Cell-penetrating peptides and thereof application

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

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AREA of TECHNOLOGY

The present invention relates to the identification and definition of the functional characteristics of the peptides of a man penetrating into the cells (CPP), and their application, in particular, as carriers during transfection.

PRIOR art

The plasmatic cell membrane is an effective barrier preventing entry into the cell of the majority of molecules, which are not subject to active transport, but also the most difficult target delivery of therapeutic substances. Only a small set of molecules with a specific molecular weight, polarity and/or net charge capable (passive) to diffuse through the cell membrane. All other molecules must be actively transported, for example by receptor-mediated endocytosis or by using the ATP-binding transport molecules. It is also possible to artificially create conditions that causes molecules to pass through the cell membrane, for example, by electroporation, cationic lipids/liposomes, microinjection, viral delivery or encapsulation in polymers. However, these methods are mainly used for the delivery of hydrophobic molecules. Additionally, significant side effects associated with these methods, and the fact that their use is mainly restricted�but systems in vitro do not allow these methods to become an effective tool for delivering drugs or other therapeutically active agents into cells for the purpose of prevention or treatment of medical conditions.

In particular, the need to target the delivery was also a significant problem in the development of drugs based on RNC (RNAi). Such agents include small RNA molecules (e.g., siRNAs, miRNAs or sh) that inhibit expression of genes that cause disease or contribute to the development of the disease. Once in 2001 the presence RNK been demonstrated in mammalian cells (Elbashir, S. M. et al. (2001) Nature 411, 494-498), it quickly became clear that this sequence-specific post-transcriptional mechanism of gene silencing can be used to develop a new class of drugs that also may be a promising treatment for diseases that until now were unavailable for therapeutic intervention (De Fougerolles, A. et al. (2007) Nat. Rev. Drug Discov. 6, 443-453).

However, as RNK occurs in the cytosol, for the manifestation of its therapeutic action any drugs based on the RNA must pass through the cell membrane. To date, we described several ways of achieving this goal, for example, the use of lipids (Schreder; A. et al. (2010) J. Intern. Med. 267, 9-21), viral media (Liu, Y. P: and Berkhout, B. (2009) Curr. Top.Med. Chem. 9, 1130-1143) and poly-nanoparticles (Howard, K. A. (2009) Adv. Drug Deliv. Rev. 61, 710-720).

Another way to move molecules across the cell membrane is the use of peptides that penetrate into the cells (CPP) (also called domains, the protein transduction (PTD) or sequences of translocation through the membrane (MTS); see the review, for example, in the publication: Fonseca, S. B et al. (2009) Adv. Drug Deliv. Rev. 61, 953-964; Heitz, F. et al. (2009) Br. J. Pharmacol. 157, 195-206).

CPP represents a heterogeneous group of peptide molecules from the point of view of their primary amino acid sequences and their structures. Known examples of CPP include translocation domain of HIV-1 TAT (Green; M. and Loewenstein, P. M. (1988) Cell 55, 1179-1188) and homeodomain Antennapedia protein of Drosophila (Joliot; A. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 1864-1868). The exact mechanism of translocation remains controversial. Mutation research Antennapedia protein revealed that the sequence of 16 amino acids, called penetration or pAntp (Derossi, D. et al. (1994) J. Biol. Chem. 269, 10444-10450), is a necessary and sufficient factor for translocation through the membrane. In the future have been developed derived from other proteins peptides CPP, such as the primary sequence of the protein HIV-1 Tat (Vives, E. et al. (1997) J. Biol. Chem. 272, 16010-16017). Developed a synthetic peptide - amphipathicity model MA peptide (Oehlke, J. et al. (1998) Biochim. Biophys. Acta 1414, 127-139).

It is shown that the connection antisense DNA or peptide nucleic acids (PNA) with peptides CPP gives the desired effect in vivo. However, it is still discussed which properties needed by the CPP peptide for the existence of translocational functions. In the General case, between the different peptides CPP was discovered a few similarities from the point of view of the sequence and/or structure. At the moment only one common property - this is a fairly high content of basic (positively charged) amino acids, which leads to a positive total charge. Thus, it is suggested that the CPP peptides first bind with negatively charged head groups of lipids or proteins (proteoglycans) in the cell membrane. However, after binding peptides remain inside membrane-connected compartments. The mechanism of absorption in the later stages is still actively debated. Assume that the CPP peptides or "retrograde" are transported in an EPR, where they fall under the scope of the existing in the cage of the mechanism of translocation (Fischer, R. et al. (2004) J. Biol. Chem. 279, 12625-12635), or they directly translucida through the membrane (Rothbard, J. B. et al. (2005) Adv. Drug Deliv. Rev. 57, 495-504). Depending on the mechanism of internalization is known to the CPP peptides are mainly located in the nucleus or in the event that they internalizers inside vesicles, remain largely within these vesicles, and only a small portion is released into the cytoplasm.

Many of the CPP peptides have serious side effects on the cells on which they act, and this is not surprising given the fact that most of the peptides from which the CPP, function as, for example, antimicrobial substances or toxins. For example, the CPP peptides can cause leakage of cytoplasm due to membrane damage, as well as to provide interfere with the normal functioning of membrane proteins. The CPP peptides can also show cytotoxic effects, example, transportan that affects Strasnoy activity (Soomets, U., et al. (2000) Biochim. Biophys. Acta 1467, 165-176). In addition, there is a growing body of evidence that many of the CPP peptides perform their function only in certain, very specific conditions that are not possible in the system in vivo. Another drawback is that, depending on the target cell, the CPP peptides may undergo rapid degradation in cells. Finally, as many of the known peptides CPP occurring proteins from non-human proteins, were regularly observed toxic and/or immunogenic effects that may prevent the application of these peptides to, for example, therapeutic celemi person.

Thus, a need remains for improved peptides that penetrate into the cells, which would overcome all the limitations mentioned above. In particular, there is a need for peptides that penetrate into the cells, which are suitable vesicles or carriers for transfection, allowing the delivery of compounds, such as therapeutic agents to target cells with high efficiency, but without showing significant cytotoxic and/or immunogenic effects.

In addition, a need remains for compositions containing such peptides CPP, as well as in the ways in which such peptides CPP used as molecular tools for diagnostic and therapeutic applications.

Accordingly, the purpose of the present invention is to offer such peptides CPP and related compositions and methods.

Summary of the INVENTION

One of the features of the present invention is that it refers to a peptide molecule, which can be internalized into the cell, wherein the peptide molecule (a) has a length of at least 10, preferably at least 15 amino acid residues; (b) contains in its primary amino acid sequence of at least 25%, preferably at least 30% positively charged amino acid �major shortcomings; and (c) internalized into the cell with efficiency component of at least 80%, preferably at least 90% of the efficiency of internalization of the TAT peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

In specific examples of an embodiment at least part of the peptide forms an alpha-helical secondary structure.

Preferably, the peptide is a mammalian peptide, in particular, preferably, is derived from the molecule man.

In additional preferred embodiments embodiment the peptide has an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 2);

(SEQ ID NO: 3);

(SEQ ID NO: 4);

(SEQ ID NO: 5);

(SEQ ID NO: 6);

(SEQ ID NO: 7);

(SEQ ID NO: 8);

(SEQ ID NO: 9);

(SEQ ID NO: 10); and wherein the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 10.

In specific preferred embodiments embodiment the peptide has the amino acid on�sledovatelnot, selected from the group consisting of:

(SEQ ID NO: 2);

(SEQ ID NO: 3);

(SEQ ID NO: 4); and wherein the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 4.

Another feature of the present invention is that it relates to a nucleic acid molecule that encodes a peptide that is specified in the document above.

Another feature of the present invention is that it refers to the vector containing the nucleic acid molecule described in this document above.

Another feature of the present invention is that it relates to the cell host containing the vector described in this document above.

An additional feature of the present invention is that it relates to a method for producing the peptide specified in this document above, including: (a) cultivating a host cell as specified in this document above, in suitable conditions; and (b) the allocation of the obtained peptide.

Another feature of the present invention is that it relates to compositions containing at least one peptide, the decree�nny in the document above, attached to any molecule selected from the group consisting of one or more nucleic acid molecules, one or more peptides or proteins, one or more small molecules and one or more nanoparticles, wherein the joining is carried out by binding selected from the group consisting of covalent binding and noncovalent binding.

In specific examples of embodiments at least one peptide in the composition is attached to one or more other peptides. Preferably, one or more other peptides to form at least partially alpha-helical secondary structure. In some embodiments, embodiments, one or more other peptides are Pro-apoptotic peptides.

An additional feature of the present invention is that it relates to a method for production of the composition specified in this document above, including: (a) creating at least one peptide that is specified in this document above; and (b) implementation of contact of at least one peptide of any molecule selected from the group consisting of one or more nucleic acid molecules, one or more peptides or proteins, one or more small molecules and one or more nanoparticles to obespechitelnye connection.

Another feature of the present invention is that it relates to a method of identifying the ability to internalization of the peptide specified in this document above, or the composition specified in this document above, including: (a) introducing a peptide or composition in one or more cells; and (b) detecting the internalization of the peptide or composition.

An additional feature of the present invention is that it relates to pharmaceutical compositions containing at least one peptide that is specified in the document above, or a composition as specified in this document above, and may further contain one or more pharmaceutically acceptable excipients and/or additives.

Another feature of the present invention is that it relates to the use of peptide is specified in the document above, or the composition specified in the above document, for transformation or transfection of one or more cells.

Another feature of the present invention is that it refers to the peptide specified in this document above, or to the composition specified in this document above, for use for the prevention and/or treatment of a condition selected from the group consisting of cancer, immune diseases, of the heart�distich diseases neurological diseases, infections and inflammatory diseases.

