, but with the sensitizer, is virtually identical to untreated cells. Additionally, the inventors tested expressio S100A4 in OHS cells with or without a sensitizer. Again, no differences in protein levels could not be observed (figa).
Example 7: Analysis of PCR with reverse transcriptase in real the nom time
In order to investigate the underlying mechanism of suppression of the gene, the inventors measured the relative levels of S100A4 mRNA using RT-PCR in real time before and after processing PNK/PCI. The aim was to explore, did molecules NCP according to the invention effect on the transcription level, or on some other level of the process of protein synthesis. As you can see on the figure of amplification, there was no distinguishing differences between the values of CTobtained from treated PNK/PCI samples and untreated controls after 96 hours after treatment (Fig). Randomised PNC used as an internal control NCP for PNC and PNC, respectively.
Previously Demidov et al. ((1995) Proc. Natl. Acad. Sci. U.S.A., 92, 2637-2641) investigated the kinetics and mechanism of binding of the NCP with the double helix of DNA. The results showed that the formation of a triple complex embedding depends on homopyrimidine NCP, communicating with homoparental target DNA. The second set, called double complex embedding can be obtained, but with homopyrimidine NCP. Conventional triplex, apparently formed only with cytosine rich homopyrimidine NCP. These results imply that a single molecule of the NCP, which is used by the authors of the invention, which is capable of re-directed the SCC to the DNA double helix, represented homoparental PNC, towards the end of the 5'-UTR. In addition, even though homoparental NCP (PNC) according to the invention is designed in order to associate it with DNA and RNA, the data FROM real-time PCR indicate that it acts at the level of translation. Other NCP with mixed bases in accordance with theory incapable of forming a triple or double complex embedding, which is necessary to block the transcription processes. This is consistent with results FROM RT-PCR real-time authors of the invention and supports theory that the NCP with mixed bases (PNC) unable to block transcriptional processes.
Example 8: Analysis of microarray
In order to investigate possible effects of inhibition of S100A4 transcription of the gene in experiments with cDNA microarrays, the inventors compared the processed PNK cells with cells exposed to the same processing at the same time, but a randomized NCP. Investigated molecules NCP directed to two target sequences of genes. All experiments were performed in two iterations. Only a small number of genes in accordance showed the relative changes in the expression of more than two times. Using hierarchical merging into clusters, defined the cluster, to the which showed the pattern counter increasing regulation in the processing of the NCP, which caused the greatest decrease levels of S100A4, and a smaller increase regulation less effective NCP. This cluster contained nine named genes (GAS2, UBE4B, FREQ, SHC1, PON3, CTSD, WNT3A, SCD and RAB6A). They represent the genes involved in the processes, including stress responses, apoptosis, and binding of calcium. Checked the levels of down-regulation of transcripts using real-time PCR. In order to demonstrate that the observed changes were the result of exposure to NCP sequence on a gene-targeted, real-time PCR carried out at each stage of the process separately, confirmed this conclusion (data not shown).
As a first illustration of the ability to use NCP/PCI/LS for the systematic suppression of genes, the inventors have investigated global changes in the level of mRNA expression using cDNA microarrays. Since the effect on gene expression add NCP and/or photochemical processing is not currently known, the inventors have conducted experiments with microarrays, using cells treated with randomized NCP as the reference channel. This was done in order to minimize the potential interfering effect of processing mode. Because a slight change in the processing time vozdeistvii so forth can still take place, the inventors have conducted real-time PCR at each stage of the process, in order to exclude that the observed changes in expression were the result of the processing, rather than a specific effect on S100A4. In particular, the PCI could lead to transcriptional changes associated with processes, including apoptosis (Ferreira S. D. et al, 2004, Lasers Med Sci 18(4): 207-12). Thus, as a General means PNK/PCI/LS, as it was thought to be not so well suited for the control of gene suppression of genes associated with these processes. In addition, the strategy has many appealing aspects to ease the design of the target sequence, the stability of the NCP, high-performance synthesis and introduction, and adjustable time delivery makes this system a good choice for the systematic suppression of cell linesin vitro. Gene suppression siPHK, as demonstrated, is a very viable strategy, but with some problems related to the design of the target sequence and stability (Amarzguioui M, et al., 2004: Biochem. Biophys. Res. Commun. 316(4): 1050-8).