Another feature of the present invention is that it relates to a method of prevention and/or treatment of a condition selected from the group consisting of cancer, immune diseases, cardiovascular diseases, neurological diseases, infections, and inflammatory diseases comprising: administration to a subject at least one peptide that is specified in the document above, or the composition specified in the document above.

An additional feature of the present invention is that it relates to a composite set containing at least one of the following: (a) a peptide specified in this document above; (b) a nucleic acid molecule indicated in this document above; (c) the vector specified in this document above; (d) a host cell that is specified in this document above; and (e) the composition specified in the document above.

Other variants of embodiment of the present invention will be apparent from the detailed description below.

DESCRIPTION of GRAPHIC MATERIALS

FIGURE 1. Bioinformatic method for identifying peptides that penetrate into human cells.

A schematic view of the applied screening procedures. Were selected all database items SwissPot, related to human proteins, along with their description and annotation of Gene ontology (GO) (A), and analyzed with the use of a sliding window size of 30 amino acid residues (B). For identification of the suspected peptides that penetrate into the cells (CPP) used bioinformatic filters (C): first, I selected only the peptides containing 10 or more positive charges; secondly, were selected only extracellular proteins with a low probability of existence of immunogenicity (stages 2 and 3"). To reduce the number of peptides selected several approaches: the combination of the maximum value of the isoelectric point (IEP) and the higher degree of hydrophobicity, sequence similarity with the sequence derived from the HIV TAT peptide, as well as a thorough study of the literature and the results of BLAST analysis.

FIGURE 2. Chart showing the hydrophobicity and IEP for 500 randomly selected peptides.

The chart showed the hydrophobicity and IEP for 500 peptides randomly selected from the set consisting of 10.5×10630-dimensional peptides belonging to the person. As controls are added TAT peptides, poly-Arg, REV, Protamine and INF7 (unfilled squares). Non-toxic and incapable of transfection peptides are indicated as filled black circles, transfairusa toxic peptides designated �aracharkneri unfilled circle toxic peptides - unfilled circle with an inscribed cross and non-toxic transfairusa peptides - unfilled circle.

FIGURE 3. Chart showing the toxicity and transfection ability to for all analyzed peptides.

All peptides were analyzed for the ability to carry out the transfection siRNAs, and their toxicity was plotted as the average value of the levels of GAPDH mRNA at a concentration of 20 μm each of Aha1 siRNAs and siPHK luciferase (y-axis, "Toxicity"), and to the relative differences in the Aha1/GAPDH in transfection siPHK luciferase and Aha1/GAPDH transfection with Aha1 siRNAs at a concentration of 20 μm each (x axis, "Transfection"). Thresholds for toxicity (the average content of GAPDH mRNA at the level of 70%) and transfection (>TAT, 18%) indicated by the red dotted lines. The dotted lines divide the graph into 4 quadrants: upper left - notransferase non-toxic peptides; upper right - transfairusa non-toxic peptides; bottom left - toxic peptides; bottom right - transfairusa toxic peptides. Inset shows toxic peptides that fall into the range beyond the chart.

FIGURE 4. Analysis of peptide WNT16.

WNT16 is an example of a non-toxic, incapable of transfection (i.e., notransferase) peptide. Used �from the same experimental approach, in the case of Fig.3. To demonstrate the dose-dependent effects of the value of the content of mRNA obtained at a concentration of 1 µm, were taken as 100%. Viability is expressed as the percentage relative to the control (medium without additives). Data for Aha1 siRNAs are indicated by black shaded squares; data siRNAs for luciferase (Luc) are shown as unfilled circles.

FIGURE 5. Analysis of peptides BPIL3 and FALL.

BPIL3 is an example of a toxic incapable peptide transfection. Used the same experimental approach as in the case of Fig.3. (A). Note that the levels of Aha1 and GAPDH demonstrate similar behavior, while GAPDH shows a higher sensitivity. This is the explanation of the increase of the values of Aha1/GAPDH. To demonstrate the potential therapeutic window for toxic peptide that has the ability to transfection, which is masked by its toxicity, the same analysis conducted for peptide FALL (IN). To identify dose-dependent effects of levels of mRNA for both peptides (A, B) obtained at a concentration of 1 μm was taken as 100%. Viability for both peptides (A, B) is expressed as the percentage relative to the control (medium without additives). Data for Aha1 siRNAs are indicated by black shaded squares; data for luciferase siRNAs are shown as unfilled KRU�cov.

FIGURE 6. Analysis of peptide CU025.

CU025 is an example of a toxic, capable of transfection of peptide. Used the same experimental approach as in the case of Fig.3. To demonstrate the dose-dependent effects of the value of the content of mRNA obtained at a concentration of 1 µm, were taken as 100%. Viability is expressed as the percentage relative to the control (medium without additives). Data for Aha1 siRNAs are indicated by black shaded squares; data for luciferase siRNAs are shown as unfilled circles.

FIGURE 7. Analysis of non-toxic, capable of transfection of peptides.

CPXM2, ASM3B and NRTN are examples of non-toxic, capable of transfection of peptides. Used the same experimental approach as in the case of Fig.3. As a control, was determined by the ratio of Aha1/GAPDH for peptides TAT and poly-Arg (A). As CPXM2 (B) and ASM3B (C) show concentration-dependent decrease in the level Aha1 mRNA relative to GAPDH mRNA, without affecting significantly the cell viability. A detailed analysis of NRTN (D) revealed a significant decrease in the level Aha1 mRNA relative to the level of GAPDH mRNA without significant dose-dependent effects on cell viability. (A, B, C, D) In all cases to demonstrate dose-dependent effects of mRNA levels obtained at a concentration of 1 µm, were taken as 100%. The viability �of iraena as the percentage relative to the control (medium without additives). Data for Aha1 siRNAs are indicated by black shaded squares; data for luciferase siRNAs are shown as unfilled circles.

FIGURE 8. Analysis of the electrophoretic mobility shift of the selected peptides in the gel.

To demonstrate the formation of corresponding complexes of TAT peptides (A), NRTN (B) and WNT16 (D) were incubated with 500 µg of duplex siRNAs with the peptide taken in a specified molar ratio, in a period of one hour and analyzed using electrophoresis in agarose gel and staining with ethidium bromide. This analysis demonstrates the formation of complexes between siRNAs and peptides TAT, NRTN and WNT16, respectively. To demonstrate the effect of the NRTN peptide on the availability of siRNAs for ethidium bromide complex is incubated in the presence or in the absence of proteinase K (C).

FIGURE 9. Mediated by NRTN peptide transfection enough to influence cellular phenotype.

The specificity of the NRTN peptide to the sequence of the siRNAs were studied by means of analysis of the ability of the peptide to transferout duplex siRNAs, which serves as a target Eg5 mRNA man. The induction of apoptosis caused by Eg5 knockdown was determined by analysis of cytotoxicity Cyto Tox-Glo (Promega Inc.) To demonstrate the dose-dependent effects of the value of the content of mRNA obtained at a concentration of 1 µm, were taken as 100%. Induction of apoptosis is expressed� as the percentage relative to the control (medium without additives). Data for Aha1 siPHK are indicated by black shaded squares; data for luciferase siRNAs are shown as unfilled circles.

FIGURE 10. Alignment of sequences of GDNF and NRTN.

Alignment of the amino acid sequences of rat GDNF and NRTN people have demonstrated that they are related proteins. Identical amino acids are shown in light grey on a white background, similar amino acids are in black on a light gray background and differing amino acids in black on a white background. In GDNF rats designated segment corresponding to alpha-helical structure. Rectangular frame composed of NRTN isolated peptide, which was used in the experiments.

FIGURE 11. NTRN contains alpha-helical structural element.

Analysis of peptides FALL (A), NRTN (B) and TAT (C) using spectroscopy circular dichroism in the UV range. Spectra were obtained in the range from 195 nm to 260 nm with a step of 0.1 nm and a bandwidth of 1 nm, using a solution of peptide (0.1 mg/ml in the absence ("H2O") or in the presence of 10%, 25% and 50% of triptoreline (TFE), respectively.

FIGURE 12. NRTN, TAT and FALL function as the peptides that penetrate into the cells.

The use of fluorescence-activated cell sorting (FACS) analysis of TAT peptides (A), WNT16 (B) FALL (C) and NRTN (D) containing an N-terminal FITC-label. Cells were incubated for 3 h in �richtsteig peptide, was treated with proteinase K for 30 min. and analyzed with the use of FACS to detect the internalized peptides in the channel corresponding to FITC. The black lines represent the signals at the concentration of 1 μm, light gray lines show the signals at the concentration of 5 µm and dark gray lines show the signals at the concentration of 10 µm.

FIGURE 13. NRTN shows the activity in the presence of serum.

Conducted an analysis of the ability of NRTN to carry out the transfection of siRNAs duplexes in the absence (A) and presence (B) of serum in the medium for transfection. Used the same experimental approach as in the case of Fig.3. Analysis was performed either in normal culture medium RPMI 1640 containing 10% FCS (serum of fetuses of cattle) (B), or by incubation for three hours in OptiMEM medium for cultivation with reduced levels of serum, followed by replacement of the medium with normal growth medium (A). To demonstrate the dose-dependent effects of the value of the content of mRNA obtained at a concentration of 1 µm, were taken as 100%. Data for Aha1 siRNAs are indicated by black shaded squares; data for luciferase siRNAs are shown as unfilled circles.

FIGURE 14. Absorption NRTN endothelial cells in the human brain.

HRP conjugated by fluorescein isothiocyanate (FITC) peptides NRN (with N-terminal and C-terminal conjugation, respectively) were incubated with endothelial brain cells hCMEC/D3 at a concentration of 5 µm for 1 h at 37°C. then cells were washed and fixed. Images were obtained using fluorescence microscopy. In both cases, the peptide was located in intracellular endosomal structures.

FIGURE 15. NRTN-mediated uptake by cells of Pro-apoptotic Nur77 peptide.

Breast cancer cells human MCF-7 were incubated for 24 h in the presence of different concentrations of the NRTN peptide (squares), Nur (circles) and NurNRTN (triangles). The corresponding amino acid sequence is shown in the lower part of the figure. To assess the viability of the cells (and, thus, induction of apoptosis) used the same experimental approach as in the case of Fig.3.

FIGURE 16. NRTN-mediated uptake by cells of Pro-apoptotic peptide 4E-BP1.

Breast cancer cells human MCF-7 were incubated for 24 h in the presence of different concentrations of peptides 4E-BP1 (circles), TAT4E-BP1 (squares) and NRTN4E-BP1 (triangles) (upper part of the figure). Additionally, we compared the respective effects of the two chimeric peptides (solid lines) with their inactive versions (TATinact4E-BP1 and NRTNinact4E-BP1; dotted lines), respectively (the middle part of the figure). The corresponding amino acid sequence is shown�; and in the lower part of the figure. To assess the viability of the cells (and, thus, induction of apoptosis) used the same experimental approach as in the case of Fig.3.

DETAILED DESCRIPTION of the INVENTION

The basis of the present invention is unexpectedly discovered the fact that by using a combination of bioinformatic screening and subsequent experimental evaluation of candidate peptides can be identified several peptides CPP, which demonstrate improved functional profile, compared with the previous "gold standard" - a reference to the TAT peptide, in particular, a higher overall efficiency of transfection, a higher transfection activity in the presence of serum, as well as a lower degree of cytotoxicity. It is noteworthy that these peptides CPP does not show significant similarity in their primary amino acid sequences. Thus, these peptides can serve as modules in the development of new effective agents for the delivery of the purpose of use in therapeutic intervention.

The present invention, an illustrative description of which is given hereinafter, can be applied in practice in the absence of any element or elements, limitation or limitations, not specifically disclosed in this document.

In cases where in the present description and the claims use the term �containing", does not exclude the presence of other elements or steps. For the purposes of the present invention, it is assumed that the term "consisting of" is the preferred option embodiment of the term "containing". If later in this document indicates that the group contains at least a certain number of embodiments, it should also be understood as the disclosure group, which preferably consists only of these embodiments.

In case if you are using a singular noun, it includes the plural of that noun unless specifically stated otherwise.

If provided in the context of the present invention, numerical values to the person skilled in the art will understand that the technical action of the considered features is guaranteed to be within the range defined with some precision, which in the typical case involves a deviation from a given numeric values by ±10% and preferably ±5%.

Additionally, the terms "first", "second", "third", "(a)", "(b)", "(c)" etc. in the description and in the claims are used to separate from each other similar elements and not necessarily for describing a sequence or chronological order. It is understood that thus the terms are vtimezone�the time used under appropriate conditions, and that variants of embodiment of the invention described in this document, can be performed in sequences other than those described or illustrated in this document.

Additional definitions of terms will be given, in the context in which these terms are used. The following terms or definitions are presented solely to facilitate understanding of the invention. These definitions should not be interpreted as having a smaller volume than the volume which is understood by the person skilled in the art.

The first feature of the present invention is that it refers to a peptide molecule, which can be internalized into the cell, wherein the peptide molecule is:

(a) has a length of at least 10, preferably at least 15 amino acid residues;

(b) comprises in its primary amino acid sequence of at least 25%, preferably at least 30% positively charged amino acid residues; and

(c) internalized into the cell with efficiency component of at least 80%, preferably at least 90% of the efficiency of internalization of the TAT peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

The term "peptide molecule" (also referred to in this document as "peptide") when used in this �the document having means any natural or synthetic origin (e.g., obtained using chemical synthesis or recombinant DNA technology) linear macromolecules containing many natural or modified amino acid residues, connected by peptide bonds. Such peptides can form oligomers consisting of at least two identical or different peptide molecules.

The peptides according to the invention have a length of at least 10 amino acid residues (e.g., 10, 11, 12, 13 or 14 amino acid residues) and preferably have a length of at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, or at least 45 amino acid residues. In particular examples of embodiment of the peptides according to the invention have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44 or 45 amino acid residues.

The term "natural amino acid residues when used in this document means any of the 22 "canonical" amino acids, which in nature are embedded in the peptides. Of these twenty-two of the twenty amino acids directly coded with the universal genetic code. The remaining two, selenocysteine and pyrroles�n, built into proteins by unique mechanisms of synthesis. In the typical case, the amino acid residues of the peptide according to the invention are presented in the form of L-isomers. In some embodiments, embodiments, one or more amino acid residues of the peptide according to the invention are presented in the form of D-isomers. The term "modified amino acid residue" when used in this document refers to non-canonical amino acids, such as posttranslational modified amino acid. Examples of posttranslational modifications include, among other things, phosphorylation, glycosylation, acylation (e.g., acetylation, monitorowanie, palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitylation, changing the chemical nature (e.g., gemidiriya, delicioasa on the mechanism of β-elimination) and structural changes (e.g. formation of disulfide bridges).

The amino acid sequence of peptides that are specified in this document are presented in accordance with generally accepted rule, from amino (M)-end to the carboxy (C)-the end. However, the corresponding "reverse" peptides are also included in the scope of the present invention. The term "reverse peptide" when used in this document refers to the peptides with Taco� same sequence, that and their "normal" counterparts, but presented in reverse orientation, i.e., from C-to N-Terminus. For example, "normal" TAT peptide has an amino acid sequence GRKKRRQRRRPPQ. The corresponding "reverse" the TAT peptide has an amino acid sequence QPPRRRQRRKKRG.

The peptides of the present invention contain in their respective primary amino acid sequences (i.e., along its entire length) of at least 25%, preferably at least 30% positively charged amino acid residues. The term "positively charged amino acid" (in this document also referred to as "basic amino acids") when used in this document denotes the set of residues of lysine (K), histidine (H) and arginine (R) present in a particular peptide. In particular examples of embodiment of the peptide according to the present invention comprises in its primary amino acid sequence 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of positively charged amino acid residues. In other embodiments, the embodiment of the peptides according to the invention contain in their respective primary amino acid sequences of at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or at least 60% of positively charged amino acid residues.

The term "may byinternational into the cell" when used in this document refers to the ability of the peptides to pass through the cell membrane (including, among other things, the outer "limiting" the cell membrane (also commonly referred to as "plasma membrane"), endosomal membrane and the membranes of the endoplasmic reticulum) and/or to control the passage of the agent or the load through the cell membrane. A passage through the cell membrane, called in this document "penetration into the cell". Accordingly, peptides having specified the ability to pass through cell membranes, called in this document "peptides that penetrate into the cells". In the context of the present invention means any possible mechanism of internalization, including both energy-dependent mechanisms (i.e., active) transport (e.g. inditos), and the mechanism is non-volatile (i.e., passive) transport (e.g., diffusion). When used in this document, the term "internalization" should be understood as including the localization of at least some of the peptides that passed through the plasma cell membrane, in the cytoplasm (as opposed to localization in other cell compartments, such as vesicles, endosomes or the core). In specific examples of this embodiment involves the transport mechanism ensures that at least 0.01 percent, at least 0.05%, at least about 0.1%, at least 0.5%, at IU�e 1%, at least 2%, at least 5% or at least 10% of the internalized peptides or compositions were localized in the cytoplasm.

The peptides of the present invention internalizers into the cell with efficiency component of at least 80%, preferably at least 90% of the efficiency of internalization of the TAT peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1; see also the publication: Vives; E. et al. (1997), supra). In other words, the characterization of the functional activity of the peptides is given in comparison with the reference peptide (TAT peptide represents the "gold standard" for peptides that penetrate into the cells). In particular examples of embodiment of the peptides according to the invention internalizers with efficiency equal to 80%, 85%, 90% or 95% of the efficiency of internalization of TAT peptide. In a specific preferred examples of embodiment of the peptides according to the invention internalisers at least with the same efficiency (i.e., 100%) as the TAT peptide. In particular, preferably, the peptides according to the invention internalizers with higher efficiency (i.e., more than 100% or at least 101%) than the TAT peptide, for example, with efficiency component 105%, 110%, 115%, 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% of the efficiency of internalization of TAT peptide.

The term "efficiency of internalization" when used�the stripes in this document should be understood in a broad sense. This term refers not only to the degree to which the peptide according to the invention passes through the plasma membrane of cells (i.e., to the properties of the actual internalization), but also to the efficiency with which the peptide controls the passage of the agent or the load cell through the plasma membrane (i.e., its ability to carry out transfection; in this document it is also called "transfection efficiency"). In this area a number of methods of determination of properties of internalization and/or ability to transfection of this peptide, for example, apparently detected by attaching a label such as a fluorescent dye) to the peptide (and/or to the load, which will transferrates) or the peptide compounds with a reporter molecule that allows detection after the uptake of the peptide by cells, e.g., using FACS analysis or by using specific antibodies (see, e.g. Ausubel, F. M. et al. (2001) Current Protocols in Molecular Biology, Wiley & Sons, Hoboken, NJ, USA). The person skilled in the art also know how to choose appropriate ranges of concentrations of the peptide and, if applicable, the load that will be applied in such ways; this choice may depend on the nature of the peptide, the size of the load, the type of cells, etc.