The ability of S100A4 to modulate gene expression is unknown, but because it is a protein, which is believed to involved in the reverse engineering of the cytoskeleton, and as such it is the protein of the cell structure, not a regulator of transcription, are expected to be relatively minor effects. Accordingly, the observed changes in levels of transcription are relatively minor and in respect of amplitude, and the number of affected genes. Among the genes for which one can observe a strong change, frequeny is especially interesting, being a calcium-binding protein with four binding domains (polyclonal antibody is available on www.abcam.com).
Example 9: Genetic inhibition of the tyrosinase gene (TYR) in cell lines of melanoma (FEMX V)using NCP/PCI
The inventors have designed NCP to various genes involved in the biosynthesis of melanin; including tyrosinase (TYR), with tyrosinase associated protein 1 (TRP-I) and the transcription factor of microphthalmia (MITF). The goal of the authors of the invention is to suppress all three genes when using the NCP/PCI. TYR, TRP-I and MITF are connected to each other, which makes them interesting as a model system. As these genes are related to each other, it will be very interesting to investigate the possible influence on the levels of protein TYR/TRP-1, when the inventors will shut down MITF.
Cell line and culturing conditions
FEMX cells V (melanoma) were collected in the Norwegian Radium Hospital. Cells were cultured in medium RPMI-1640 (Bio Whittaker, Verviers, Belgium). Environment used without antibiotics, but to what t 10% serum embryo calf (FCS; PAA Laboratories, Linz, Austria) and 2 mm L-glutamine (Bio Whittaker, Verviers, Belgium). Cells were grown and incubated at 37°C in humidified atmosphere containing 5% CO2. The cells were tested and found that they are negative against infectionMycoplasma.
Specific for tyrosinase gene (TYR), the NCP, including randomized NCP received from Oswell DNA Service (Southampton, UK). Modifications were carried out on both ends (FAM and sequence NLS). The target to the start AUG codon was chosen based on previous studies on the inhibition of the NCP. Sequences were aligned against a database of human genome BLAST search to eliminate sequences with significant homology to other genes.
Used the following sequence NCP:
Each molecule NCP noted FAM N-end (5'end) and the NLS peptide on the C-end (3'end), i.e. FAM-L-L-PNK-L-L-PKKKRKV, where L represents a linker (2-aminoethoxy-2-toxicsuse acid (AEEA)).
Royal solutions (1 mm) was prepared by dissolving NCP 0.1% triperoxonane acid and before use was heated to 50°C in order to ensure that the NCP was dissolved completely. Before using NCP was further diluted to working solutions (100 μm) in sterile water and stored at -20°C.
Molecules NCP who drove in FEMX cells V, using the method of PCI, as described in example 6, and the protein levels were determined using Western blotting.
The results, as shown in Fig demonstrate that TYR can be suppressed by way PNK/PCI, however, further optimization will lead to a stronger effect of gene suppression. In particular, track number 7 shows decreasing regulation of protein TYR after incubation with 10 mm NCP for directional migration to the area of the start codon.
1. The method of introduction of the molecules of the NCP in the cytosol of mammalian cells, involving contacting the specified cells with molecules NCP and photosensitizing agent and irradiation of the cells with light of a wavelength effective to activate the photosensitizing agent, where these molecules NCP conjugated to a positively charged peptide.
2. The method of introduction of the molecules of the NCP in the nucleus of mammalian cells, involving contacting the specified cells with molecules NCP and photosensitizing agent and irradiation of the cells with light of a wavelength effective to activate the photosensitizing agent, where these molecules NCP conjugated to a positively charged peptide.
3. The method according to claim 1 or 2, in which these molecules NCP vary in length less than 25 grounds.
4. The method according to claim 1 or 2, in which these molecules NCP are antimicrob the e molecule, complementary gene molecules or probes.
5. The method according to claim 1 or 2, in which the introduction of these molecules NCP is at a concentration that causes a decrease in the expression of the target genes by more than 10% after incubation with cells for 24 hours
6. The method according to claim 1 or 2, wherein said photosensitizing agent is selected from TPPS4, TPPS2a, AlPcS2a, TRS2A5-aminolevulinic acid and esters of 5-aminolevulinic acid.
7. The method according to claim 1 or 2, wherein said positively charged peptide is a polymer of consecutive amino acids.
8. The method according to claim 1 or 2, wherein said positively charged peptide ranges in length from 3 to 30 amino acids.
9. The method according to claim 1 or 2, wherein said positively charged peptide anywhereman directly with the molecules of the NCP through covalent binding.
10. The method according to claim 1 or 2, wherein said positively charged peptide has a charge of +1 to +10, preferably from +3 to +6.
11. The method according to claim 1 or 2, wherein said positively charged peptide contains a sequence of Xn(Y)m-Xowhere X is a neutral residue and Y is a positively charged residue, which may be the same or different in each position, and n, m and o are from the Oh integers ≥1.
12. The method according to claim 11, in which Y is the same in each position and represents K, R or N.
13. The method according to claim 1 or 2, wherein said positively charged peptide represents SEQ ID NO: 7 MSVLTPLLLRGLTGSARRLPVPRAKIHSL, SEQ ID NO: 6 AKL or SEQ ID NO: 5 GHHHHHG.
14. The method according to claim 1 or 2, in which more than one type of molecules NCP injected simultaneously, where each type has a different sequence.
15. The method according to claim 1 or 2, in which one or both of the photosensitizing agent and the molecule NCP is attached to, or associated with, or conjugated with one or more molecules of the carrier or molecules for directional migration.
16. The method according to item 15, in which the specified media molecule, or a molecule for the directional migration is a polycation, a cationic lipid, lipofectin or peptide.
17. The method according to claim 1 or 2, wherein said photosensitizing agent and these molecules NCP sent in a cage together or sequentially.
18. The method according to claim 1 or 2, wherein said method is carried out by contacting the specified cells with a photosensitizing agent, contacting the specified cells with molecules of the NCP, which must be entered and irradiation of the specified cell with the light of a wavelength effective to activate the photosensitizing agent, where the specified irradiation is carried out until kletocnah the capture of these molecules NCP in the intracellular compartment, containing the specified photosensitizing agent, preferably before cell capture these molecules into any intracellular compartment.
19. The method according to claim 1 or 2, in which the irradiation is carried out up to 60 minutes
20. The method according to claim 1 or 2, carried out in vitro or ex vivo.
21. A method of inhibiting the transcription or expression of the target genes through the introduction of the molecules of the NCP in the cell that contains the specified gene target, the method according to any one of claims 1 to 20, in which the specified molecule NCP specifically binds to a specified gene target or by-product of its replication or transcription.
22. The method of determining or estimating the level of the target genes or product its replication or transcription in the cell, where the method includes the introduction of the molecules of the NCP in the cell that contains the specified gene target, or the product of its replication or transcription, the method according to any one of claims 1 to 20, in which the specified molecule NCP specifically binds to a specified gene target or by-product of its replication or transcription, and evaluation levels associated NCP to determine the presence or level of a specified gene target, or the product of its replication or transcription.
23. The way of making reparation of the target genes, preferably the defective gene in the cell, where the method provides for the introduction of molecules NCP method is the yubom one of claims 1 to 20, and the introduction of the donor molecules of the oligonucleotide, containing the desired sequence in the cell that contains the specified gene target, in which the molecule NCP specifically binds to a specified gene target, to form a fastening NCP.
24. The method of in vitro diagnosis of the disease, condition or violation, providing for the introduction of molecules NCP in the cell according to the method according to any one of claims 1 to 20, in which the specified molecule NCP specifically binds to a gene target or by-product of its replication or transcription, which indicates the presence of specified disease, condition or violation, and assessment of the level associated NCP to determine the presence, stage or prognosis of developing this disease, condition or violation.
25. A method of treating diseases in which favorable downward regulation, repair or mutation of one or several genes, providing for the introduction of molecules NCP in the cell by the method according to any one of claims 1 to 20, in which the disease preferably is a malignant tumor, cystic fibrosis, cardiovascular disease, viral infection, diabetes, amyotrophic lateral sclerosis, Huntington's disease or Alzheimer's disease.
26. Cell or population of cells containing molecule NCP, which was internalizeone in the cytosol or the nucleus of the specified cell method is m according to any one of claims 1 to 20, where this molecule NCP conjugated to a positively charged peptide and where the cell is intended for use in the treatment or prevention of diseases, disorders or infection in the patient.