In additional embodiments, is embodied�Oia peptides of the present invention do not show significant cytotoxic and/or immunogenic action against their respective target cells after internalization, that is, they do not have a hindering effect on cell viability (at least at concentrations that are sufficient for mediating cellular transfection and/or penetration). The term "minor" when used in this document should be understood in the sense that less than 50%, preferably less than 40% or 30% and in particular less than 20% or 10% of target cells killed after internalization of the peptide according to the invention. In other embodiments, the embodiment of cytotoxic (and/or immunogenic) the effects of peptides after internalization into the cell, the same or less pronounced than the corresponding effects of the TAT peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1). In specific examples of an embodiment of cytotoxic (and/or immunogenic) the effects of peptides after internalization into the cell, is the same or less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the actions provided by the TAT peptide. The person skilled in the art well known methods for determining cytotoxicity of the compound and/or the viability of the target cells on which to apply connection (see, for example, Ausubel, F. M. et al. (2001), supra). Appropriate analysis�algebraic sets are available for purchase from different suppliers.

In specific examples of realization of potential cytotoxic and/or immunogenic activity, inherent to the peptide according to the invention may be disguised by applying a peptide one or more modifications, for example, by chemical synthesis or recombinant DNA technology. Such modifications may be related, for example, with the addition, removal or substitution of functional groups or modify the provisions of such functional groups. The person skilled in the art are well aware of how this "masking" can be achieved in the case of this peptide.

In additional embodiments, embodiments peptide molecules of the invention contain at least one structured domain, i.e., the element that forms (i.e., placed in a certain way) stable secondary structure, namely, a certain spatial arrangement of amino acid residues that are localized next to each other in linear sequence. Often steric interactions between amino acid residues are of a normal recurring nature, which leads to the formation of periodic structures, well known to specialists in this field, such as α-helix (similar to the rod tightly coiled spiral structure) and β-structure (elongated area, a lot of the�their plots possibly form a parallel or antiparallel β-pleated sheets). In the framework of the present invention the peptide molecule may contain one structured domain, surrounded by flat fields, or may contain two or more such structured domains (of the same type or of different types, for example, two α-helix or one α-helix and β-structure), separated from each other. In some embodiments, embodiments of the peptide molecule, forms a secondary structure along its entire length (i.e., does not contain unstructured regions).

In the preferred examples of embodiment at least part of the peptide molecules described in this document forms an alpha-helical secondary structure. α-Helical element may comprise at least 4, or 6 amino acid residues, and preferably at least 8 or 10 amino acid residues. In specific examples, embodiments of the peptide molecule according to the invention contains a single α-helical element as the only secondary structure.

In the preferred embodiments peptide molecules the present invention are derived from molecules to mammals, i.e., they originate from organisms such as mouse, rat, hamster, rabbit, cat, dog, pig, cow, horse or monkey. In particular, preferably,the peptide molecules derived from molecules to man, i.e., they are derived from human sequences, or a sequence of a man. The term is derived from sequences of a person" when used in this document indicates the sequence derived from the human molecules bearing a minor modification (e.g., one or more amino acid substitutions) in comparison with human sequences of natural origin. The term is a sequence of a person" when used in this document refers to a sequence identical to the sequence of human natural origin (i.e., not containing variants or modifications of the sequence).

In additional preferred embodiments embodiments of the peptide molecule according to the invention has an amino acid sequence selected from the group consisting of:

(NRTN peptide; SEQ ID NO: 2);

(peptide SRM; SEQ ID NO: 3);

(peptide ASM3B; SEQ ID NO: 4);

(FGF peptide 12; SEQ ID NO: 5);

(peptide CU025; SEQ ID NO: 6);

(peptide IGS10; SEQ ID NO: 7);

(peptide SRM; SEQ ID NO: 8);

(peptide CD026; SEQ ID NO: 9);

(peptide FALL39, var.1 (also called peptide FALL; SEQ ID NO: 10);

And wherein the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 10.

In specific preferred embodiments embodiment the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; and wherein the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 4.

The term "percent (%) sequence identity" when used in this document describes the number of matching identical amino acids in two or more aligned amino acid sequences compared to the number of amino acid residues comprising the full length amino acid sequences that serve as sample for comparison. In other words, when applying the alignment of two or more sequences or sub-sequences (i.e., derived from fragments or truncated variants) you can determine the percentage of amino acid residues to�which are the same, when (sub-)sequences are compared and aligned to achieve the maximum degree of compliance over the window of comparison, or over a specified area according to the assessment with the use of well-known specialists in this field of the algorithm of sequence comparison, or when aligning manually and visual assessment. Therefore, the above definition is applicable not only to a full-sized sequences with SEQ ID NO: 2 through SEQ ID NO: 10, but also to any shortened variant amino acid sequence with a length of at least 10, preferably at least 15 residues in any sequence with SEQ ID NO: 2 through SEQ ID NO: 10.

To assess the level of identity between two protein sequences can be aligned electronically using suitable computer programs known to specialists in this field. Such programs include, among others, BLAST (Altschul, S. F. et al. (1990) J. Mol. Biol. 215, 403-410), FASTA (Lipman, D. J. and Pearson, W. R. (1985) Science 227, 1435-1441) or program using the algorithm of Smith-Waterman (Smith, T. F. and Waterman, M. S. (1981) J. Mol. Biol. 147, 195-197). These programs, in addition to providing a pairwise sequence alignment, also determine the level of sequence identity (usually in the form of percent identity) and the probability that the alignment was occasionally�m phenomenon (factor p). In the case of amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, and expectation (E) equal to 10. In the matrix of substitutions BLOSUM62 (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919) applies the alignment value (In) of 50, expectation (E) equal to 10, M=5, N=4 and a comparison of both strands. Such computer programs, as CLUSTALW (Higgins, D. et al. (1994) Nucl. Acids Res. 2, 4673-4680) may be used to align more than two sequences. In addition, the program CLUSTALW when calculating the degree of identity takes into account the gaps in the sequences.

If the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 10, it is a variant of the embodiment of the present invention. Type present amino acid modifications (e.g., addition, insertion, deletion and substitution of one or more amino acid residues or a combination of these changes) does not matter. In specific examples of implementation "derived amino acid sequences have at least 70%, at least 72%, at least 74%, at least 76%, or at least 78% sequence identity relative to any� of the sequences with SEQ ID NO: 2 through SEQ ID NO: 10. Preferably, the derived amino acid sequences have at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% overall sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 10.

The second feature of the present invention is that it relates to a nucleic acid molecule encoding the peptide molecule, specified in the document above.

The term "nucleic acid molecule" when used in this document means any nucleic acid encoding the peptide according to the invention. Examples of such nucleic acid molecules include nucleic acids of natural origin, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as well as artificially created nucleic acid that has been chemically synthesized or produced by recombinant technology genes, including, for example, analogues of nucleic acids, such as, among others, and peptide nucleic acid (PNA) or locked nucleic acids (LNA) (see, e.g., Sambrook, J., and Russel, D. W. (2001), Molecular cloning: A laboratory manual (3rd Ed.) Cold Spring Harbor, NY, Col Spring Harbor Laboratory Press). Specific examples of nucleic acids of natural origin include DNA sequences, such as genomic DNA or cDNA molecules, and RNA sequences, such as molecules garns, mRNA or rRNA, or complementary nucleic acid sequences. Such nucleic acids can be of any length and may be single-stranded and double-stranded molecules. In a typical case, the target nucleic acids according to the invention have a length of from 30 to 5000 nucleotides, e.g., from 30 to 3000 nucleotides, from 45 to 2000 nucleotides, 60 to 1000 nucleotides, or from 75 to 500 nucleotides. When used in this document, the term "nucleotide" should be understood to refer to both ribonucleotides and deoxyribonucleotides (i.e., to molecules RNA and DNA).

Preferably, the nucleic acid molecule of the present invention is an integral part of genetic structures (also often referred to as "gene expression cassette"), which ensures its expression. Genetic design is called "capable of expressing a nucleic acid molecule" or capable "to ensure the expression of a nucleic acid sequence (i.e., nucleotide sequence)" if it contains the elements of the sequence, which include information, Cass�concerning the regulation of transcription and/or translation, and if such sequences "functionally connected" to the nucleotide sequence that encodes the peptide. The functional connection is a connection, wherein the regulatory sequence elements and sequence that will Express (and/or sequence that will Express in conjunction with each other) are connected in such a way that possible gene expression.

The precise nature of the regulatory regions needed for gene expression may vary between species, but in General these areas contain a promoter that in prokaryotes includes both the actual promoter, i.e., the DNA elements that control initiation of transcription, DNA elements, which after transcription into RNA will serve as a signal the initiation of translation. Such promoter region typically include a 5'-non-coding sequences involved in the initiation of transcription and translation, such as -35/-10-the boxes and the item Shine-Dalgarno in prokaryotes or TATA-box, CAAT sequence and the 5'-kiperousa elements in eukaryotes. These areas may also include enhancer or repressor elements, as well as the transmitted signal and leader sequences for targeting of the native polypeptide to a specific compartment of the host cell. Suitable� prokaryotic promoters include, among other things, the lacUV5 promoter, trp, tet and tac from E. coli and the promoter of phage T7. Suitable eukaryotic promoters include, among others, early and late SV40 promoters, the promoters RSV and CMV and promoters of yeast A0X1 and GAL1. In addition, the 3'-non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation or etc. However, if these sequences termination do not provide a satisfactory functioning in the host cell, they can be replaced by the signal sequence is functionally active in the cell. Specialist are well aware of these regulatory elements, and he is able to choose such elements suitable for expression of the nucleic acid molecule under specified conditions.

Nucleic acid molecules according to the invention may, as part of a gene expression cassette, can also be part of a vector or other media for cloning. Accordingly, an additional feature of the present invention is that it refers to the vector containing the nucleic acid molecule of the present invention.