27. A composition comprising a molecule NCP and photosensitizing agent, for use in the treatment or prevention of diseases, disorders or infections, for which typical abnormal expression of the gene or in which favorable downward regulation, repair or mutation of one or more genes, where the composition is intended for use in the method, which contains the introduction of the molecules of the NCP in the cytosol or the nucleus of the cell, involving contacting the specified cell with the molecule NCP and photosensitizing agent and irradiation of the cells with light of a wavelength effective to activate the photosensitizing agent, and in which the molecule NCP conjugated to a positively charged peptide, preferably where the specified molecule NCP is the length of less than 25 bases, and/or the specified molecule NCP is an antisense molecule complementary to the gene molecule or probe, and/or the positively charged peptide is a polymer of consecutive amino acids, and/or the positively charged peptide ranges in length from 3 to 30 amino acids and/or the positively charged peptide anywhereman directly with the molecules of the NCP through covalent binding, and/or the positively charged peptide has a charge of +1 to +10, preferably from +3 to +6, and/or the positively charged peptide contains a sequence of Xn(Y)m-Xowhere X is a neutral residue and Y is a positively charged residue, which may be the same or different in each position, and n, m and o are integers ≥1, and/or Y are the same at each position and represents K, R or N, and/or the positively charged peptide represents SEQ ID NO: 7 MSVLTPLLLRGLTGSARRLPVPRAKIHSL, SEQ ID NO: 6 AKL or SEQ ID NO: 5 GHHHHHG.
28. A composition comprising a cell or population of cells p, for use in the treatment or prevention of diseases, disorders or infections, for which typical abnormal expression of the gene or in which favorable downward regulation, repair or mutation of one or several genes.
29. The composition according to item 27 or 28 for use in therapy of a malignant tumor or gene therapy.
30. The use of molecules NCP in the preparation of drugs for treatment or prevention of diseases, disorders or infections in a patient by modifying the expression of one or more of target genes in the specified patient, where the medicinal product is intended for use in the method, which which contains the introduction of the molecules of the NCP in the cytosol or the nucleus of the cell, involving the contacting of the specified cell with the molecule NCP and photosensitizing agent and irradiation of the cells with light of a wavelength effective to activate the photosensitizing agent, where this molecule NCP conjugated to a positively charged peptide and, where preferably the specified molecule NCP is the length of less than 25 bases, and/or the specified molecule NCP is an antisense molecule complementary to the gene molecule or probe, and/or the positively charged peptide is a polymer of consecutive amino acids, and/or the positively charged peptide ranges in length from 3 to 30 amino acids, and/or the positively charged peptide anywhereman directly with the molecules of the NCP through covalent binding, and/or the positively charged peptide has a charge of +1 to +10, preferably from +3 to +6 and/or the positively charged peptide contains a sequence of Xn(Y)m-Xowhere X is a neutral residue and Y is a positively charged residue, which may be the same or different in each position, and n, m and o are integers ≥1, and/or Y are the same at each position and represents K, R or N, and/or specified positive ZAR is defined peptide represents SEQ ID NO: 7 MSVLTPLLLRGLTGSARRLPVPRAKIHSL, SEQ ID NO: 6 AKL or SEQ ID NO: 5 GHHHHHG.
31. The use of cells or population of cells p in the production of the composition or the medicinal product for the treatment or prevention of diseases, disorders or infections by altering the expression of one or more of target genes in the specified patient.
32. The application of article 30 or 31, in which the specified drug is a tool for gene therapy or therapy of malignant tumors.
33. Method for the treatment or prevention of diseases, disorders or infection in the patient, providing for the introduction of molecules NCP in one or more cells in vitro, in vivo or ex vivo according to the method according to any one of claims 1 to 20, and where necessary the introduction of these cells indicated patient.
34. The method according to p, wherein said method is used for the treatment of malignant tumors or used in gene therapy.
35. The use of cells or populations of cells in p as a tool for screening.
36. The in vitro method of screening cells with altered patterns of gene expression, including:
a) analysis of expression of the target genes, or one or more additional genes cells defined in p where the specified NCP specifically binds to a specified gene target or by-product of its replication or transcription and alters the expression of ucasinogameo or more genes; and
b) comparing expression of a specified gene target, and/or one or more additional genes with the expression of the indicated genes in control cells, preferably cells of the wild type.
37. The method according to p in which the expression of a specified gene target is reduced by less than 80% of the control (wild-type) levels.
SUBSTANCE: there is offered an improved method for recovery of protein expressed from an open reading frame 2 of porcine circovirus type 2. The method involves the stages of introduction of recombinant baculovirus containing coding sequences of the open reading frame 2, in an insect cell contained in a culture medium. It is followed by expression of the open reading frame 2 by baculovirus and recovery of the expressed protein from a supernatant. Recovery is carried out approximately from the 5th day following cell infection to enable significant amounts of the recombinant protein to be expressed and secreted from the cells in the culture medium.
EFFECT: such methods avoid expensive and labour-intensive recovery procedures requiring separation and recovery of the recombinant protein from an internal cell space.
32 cl, 3 dwg, 24 tbl, 5 ex
SUBSTANCE: offered test system for quantitative determination of Streptococcus agalactiae includes species-specific primers having nucleotide sequences 5'-CAGTTGAATCCAAATGTTACGG-3' and 5'-TAATGCTGTTTGAAGTGCTG-3', and a probe having a nucleotide sequence 5'-CAACAAGTTGATCAAGAGATTGTAACATTACAAGCA-3'. A DNA target for specific amplification is a gene cfb Streptococcus agalactiae fragment between 685 and 762 nucleotide residues of its complete sequence.
EFFECT: invention allows quick and high-specific detection of Streptococcus agalactiae in samples of a biological material and determination of the definition of its quantitative content.
2 dwg, 2 tbl, 1 ex
SUBSTANCE: there are offered synthetic oligonucleotide primers having the following base composition: (SEQ ID NO: 5) gaagggtgttcggggccgtcgcttagg and (SEQ ID NO: 6) ggcgttgaggtcgatcgcccacgtgac and complementary for a genome IS900 region specific for M.paratuberculosis that is a paratuberculosis agent. There is offered a one-round method for detecting DNA of Mycobacterium paratuberculosis that is a paratuberculosis agent, assisted by oligonucleotide primers (SEQ ID NO: 5) gaagggtgttcggggccgtcgcttagg and (SEQ ID NO: 6) ggcgttgaggtcgatcgcccacgtgac by polymerase chain reaction (PCR). The method includes DNA recovery, DNA amplification on oligonucleotide primers, transfer of the amplification product on gel followed by result detection in a transilluminator; a positive reaction enables synthesising a fragment matched with size 413 bps.
EFFECT: invention enables instant diagnostics of paratuberculous infection.
3 cl, 1 tbl, 4 ex
SUBSTANCE: method includes separation by centrifugation of heparinised mononuclear cells of umbilical blood (further UB) in gradient of density on Ficoll-Paque™PLUS. After that realised are thorough washing in PBS+0.1 % BSA medium and denaturation at temperature 82°C of suspension, consisting of UB cells and control cell culture 1301, supported in RPMI medium with addition of 10% bull serum of glutamine and penicillin with streptomycin. After that, analysed and control cells are distributed in two pairs of test tubes, adding to them up to 0.5 ml PBS 0.1% BSA solution, centrifuging with acceleration 500 g for 5 minutes. Then in two rest tubes with cells poured is hybridisation buffer, and into the second pair - poured is hybridisation buffer with peptide-nuclein probe. After that, they are incubated in darkness for 2-20 hours, washed, mixed, centrifuged, stained in solution of propidium-iodide and RNK A. After that, analysis of cell samples is carried out with determination of their relative length telomeres depending on length of telomeres of cell culture 1301, taking into account DNA index.
EFFECT: invention makes it possible to increase quality of determination of properties of transplant, samples of hemopoetic stem cells of umbilical blood for transplantation.
SUBSTANCE: method of predicting development of arterial hypertension in pregnant women consists of realisation of DNA separation from peripheral venous blood, carrying out polymerase chain reaction, finding out data about presence of polymorphism of α-adducin 1 gene. If carrying genotype 460WW of gene of α-adducin 1 ADD1 G460W is detected, conclusion about risk of hypertension development in pregnant women is made.
EFFECT: increased efficiency of predicting arterial hypertension development in pregnant women.