The vector according to the invention can be, for example, a plasmid, kosmidou, fahmida, a virus, a bacteriophage, an artificial chromosome or other media commonly used in genetic engineering Preferably the vector is an expression vector that is able to provide the expression of a nucleic acid molecule according to the invention. Such an expression vector may include, in addition to the regulatory sequences described above and nucleic acid sequence to be expressed, at least one origin of replication and regulatory sequences derived from a species similar to the body-master, whose cells are used for expression, and one or more selectable markers, providing a selectable phenotype of transfected cells. Specially designed vectors (i.e., Shuttle vectors) containing more than one origin of replication, allow a transfer between different hosts, for example, between bacterial cells and fungi cells, or between bacterial cells and animal cells. Suitable oridzhiny replication for prokaryotic cells include, for example, ColE1 and M13. An example of the origin of replication in vectors for mammalian cells, SV40 is. Suitable prokaryotic selectable markers include, among others, genes for resistance to kanamycin, ampicillin and tetracycline. In the case of eukaryotic cells examples of selectable markers that can be applied, TPU� dihydrofolate reductase gene and the gene glutamylcysteine. Ways that you can apply to design and/or modification of recombinant vectors are well known to those skilled in the art (see, for example, Sambrook, J., and Russel, D. W. (2001), see above).

A large number of suitable vectors are commercially available and well known to specialists in this field who is also able to determine which vectors are suitable for expression of interest nucleic acid molecule under specified conditions. Examples of such vectors include, among others, prokaryotic vectors, such as vectors of the pUC series, pBluescript, pET series vectors, pCRTOPO, lambda gt11 vectors series pBBR1-MCS and pBC2, and vectors compatible with expression in mammalian cells, such as pCEP4, pXT1, pSG5, pRSVneo, pSV2-dhfr, with pcDNA3 pSIR and pIRES-EGFP. Examples of plasmid vectors suitable for gene expression in Pichia pastoris include, among other things, pAO815, pPIC9K and pPIC3.5K.

In accordance with another embodiment, the nucleic acid molecule of the present invention described in this document can also be inserted into vectors such that there is a connection with another nucleic acid molecule, and is formed during translation of the chimeric product. The other nucleic acid molecule may encode a peptide or protein that increases the solubility and/or facilitates the purification of the peptide according to the invention. Prima�s such vectors include pET32, pET41 and pET43.

Another feature of the present invention is that it relates to the cell host containing the vector described in this document above.

The introduction of the vector in the form of nucleic acid in a host cell can be effected using various methods of transformation, transduction or transfection, which are well known to those skilled in the art (see, for example, Sambrook, J., and Russel, D. W. (2001), see above).

In the framework of the present invention, the embedded vector can replicate and persist in the host cell in the form of independent genetic units or it may stably integrate into the genome of the host cell by genetic recombination. Such recombination may occur either at random positions in the genome of the mechanism of homologous recombination, either in specific locations in the genome of the mechanism of homologous recombination, or involving the site-specific integrase.

A host of the present invention may be a prokaryotic or eukaryotic cell, wherein preferable is a eukaryotic cell. Suitable prokaryotic cells-owners include, among others, strains of Escherichia coli (E. coli) (e.g., BL21, DH5α, XL-1-Blue, JM105, JM110 and Rosetta®), Bacillus subtilis, Salmonella spec., and Agrobacterium tumefaciens. Suitable eukaryotic�Chia the host cell include, among other things, yeast (e.g., Pichia pastoris and Saccharomyces cerevisiae), insect cells (such as cells of Drosophila S2 melanogaaster and Sf9 cells Spodoptera frugiperda) cells and plants. Preferably used in the present invention, eukaryotic cells-the hosts are mammalian cells, particularly human cells.

Suitable mammalian cells include, among others, immortalisation cell lines, such as human cells, Hela, HEK293, H9, MCF7 and Jurkat, NIH3T3 mouse cage, C127 and L, the cells of monkey COS1 and COS7 cells, quail QC1-3 cells and Chinese hamster ovary (CHO). All these cells-the hosts can be obtained from depositories such as the American type culture collection (Manassas, VA, USA) or the German collection of microorganisms and cell cultures (Braunschweig, Germany), as well as from various commercial suppliers. Also in the scope of the present invention includes a primary culture of mammalian cells, i.e., cell culture obtained directly from the organism (at any stage of development, including, inter alia, the blastocyst stage, embryonic, larval stage and adult organisms). Examples of suitable primary cell cultures include culture of cardiomyocytes, hepatocytes, fibroblasts, neurons, and stem cells. In the scope of the present invention also includes immortalisation stable�s cell line, obtained from primary cell lines.

In some embodiments embodiment, the cell-the owner of the present invention is part of a multicellular organism. In other words, the invention also relates to transgenic organisms containing at least one host cell, described in this document. Preferably, the transgenic organism is a mammal, e.g., mouse, rat, hamster, rabbit, cat, dog, pig, cow, horse, monkey or human.

An additional feature of the present invention is that it relates to a method of producing a peptide described in this document above, including:

(a) cultivating a host cell according to the invention in suitable conditions; and

(b) the allocation of the obtained peptide.

There are a great number of suitable methods for producing peptides in the corresponding cells of the host. In the case of single cells, e.g., prokaryotic cells or cell lines of mammals, the person skilled in the art can apply various conditions of cultivation. In one embodiment, the generated peptide harvested from the culture medium, lysates or extracts of cultured cells or from selection (biological) membranes with the use of conventional methods (see, e.g., Sambrook, J., and Russel, D. W. (2001), supra). If cle�ka-master is part of a multicellular organism, fraction of these cells can serve as a source for the isolation of peptide according to the invention.

Appropriate culture medium and culturing conditions to the above-described host cells are well known to specialists in this field. For example, suitable conditions for the cultivation of bacteria include the growing conditions in the aeration in Luria-Bertani (LB). With the aim of increasing the yield and solubility product expression environment you can buffer or make suitable additives known to those skilled in the art (such as chaperones, tRNA for rare codons, prosthetic groups, co-factors, metal ions, etc.) In the typical case of E. coli can be cultured at temperatures from 4°C to 37°C, the specific temperature value or a sequence of change of temperature depends on the molecule that will Express. In the General case, the person skilled in the art also understand that these conditions may need to be adapted to the needs of the host cell and the requirements expressed peptide or protein. If for the regulation of interest the expression of the nucleic acid molecule in the host cell used inducible expression system (for example, tetracycline inducible system Tet-On/Tet-Off or system induced by ecdysone), the expression can� to induce the addition of the appropriate inducing agent.

Depending on the applied type of cells and their specific requirements, growth conditions, culture of mammalian cells can be cultured, for example, in the medium RPMI 1640 or DMEM (Wednesday Needle in the modification of Dulbecco) with addition of 10% (vol./about.) FCS (serum of fetuses of cattle), 2 mm L-glutamine and 100 units/ml penicillin/streptomycin. In accordance with another variant, it is possible to apply a nutrient medium with a low concentration of serum, such as OptiMEM. Cells can be incubated at 37°C in a water-saturated atmosphere with 5% CO2.

Suitable environment for the culture of insect cells include, among others, environment TNM with the addition of 10% FCS, or medium SF900. The insect cells are usually grown at 27°C in the form of adhesion or suspension cultures.

Suitable expression protocols for both prokaryotic and eukaryotic cells are well known to those skilled in the art (see, for example, Sambrook, J., and Russel, D. W. (2001), supra). Appropriate analytical systems, kits, and reagents are commercially available from different suppliers.

In accordance with another method of obtaining peptide molecules according to the invention is used mRNA translation in vitro. Suitable cell-free systems broadcast in vitro include, among other things, the lysate of rabbit reticulocytes, wheat germ extract, microsomal� membrane cells of the pancreas of the dog the S30 extract from E. coli, as well as combined systems for transcription and translation. Appropriate analytical systems available for purchase from different suppliers.

Methods for isolating the obtained peptide is well known to specialists in this field and include, among others, ion exchange chromatography, affinity chromatography, gel filtration (size exclusion chromatography), high performance liquid chromatography (HPLC), bratovanova HPLC, disc-electrophoresis in gels and immunoprecipitation (see, e.g., Sambrook, J., and Russel, D. W. (2001), see above).

Another feature of the present invention is that it relates to the composition (also called in this document") containing at least one peptide that is specified in the document above, attached to at least one other portion (also called in this document "carrier"), wherein at least one other fragment preferably is a fragment selected from the group consisting of one or more nucleic acid molecules, one or more peptides or proteins, one or more small molecules and one or more nanoparticles, in this case, the joining is carried out by binding selected from the group consisting of covalent bonding and non-covalent binding�Oia.

The term "accession" when used in this document should be understood in the broadest sense, i.e., it refers to any type of molecular interaction between two or more connections. The term "covalent binding" refers to the form of intramolecular formation of a chemical bond characterized by the commodification of one or more pairs of electrons between two components, resulting in a mutual attraction that holds formed in the result of the molecule as a whole. The term "non-covalent binding" refers to the various interactions that are not covalent in nature, between molecules or parts of molecules that provide the retention of molecules or parts of molecules together, usually in a specific orientation or conformation. Such non-covalent interactions include, inter alia, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals interactions, and dipole-dipole connection. In the case of covalent binding peptide according to the invention can be connected to at least one other fragment directly or through a linker molecule, which is used for physical separation of the peptide according to the invention and at least one other fragment, and thereby ensures that no space limitations are a primary� functioning of both components because of their proximity to each other. Depending on at least one other fragment, the linker can be, for example, a peptide linkage, an amino acid, a peptide corresponding length or a different molecule, providing the required properties. In specific examples, embodiments, the linker is a residue of lysine or arginine ε-amino group of which is suitable for attachment of peptides, such as described in this document, with the various other fragments. Specialist in the art knows how to construct a suitable linker molecules, in particular, the linker peptides based on known knowledge. For example, peptide linkers can choose from the database of the LIP (Loops in Proteins (Loops in proteins")) (Michalsky, E. et al. (2003) Prot. Eng. 56, 979-985). Such a linker can be attached to the N - or C-Terminus or, if deemed preferable, it also does not terminal amino acid residue of the peptide of the present invention.