1 ex, 1 tbl, 2 dwg
SUBSTANCE: invention represents primer sets for carrying out LIMP or PCR used for Saccharomyces pastorianus detection. Also, there are presented sets for Saccharomyces pastorianus detection containing a primer set according to the invention in a combination with a primer set for carrying out LAMP used for Saccharomyces bayanus detection, and also in a combination with a primer set for carrying out LAMP used for Saccharomyces cerevisiae and Saccharomyces pastorianus detection. There are presented methods for Saccharomyces pastorianus detection.
EFFECT: invention provides precise, quick and easy identification of Saccharomyces pastorianus yeast by means of PCR or LIMP.
19 cl, 4 dwg, 5 tbl, 4 ex
SUBSTANCE: what is presented is a method for Apo2L/TRAIL sensitivity prediction of a malignant tissue or cell sampled from a mammal, involving the stages as follows: sampling a malignant tissue or cell from a mammal; analysing the sample malignant tissue or cell for detecting expression of one or more biomarkers selected from a group of fucosyl transferase 3, fucosyl transferase 6, sialyl-Lewis A and/or X antigen (antigens) where expression of one or more specified biomarkers is an indicator of the fact that the specified sampled tissue or cell is sensitive to apoptosis-inducing activity Apo2L/TRAIL. Also, what is described is a method of apoptosis induction in the sampled malignant tissue or cell of a mammal. What is offered is a method of treating a malignant tumour in a mammal. The inventions enables using the detection of expression of one or more biomarkers as the indicator of the fact that a sample is sensitive to apoptosis-inducing agents, such as Apo2L/TRAIL and DR5 agonist antibodies. Specific biomarkers to be examined include fucosyl transferases, particularly fucosyl transferase 3 (FUT3) and/or fucosyl transferase 6 (FUT6), as well as sialyl-Lewis A and/or X antigens.
EFFECT: method improvement.
35 cl, 22 dwg, 1 ex
SUBSTANCE: cell suspension under investigation is incubated with biochip, containing immobilised on biochip antibodies, which have specificity to superficial antigens of investigated cells. After incubation, biochip is washed from non-specifically bound cells. Cells, which remain bound with biochip, are subjected to processing with labelled polynucleotide probes of one or several types with further hybridisation. Reading and processing of results are performed by presence of cell binding in area of biochip sites, containing immobilised antibodies, presence in them of determined superficial antigens is detected, and presence and character of binding of labelled polynucleotide probes are used to determine genetic signs in the same cells.
EFFECT: method application makes it possible to increase quantity of simultaneously determined superficial antigens on different cells with application of non-conjugated with label antibodies, simultaneously reducing number of used antibodies.
9 cl, 2 dwg, 2 ex
SUBSTANCE: what is offered is applying an analysis of p53(TP53) gene status and/or expression level as a biomarker while evaluating sensitivity of an individual suffering a proliferative disease to treatment by an mTOR inhibitor combined with a cytotoxic agent or while selecting individuals sensitive to the specified combined therapy for the following treatment of the disease by this method. Thus sensitivity to treatment of the proliferative disease by the mTOR inhibitor combined with the cytotoxic agent is predicted if wild-type functionally active p53 gene is found in a sample taken from the patient.
EFFECT: higher analysis accuracy.
14 cl, 5 ex
SUBSTANCE: what is offered is a method of structure stabilisation of thrombin binding DNA-aptamers, and also DNA-aptamers stabilised in such a way. The presented method provides formation of an additional base-stacking system by means of heterocycles or their analogues by means of increasing a surface of an aromatic system of heterocycles or their analogues, owing to using methods of determining a tertiary structure or molecular simulation with stating the fact of contact formation of the aromatic system of heterocyclic bases or their analogues with a G-quadruplex quartet which is related to a lateral loop.
EFFECT: method allows more effective assembly of antithrombin DNA-aptamers and improved structural stability under physiological conditions.
7 cl, 7 dwg, 1 tbl, 2 ex
SUBSTANCE: polypeptide, which is used in composition of pharmaceutical composition and in sets for screening of adhesion inhibitors of platelet adhesion or aggregation, is obtained in recombinant way applying matrix of cDNA of Anopheles stephensi salivary gland.
EFFECT: invention makes it possible to obtain polypeptide which possesses inhibiting activity with respect to platelet aggregation or inhibiting activity with respect to platelet adhesion.
10 cl, 4 dwg, 5 ex
SUBSTANCE: method provides phagocyte recovery from a cell mixture. The recovered cells are mixed with medium 199 in 18 bottles, and the cells are attached to glass at room temperature for 60 minutes. In 9 bottles, a culture medium containing medium 199, L-glutamine and mixed human serum heated for 30 min at 52°C is added to the cells in the preset amounts to produce cell samples. A culture medium of said composition containing 0.01% of zymosan particles is added to another 9 bottles with the cells to produce the cell samples. The prepared cell samples are divided into three portions. One one-third potion of zymosan and zymosan-free samples are frozen. The second portion of the zymosan and zymosan-free cell samples are cultivated at temperature 37°C for 4 to 6 h to be frozen, and the third portion of the zymosan and zymosan-free samples are cultivated at temperature 37°C for 18 to 24 h to be frozen respectively. All 18 bottles are exposed to simultaneous multiple freezing to -10°C and thawing at room temperature to complete recovery of intracellular lysozyme with determination of amount by a micromethod and calculation of synthesised lysozyme and a phagocyte activation value (PAV) by formula: PAV=Z Lsynth - Z-free Lsynth, mcg/ml, where Z Lsynth is a difference of lysozyme amount in the cultivated zymosan cells and lysozyme amount in the uncultivated zymosan cells, mcg/ml; Z-free Lsynth is a difference of lysozyme amount in the cultivated zymosan-free cells and lysozyme amount in the uncultivated zymosan-free cells, mcg/ml; the phagocyte activation value for long-term activation mechanism assessment is calculated by said formula by results of cell cultivation for 4 to 6 hours, and the phagocyte activation value for long-term activation mechanism assessment is calculated by said formula by results of cell cultivation for 18 to 24 hours.
EFFECT: invention allows more precise phagocyte activation assessment.
4 tbl, 2 ex
FIELD: oil and gas production.
SUBSTANCE: samples from oil contaminated surface are chosen for selection of strains of micro-organisms-destructors of oil and oil products. There are selected pure cultures of hydrocarbon containing bacteria and they are cultivated on dense growth medium. There is determined catalase activity of grown strains of micro-organisms. Further, they are used for preparation of one billion microbe suspension which is mixed with Raymond liquid medium at ratio 1:150. As a sole source of carbon there is added oil or oil products from a place of contamination at 1 cm3 per 1 dm3 of Raymond medium and there is carried out incubation during 12 days. Further, culture is sown on dense growth medium and cultivated. Upon completion of cultivation there is determined catalase activity of studied strains. At its decrease in comparison to a source at 30 % and more analysed strain of micro-organism is chosen as active destructor of oil and oil product.
EFFECT: selection of strains of micro-organisms-destructors among aboriginal micro-flora most actively decomposing oil and oil products facilitating efficient measures for purification of water and soil ecotopes contaminated with oil and oil products.
SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.
EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.
SUBSTANCE: immunoglobulin work solution migration time specific to each series and manufacturer is set; specific immunoglobulin is used as a reference mark (control). Further, an analysed sample suspected for the presence of sporous bacteria is prepared for an antigen-antibody reaction by adding the specific immunoglobulin work solution in a phosphate buffer to the sample. In reaction, a complex of bacterium in the sporous form and specific immunoglobulin is produced. It is followed with capillary electrophoresis, UV detection, complex migration time test in the capillary electrophoresis system to be compared with the migration time in the capillary electrophoresis system of the specific immunoglobulin (control). If the complex migration time exceeds the specific immunoglobulin migration time by 50 seconds, the presence of bacteria in the sporous form is detected in the sample. Also, the analysis conditions and parametres are described.
EFFECT: invention allows the high-reliable specific identification of bacteria in the sporous form.
6 cl, 2 dwg, 1 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and specifically to methods of determining degree of sensitivity of bacteria to damaging effect of bacteriolytic enzymes. The method involves growing bacteria on dense culture medium for 20-24 hours at temperature 37 °C. The obtained culture of bacteria is washed with 0.9% NaCl solution and centrifuged. After centrifuging, the bacteria cells are resuspended in a buffer whose pH is optimum for the enzyme with subsequent measurement of optical density of the suspension at wavelength 540 nm, setting the density on the level of 0.5 units and determination of the absolute quantity of bacteria in 1 ml of the suspension. 0.2 ml lysozyme with concentration of 10 mcg/ml or lysostaphin in concentration of 2.5 mcg/ml is added to 2.8 ml of the obtained suspension. The suspension of cells and enzyme solution are mixed. Optical density is measured every 10 second for 600 seconds from the moment the enzyme solution is added, with subsequent evaluation of bacteria sensitivity on the section with maximum rate of reaction for its characteristic with number of bacteria cells lysed in unit volume (ml) per unit time (sec).