In preferred embodiments at least one peptide, such as described in this document above, attached to at least one other fragment via noncovalent interactions, for example, by (reversible) formation of the complex.

In other preferred embodiments, embodiments at least one peptide, such as described in this document above, attached to at least one other fragment�have through covalent interactions, for example, in the form of the chimeric molecule. The term "chimeric molecule" when used in this document means at least a two-part molecule that contains a peptide according to the invention, connected to at least one other fragment to form a single molecule. Peptide and at least one other fragment can be separated by a linker, as described above, or can be connected directly. At least one other fragment can be attached to the peptide according to the invention on the N-end C-end or to any amino acid different from the terminal amino acids, it is preferred to attach to the N-Terminus. The fragment is already contained in the composition of the chimeric molecules can be attached to additional pieces. Specialist known methods of analysis for determining the optimal order and/or combinations of fragments in the chimeric molecule according to the invention. In the typical case, if the chimeric molecule contains a peptide according to the invention and at least one other peptide, such chimeric molecules are not included in this term if the connection leads to the formation of peptides of natural origin. Such a chimeric molecule can be obtained and allocated according to the methods described above for obtaining the peptides of the invention.

A composition according to the invention can contain one or more peptide�in, such as described in this document. In the case of a plurality of at least two peptides, they may be of the same type or of different types. Conversely, if at least one peptide according to the invention attached to two or more fragments, these fragments can be the same type or different types (for example, two nucleic acid molecules or one molecule of nucleic acids and one peptide molecule). In specific examples of an embodiment of a single peptide of the invention attached to the set of two or more other fragments. In other embodiments, the embodiment of the set of two or more peptides according to the invention attached to a single another fragment.

In preferred embodiments, at least one other portion selected from the group consisting of one or more nucleic acid molecules, one or more peptides or proteins, one or more small molecules and one or more nanoparticles.

The composition of the present invention may also contain additional components, such as agents to stabilize the connection between one or more peptides described in this document, and at least one other fragment (e.g., chelating agents); agents for the protection of the composition (e.g., from cellular nucleases); or agents for the compensation�AI total charge of the composition to facilitate uptake by cells.

The term "small molecule" when used in this document should be understood in the broadest sense, and includes not only low-molecular-weight organic compounds, but also labels and reporter molecules (see below), haptens (i.e., small molecules that are able to induce an immune response only when they join the larger media), such as hydralazine, urushiol, fluorescein, Biotin and digoxigenin, and aptamers.

The term "nanoparticles" when used in this document refers to a microscopic particle whose size in at least one dimension less than 100 nm. In the typical case, the nanoparticles have a diameter in the range from 50 nm to 500 nm (i.e., from 0.05 µm to 0.5 µm), retain a stable structure under physiological conditions and is able to hold smaller molecules such as drug substances or other bioactive agents, which can then be delivered to the desired area. Many nanoparticles (or nanocarriers) are temperaturesalinity and/or sensitive to the pH value, i.e., they release their load during the heating and/or pH change. Such nanocarriers protect inside them compounds from degradation and digestive juices until, until they are released.

Specific examples of the incarnation at m�re one peptide according to the invention, which is the composition that is attached to one or more nucleic acid molecules. Preferably, at least one peptide according to the invention, attached to one or more molecules of nucleic acid has the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and wherein the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 4.

One or more nucleic acid molecules which are attached to at least one peptide, such as described in this document may be DNA molecules or RNA molecules of natural or synthetic origin, having any length (including aptamers), and can be single-stranded or double-stranded. In variants of the embodiment with more than one nucleic acid molecule attached to at least one peptide, this nucleic acid molecule can be of the same type (i.e., having identical nucleotide sequence) or different types. In the typical case, one peptide of the invention attached to a single nucleic acid molecule.

In specific examples, embodiments, one or NESCO�Ko of the nucleic acid molecules are RNA molecules in the typical case - molecules, small non-coding RNA (i.e., RNA that is not translated into a peptide or protein, such as snrna magrsc, st, siRNAs, miRNAs and sh), and preferably an RNA molecule selected from the group consisting of molecules, siRNAs, miRNAs molecules and molecules sh.

The term "molecule miRNAs" (or "miRNAs") when used in this document has its usual meaning adopted in this area (see, for example, in the publications: Bartel, D. P. (2004) Cell 23, 281-292; He, L. and Hannon, G. J. (2004) Nat. Rev. Genet. 5, 522-531). Accordingly, the term "microRNA" refers to endogenous RNA molecule, derived from the genomic locus, which was processaway from transcripts that can form local structure, characteristic of RNA-precursors of miRNAs. Mature miRNAs usually has a length of 20, 21, 22, 23, 24 or 25 nucleotides, although it may also be a different number of nucleotides, e.g., 18, 19, 26 or 27 nucleotides.

The sequence that encodes the miRNA has the ability to mate with flanking genomic sequences, the Mature miRNAs involved in the formation of imperfect duplex (also called in this document the structure of the stem-loop or hairpin structure or pre-miRNAs), which serves as an intermediate product in the processing of miRNAs from a longer transcript of the predecessor. This p�ocessing in the typical case occurs by the sequential action of two specific endonucleases, called Drosha and Dicer, respectively. As a result of the action of Drosha from the primary transcript (called "pri-miRNAs") is formed precursor miRNAs (also referred to in the General case of "pre-miRNAs"), which in the General case folds into a hairpin structure, or the structure of the stem-loop. From this precursor miRNAs under the action of Dicer cut duplex miRNAs that contains Mature miRNAs in one shoulder hairpin structure or the structure of the stem-loop and having a similar size of the segment (usually denoted by miRNAs*) in the other shoulder. After that miRNAs are sent to its target mRNA to carry out its functions, miRNAs* in many cases are degraded. Depending on the degree of complementarity between miRNAs and target mRNAs, miRNAs can run various regulatory processes. Target mRNAs that are highly complementary miRNA are cleaved specifically with the participation mechanisms, identical RNAi (RNC), a miRNAs perform the role of small interfering RNAS (siRNAs). Target mRNAs that are less complementary miRNAs or sent to a metabolic pathway of degradation in the cell and/or their translation is repressed. However, the mechanism by which miRNAs inhibit the translation of their target mRNAs, is still a contentious issue.

In some embodiments, embodiments, one or more of�about molecules of nucleic acid, attached to at least one peptide, such as described in this document are Mature miRNAs molecules. In other embodiments, the embodiment is applied, the molecules of precursor miRNAs. The term "precursor miRNA (or miRNA precursor" or "pre-miRNAs") when used in this document refers to the portion of the primary transcript miRNA from which the result of processing is formed of Mature miRNAs. In the typical case, the pre-miRNA folds into a stable hairpin structure (i.e., duplex) or structure of the stem-loop. Hairpin structure has a length in the range from 50 to 120 nucleotides, typically from 55 to 100 nucleotides, and preferably from 60 to 90 nucleotides (including residues of nucleotides, mating with miRNAs (i.e., the "stem") and any intervening segments (i.e., "loop"), but excluding located in more distal sequences).

The term "molecule siRNAs" (or "siRNAs") when using this document also has its usual value accepted in this field (see, for example, in the publications: Dorsett, Y. and Tuschl, T. (2004) Nat. Rev. Drug Disc. 3, 318-329; Rana, T. M. (2007) Nat. Rev. Mol. Cell Biol. 8, 23-36). Accordingly, the term "siRNAs" refers to double-stranded RNA molecule, typically having 2 unpaired nucleotide at its 3'end and a phosphate group at its 5'-end. Mature siRNAs typically has a length of 20, 21, 22, 23, 24 Il� 25 nucleotides, although you can also have a different number of nucleotides, e.g., 18, 19, 26 or 27 nucleotides. In the framework of the present invention can also be applied molecules precursor of siRNAs with a length of 49 nucleotides. Mature siRNAs formed from such a precursor as a result of processing involving Dicer.

Traditionally, the term "siRNAs" was used to indicate interfering RNA, which are exogenously introduced into the cell. However, in different organisms were detected endogenous siRNAs that are at least four classes: TRANS-acting siRNAs (tasirnas) associated with repetitions of siRNAs (rasi), scanning small (scn)RNAS and Piwi interacting with (pi)RNA (for a review see, for example, in the publication: Rana, T. M. (2007), see above).

One chain siRNAs is part ribonucleoprotein complex, known as appliance, which induced RNA silencing (RISC). The RISC complex uses this circuit siRNAs to identify a target mRNA molecules that are at least partially complementary to the included thread siRNAs, and then cleaves the target mRNA. Thread siRNAs, which is part of the RISC complex, called the thread-guide or antisense strand. Another thread siRNAs, called thread-passenger or semantic thread is removed from siRNAs, and it is at least partially homologous to the target mRNA. Special�students in this field will be clear that is, in principle, any thread siRNAs can enter the RISC complex may also function as a filament guide. However, the structure features of siRNAs (e.g., reduced stability of duplex siRNAs with 5'-end of the thread, which should serve as a guide) can help to integrate into the RISC complex. guide. Antisense thread siRNAs is an active agent that is performing in the role of siRNAs guide, in the sense that antisense thread is included in the composition of the RISC complex, thus allowing the RISC complex to target specific mRNA having at least partial complementarity with the antisense thread siRNAs, with the aim of cleavage or inhibition of translation. Mediated complex RISC cleavage of mRNA having a sequence at least partially complementary to the thread-guide, leads to lower equilibrium level of this mRNA and the corresponding protein.