EFFECT: invention enables to carry out sound correction of a system of treatments with a positive clinical result.
1 dwg,1 ex
SUBSTANCE: method includes incubation of microorganism E. coli in soil suspension, containing investigated substance, taken at the level of its limit permissible concentration (experiment) and without it (control) with further comparison of lipase activity of bacteria from control and experimental samples and in case of its reduction compared to control one, this concentration of substance is considered to be dangerous.
EFFECT: method makes it possible to increase sensitivity, accuracy, reduction of time required for sanitary-hygiene normalising of hazardous substances in soil with simultaneous simplicity and affordability of this method for wide practical use.
1 tbl, 5 ex
SUBSTANCE: tested culture of B. pseudomallei or B. Mallei in dose 104 mc is inoculated on Muller Hinton medium, disc, usually applied for treatment of acute forms of melioidosis (ceftazidime, meropenem, ciprofloxacin, doxycycline, rifampicillin, chloramphenicol, co-trimoxazole) are added and cultivated at 37°C for 24 hours. Simultaneously the same concentrations of tested cultures are inoculates on the same medium with addition of 10% of blood of laboratory animals - golden hamsters, discs are applied and cultivated at 37°C in atmosphere, containing 5% CO2, for 24 hours, after which results are counted. As index of antibacterial medication activity serves preservation of activity in conditions close to in vivo, at the level of bacteria growth indexes in standard conditions, zone of growth retardation essentially exceeding index of bacteria sensitivity to said antibacterial medication. Simultaneously determined is comparative efficiency of medications in experiments on treatment of experimental animals.
EFFECT: invention allows to determine which antibacterial medication is efficient for treatment of diseases caused by pathogenic burkholderias more accurately.
2 cl, 2 tbl, 2 ex
SUBSTANCE: cell material containing marrow stem cells in a liquid culture medium is placed over a prepared agar medium containing marrow cells producing homing factors. After incubation of the prepared two-layer culture, inadherent cells are transferred to a semi-viscous culture medium and incubated in a mode required for colony-formation. Then by recording the difference of stem cell count in the cell material in the reference, and after placing on the agar medium containing homing factors, stem cells migrated by stem cell homing factor are counted.
EFFECT: invention allows simplifying the method for determination of production of stem cell homing factors due to the use of the agar medium as a semipermeable membrane and the introduction of colony-forming ability change of the cell material as an evaluation criterion of production of stem cell homing factors.
1 tbl, 1 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and specifically to a method of determining death rate of microbes. The method enables determination of effectiveness of antimicrobial agents by determining death rate of microbes. The method involves determination of the death rate of microbes by calculating the logarithm of ratios of time values at which the microbes die, where the said logarithm is standardised on a time interval and the said time values are spaced apart by the said interval.
EFFECT: invention cuts on time, increases accuracy and simplifies determination of the death rate of microbes.
2 cl, 2 dwg, 2 ex
SUBSTANCE: invention refers to biotechnology and genetic engineering, namely to a genetically engineered construct pGoatcasGCSF for human granulocyte colony-stimulating factor (GCSF) expression . The offered invention can be used for producing transgenic animals that are human granulocyte colony-stimulating factor producers. It involves creating the genetically engineered construct pGoatcasGCSF of the size 6386 bps, and a specified nucleotide sequence shown in SEQ ID 1. The genetically engineered construct pGoatcasGCSF includes a 5'-regulatory sequence of the goat CSN1S1 asi-casein gene of the size 3387 bps connected with the full-size human GCSF gene of the size 1485 bps, and a 3'-flanking region of the cow gene CSN1S1 of the size 1514 bps.
EFFECT: stable effective level of human GCSF expression in milk of the transgenic animals, eliminated possibility of ectopic transgenic expression.
9 dwg, 2 tbl, 6 ex