The term "molecule shPHK" (i.e., the molecule is a short hairpin RNA) when used in this document refers to artificially created single-stranded interfering RNA molecule containing both sense and antisense strands of duplex siRNAs consisting of the structure of the stem-loop or hairpin structure. The stem of this hairpin structure typically has a length in the range from 19 to 29 nucleotides, and loop typically has a length in the range from 4 to 15 nucleotide� (see, for example, Siolas, D. et al. (2004) Nat. Biotechnol. 23, 227-231). Usually the molecule sh encoded DNA in the composition expression vector under the control of the promoter for RNA polymerase III (e.g., U6 promoter).

In some embodiments embodiment the RNA molecules described above, the framework provides a structure that includes only ribonucleotide units. In other embodiments, such embodiments the RNA molecules contain at least one ribonucleotide unit frame and at least one deoxyribonucleotide unit frame. Additionally, the RNA molecule may contain one or more modifications of the 2'-OH group of the ribose 2'-O-methyl group or 2'-O-methoxyaniline group (also referred to as "2'-""O""methylation"), which prevent degradation by nucleases in culture medium and, importantly, also prevent endonucleolytic cleavage by a nuclease complex, exercising induced RNA silencing, which leads to irreversible inhibition of small molecule RNA. Another possible modification, which is functionally equivalent to 2'-O-methylation, includes locked nucleic acids (LNA), which are analogues of nucleic acids containing one or more nucleotide monomers, LNA, in which bicyclic furanone unit is in the "locked" state, imitating confor�ation of sugar in the RNA (see, for example, Orom, U. A. et al. (2006) Gene 372, 137-141).

In some other embodiments, embodiments nucleic acid molecules designed to attach to at least one peptide molecule according to the invention, are agents that cause silencing of the expression of endogenous miRNAs. One example of such an agent that causes silencing, are constructed by chemical methods oligonucleotides, called "antagomir", which are single-stranded RNA molecules with a length of 23 nucleotides conjugated with cholesterol (Krutzfeldt, J. et al. (2005) Nature 438, 685-689). Alternatively, such chemically modified oligonucleotides were obtained microRNA inhibitors that are able to be expressed in cells in the form of RNA, synthesized from the transgenes. These competitive inhibitors, called "sponges for microRNA" are transcripts expressed from strong promoters, containing multiple tandemly arranged binding sites for interest microRNAs (Ebert, M. S. et al. (2007) Nat. Methods 4, 721-726).

In a particularly preferred examples of embodiments at least one peptide in the composition is attached to one or more other peptides. The term "other peptide" when used in this document means that these peptides differ from the peptides, which�s can be internalized into the cell, such as described in this document (i.e., peptides that are specified in the claims).

In particular, preferably, at least one peptide according to the invention, attached to one or more other molecules of peptides has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and wherein the amino acid sequence has along its entire length at least 70%, preferably at least 80% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 4.

One or more other peptides which are attached to at least one peptide described in this document may be molecules of natural origin or artificially created molecules of any length. For example, the length of such peptides may be in the range from 2 to 500 amino acids, or from 5 to 200 amino acids. In a typical case, such peptides have a length of from 8 to 100 amino acids or from 10 to 50 amino acids. Preferably, the length of such peptides may be in the range of 10 to 40 amino acids, from 12 to 35 amino acids, or 15 to 30 amino acids. Artificially created peptide molecules can be obtained using chemical synthesis, by recombinant DNA technology, or combinations of these methods. All those FPIC�would of synthesis well known to those skilled in the art (see, for example, Sambrook, J., and Russel, D. W. (2001), see above).

In variants of the embodiment with more than one other molecule peptides attached to at least one peptide according to the invention, these molecules, other peptides may be of the same type (i.e., have identical amino acid sequence) or different types. In the typical case, one peptide according to the invention is attached to one molecule of another peptide.

In additional embodiments, embodiments, one or more other molecules of peptides contain at least one structured domain, i.e., the element that forms (i.e., placed in a certain way) stable secondary structure, namely, a certain spatial arrangement of amino acid residues that are localized next to each other in linear sequence. Often steric interactions between amino acid residues are of a normal recurring nature, which leads to the formation of periodic structures, well known to specialists in this field, such as α-helix (similar to the rod tightly coiled spiral structure) and β-structure (elongated area, many of such sites may form a parallel or antiparallel β-pleated sheets). Peptide molecules can contain one structured domain, surrounded by destruct�risovannymi areas or may contain two or more such structured domains (of the same type or of different types, for example, two α-helix or one α-helix and β-structure), separated from each other. Peptide molecules can form such secondary structure along its entire length (i.e., not contain unstructured regions). If in the composition described in this document, contains more than one molecule of other peptides, it is also possible that other molecules of peptides comprises at least one structured domain, while the rest is unstructured.

In the preferred examples of embodiment at least a portion of one or more molecules, other peptides described in this document forms an alpha-helical secondary structure. α-Helical element may comprise at least 4, or 6 amino acid residues, and preferably at least 8 or 10 amino acid residues. In specific examples, embodiments of the peptide molecule according to the invention contains a single α-helical element as the only secondary structure.

In other preferred embodiments, embodiments, one or more other peptides are Pro-apoptotic peptides, i.e., peptides that are able to induce and/or regulate apoptosis (i.e. programmed cell death). The person skilled in the art will be known protein factors that are responsible for the induction and/or mediating apoptosis, as well as suitable methods of selecting peptide sequences that possess Pro-apoptotic functional activity. For example, information on these Pro-apoptotic factors can be found in scientific publications, as well as in various databases, such as APOPTOSIS Databas" (Doctor, K. S. et al. (2003) Cell Death Diff. 10, 621-623).

In other embodiments, embodiments, one or more other peptides have a functional activity selected from the group consisting of activation/removal of repression suppressors of tumor cells, inhibition of cellular oncogenes and the inhibition of a constitutive active protein variants. In other embodiments, embodiments, one or more peptides are ligands of natural origin or synthetic ligands (e.g. agonists and antagonists) of cellular receptors, such as receptors, cytokine receptors, angiotensin, endothelin receptors, vasopressin receptors and receptors of bradykinin. Again, many of these peptides are well known to specialists in this field, or information about them can be easily found in different sources.

An additional feature of the present invention is that it relates to a method for producing a compositing�tion, specified in this document above, including:

(a) creating at least one peptide according to the invention; and

(b) implementation of contact of at least one peptide of any molecule selected from the group consisting of one or more nucleic acid molecules, one or more peptides or proteins, one or more small molecules and one or more nanoparticles, in order to ensure the implementation of the merger.

The person skilled in the art will be well-known suitable conditions for conducting reactions for the implementation of this method (see, e.g., Sambrook, J., and Russel, D. W. (2001), supra; Ausubel, F. M. et al. (2001), see above).

An additional feature of the present invention is that it relates to a method of identifying the ability to internalization of the peptide of the invention or the composition (i.e., at least one peptide attached to the load) according to the invention, including:

(a) introducing a peptide or composition in one or more cells; and

(b) detecting the internalization of the peptide or composition.

This method may be particularly preferred for assessing the possibility of using peptide or composition for medical (e.g., diagnostic) or research tasks. If found sufficient level of internalization of the peptide or composition�and and, perhaps their localization in the cytoplasm, it indicates that the corresponding connection can be used for a particular purpose.

To this end, the peptide or composition according to the invention can be attached to one or more detect labels. Labels that can be applied in accordance with the invention, include any compound that directly or indirectly causes the formation of the detected compound or signal in a chemical, physical or enzymatic reaction. Labeling and subsequent detection can be carried out by methods well known to those skilled in the art (see, for example, Sambrook, J., and Russel, D. W. (2001), supra; and Lottspeich, F., and Zorbas H. (1998) Bioanalytik, Spektrum Akademischer Verlag, Heidelberg/Berlin, Germany). Tags can be selected, among others, fluorescent labels, enzymatic labels, chromogenic labels, luminescent labels, radioactive labels, haptens, Biotin, complexes with metals, metals, and colloidal gold. All these types of labels are well known to specialists in this field, and can be purchased from various suppliers. An example of the physical reactions that mediate such marks, is the emission of fluorescence or phosphorescence when irradiated. Alkaline phosphatase, horseradish peroxidase, β-galactosidase and β-lactamase are examples of enzymatic m�current, that catalyze the formation of chromogenic reaction products and can be used in the present invention.

Another feature of the present invention is that it relates to the application of the peptide described in this document, or composition, described in this document, for transformation or transfection of one or more cells, i.e., to the use of these compounds as a carrier for delivery for the purpose of load transfer into specific target cells.

In particular examples of embodiment of the invention relates to the use of the composition described in this document above, containing at least one peptide according to the invention, which is attached to at least one compound from the group consisting of one or more nucleic acid molecules and one or more peptides or proteins for transfection and/or targeted delivery of agents to specific cells.

Another feature of the present invention is that it relates to pharmaceutical compositions containing at least one peptide molecule that is specified in this document or arrangement specified in this document (i.e., at least one peptide attached to the load), and, possibly, further comprising one or more pharmaceutically acceptable auxiliary substances�in and/or additives.

The term "pharmaceutical composition" when used in this document refers to compositions intended for administration to a subject, preferably the individual who is the man. Pharmaceutical compositions of the present invention include any pharmaceutical dosage form known in this field, such as, inter alia, capsules, microcapsules, starch capsules, dragees, pills, powders, pills, multicomponent formulations (e.g., beads, granules or crystals), aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, emulsions of the type water-in-oil, for example, ointments, and emulsions of the type oil-in-water, for example, creams, lotions and balms. Finished dosage forms can be packaged in the form of separate dosage units or in multi-dose containers.

The pharmaceutical compositions of the invention include formulations suitable for oral, rectal, nasal, local (including buccal and sublingual), peritoneal and parenteral (including intramuscular, subcutaneous and intravenous) administration or for administration by inhalation or insufflation. Administration may be local or systemic.

Pharmaceutical compositions can be prepared in accordance with well known FPIC�the means (see, for example, Gennaro, A. L. and Gennaro, A. R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA; Crowder, T. M. et al. (2003J A Guide to Pharmaceutical Particulate Science. Interpharm/CRC, Boca Raton, FL; Niazi, S. K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).

For the preparation of said compositions can be applied one or more pharmaceutically acceptable (i.e., inert inorganic or organic excipients (i.e., vehicles). For preparing, for example, dragees, tablets, capsules or granules, can be applied, for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid high molecular weight alcohols, natural and hydrogenated oils. Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for the preparation of reconstituted solutions or aerosol mixtures before application include, among others, water, alcohols, glycerin, high molecular weight alcohols and their suitable mixtures and vegetable oil. Pharmaceutical composition may also contain additives such as, for example, fillers, binders, wetting agents, sliding agents, stabilizers, preservatives, emulsifying agents and additional solvents or agents that increase the solubility, or agents, contributing to the achievement of prolonged action�me. In the latter case, it is assumed that the active peptides or compositions of the invention can be implemented in systems for slow or delayed release or targeted delivery, such as liposomes, nanoparticles and microcapsules.

Pharmaceutical composition according to the invention will be administered to a subject in a suitable dosage. Specific dosage regimen will be determined by the attending physician and will depend on clinical factors. As is well known to experts in the field of medicine, suitable for a given patient the dosage depends on many factors, including weight, sex and age of the patient, the particular compound to be administered, time and route of administration, General health, existing conditions and other medicines that are injected in parallel. A therapeutically effective amount for a given situation can be readily determined using standard experimental methods, and it can realize the ordinary Clinician or physician. In General, the dosage at regular administration should be in the range of 1 μg to 1 g per day. However, the preferred dosage may be in the range from 0.01 mg to 100 mg, more preferred dosage is in the range of 0.01 mg to 50 mg and most preferred dosage is in the range from 0.01 mg to 10 mg per �tier.

Another feature of the present invention is that it refers to the peptide specified in this document, or to the composition specified in this document, for use for the prevention and/or treatment of the condition, the condition is preferably selected from the group consisting of cancer, immune diseases, cardiovascular diseases, neurological diseases, infections and inflammatory diseases. To this end, the peptide or composition according to the invention can be prepared in the form of pharmaceutical compositions, such as described in this document above, and introduced to a subject, preferably the individual who is the person.

The term "cancer" when used in this document refers to any type or form malignant neoplasm characterized by the uncontrolled division of the target cells as a result of genetic re-programming and the ability of target cells to spread, either by direct growing into adjacent tissue through invasion or by implantation into distant region with the formation of metastases (cancer cells are transported through the bloodstream or lymphatic system). Examples include, among others, breast cancer, colorectal cancer, prostate cancer, leukemia, lymphoma, near�the blastoma, glioblastoma, melanoma, liver cancer and lung cancer.

The term "immune disease" when used in this document refers to any violation affecting the immune system. Examples of such immunological diseases include, among others, immunodeficiencies (i.e., congenital or acquired condition in which the immune system's ability to fight infectious diseases suppressed or completely absent, such as AIDS or DID), hypersensitivity (e.g. Allergy or asthma) and autoimmune diseases. The term "autoimmune disease" when used in this document should be understood as including any breach resulting from too active immune response of the body directed against endogenous substances and tissues, in this case the body attacks its own cells. Examples of autoimmune diseases include, among others, multiple sclerosis, Crohn's disease, lupus erythematosus, myasthenia gravis, rheumatoid arthritis and polyarthritis.

The term "cardiovascular disease" when used in this document refers to any violation affecting the heart and coronary blood vessels. Examples of cardiovascular diseases include, among other things, coronary heart disease, angina, arteriosclerosis, cardiomyopathy, and�fact of the myocardium, ischemia and myocarditis.

The term "neurological disorder" or "neurological disorder") when used in this document refers to any violation affecting the nervous system, including diseases of the Central nervous system (CNS) (i.e., brain and spinal cord) and peripheral nervous system. Examples of CNS disorders include, among others, Alzheimer's disease, Parkinson's disease, Huntington's disease, who awake and Tourette syndrome. Examples of diseases of the peripheral nervous system include, for example, multiple manometric and PN.

The term "infection" when used in this document refers to any infringement caused by the colonization of the host body alien pathogen, such as bacteria, viruses and fungi. Examples of bacterial infections include, among others, bacterial meningitis, cholera, diphtheria, listeriosis, pertussis, salmonellosis, tetanus and typhus. Examples of viral infections include, among other things, the common cold, flu, dengue, haemorrhagic fever Ebola, hepatitis, mumps, polio, rabies and black pox. Examples of fungal infections include, among others, dermatiti foot, blastomycosis, and candidiasis.

The term "inflammatory disease" when used in this document refers to Liu�first violation, associated with inflammation, including, for example, acne, asthma, hay fever, arthritis, inflammatory bowel disease, an inflammatory disease of the pelvic organs and graft rejection.

An additional feature of the present invention is that it relates to a method of prevention and/or treatment of a condition selected from the group consisting of cancer, immune diseases, cardiovascular diseases, neurological diseases, infections, and inflammatory diseases comprising: administration to a subject at least one peptide of the invention or the compositions according to the invention. Preferably, the subject is a human.

The final feature of the present invention is that it relates to a composite set containing at least one of the following:

(a) a peptide molecule, described in this document above;

(b) a nucleic acid molecule described in this document above;

(c) the vector described in this document above;

(d) a host cell described in this document above; and

(e) the composition described in this document above.

The various components (a) to(e) of the kit can be packaged in one or more containers such as one or more vials. For example, each component is contained in the set, can �be Packed in a separate container.

The above kit components can be supplied in a lyophilized or dry form or dissolved in a suitable buffer solution such as phosphate-saline buffer solution or buffer solution of Tris/EDTA (TE). A host of the present invention may be supplied, for example, in the form of culture, applied in the form of streaks on the Cup with agar or in any other form suitable for long term storage. Such storage methods well known to specialists in this field.

The kit may also contain additional reagents, including, without limitation, preservatives, nutrient medium and/or buffer solutions for storage and/or recovery of the above-mentioned components, solutions for washing, etc., These reagents can be supplied in combination with one or more components (a) to(e), i.e., in the same container (e.g., a peptide molecule or nucleic acid, dissolved in an appropriate buffer solution). In accordance with another variant, at least some of these additional reagents may be supplied in separate containers.

The invention is additionally described by the following figures and examples, which are intended only to illustrate specific examples of embodiments of the present invention and should not be interpreted as limiting the scope of us�Mr sage of the invention in any way.

EXAMPLES

Example 1: Materials and methods

1.1 Culture of mammalian cells

The MCF7 cells were sown at a density of 15000 cells per well in 96-well plates. Cells were incubated for 24 h at 37°C, 5% CO2and 85% humidity in RPMI medium 1640 plus 10% FCS (serum of fetuses of cattle) and L-glutamine. For analysis of the functional activity of the cells were washed in OptiMEM medium was replaced by RPMI medium 1640 in OptiMEM (all reagents were purchased in the company Invitrogen Corporation, carlsbad, CA, USA). For the formation of complexes with siRNAs peptides were incubation of 100 nm siRNAs with the indicated concentrations of peptides in OptiMEM medium for 30 min. at room temperature. The complexes were added to cells and incubated for 3 h. Then the medium OptiMEM was replaced with normal culture medium RPMI 1640. Cells were incubated for 21 h to obtain lysates for analysis with p-DNA and for an additional 45 h for analysis with sets of CellTiter-Glo or CytoTox-Glo (see below).

1.2 quantification of mRNA levels

For the purpose of quantification of cellular mRNA levels analyzed by the method of p-DNA (branched DNA), which allows to estimate the levels of individual mRNAs. For this purpose a certain number of cells that will be analyzed, seeded in 96-well plates and OST�or overnight for attachment. The next day conducted the transfection of cells with siRNAs molecules.

After another 24 hours spent quantification of individual mRNA using analytical kit QuantiGene Plex 2.0 according to manufacturer's recommendations (Affymetrix Inc., Santa Clara, CA, USA). Briefly: cell lysates was transferred into a tablet with an addictive agent in the presence of a set of gene-specific probes and incubated at 53°C overnight. After washing the wells sequentially incubated at 53°C with amplificare agent and the probe, conjugated with alkaline phosphatase (AP), and between these stages performed the flushing. It was further added fluorescent substrate for AP - dioxetane and incubated for 30 min at 53°C. the Level of luminescence was measured with the use of the reader to determine luminescence InfiniteF200 (Tecan Austria GmbH, Greding, Austria).

1.3 Analysis of cell viability

To determine the number of living cells was used for analytical kit CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation,Madison, Wisconsin, USA) according to the Protocol suggested by the manufacturer. Cells were incubated for 48 h in the presence of peptides and siRNAs. Then spent the lysis of the cells and induced fluorescent signal that is proportional to the amount of ATP present, which, in turn, proportional to the number of Pris�according to living cells. The level of luminescence was analyzed with a reader to determine the luminescence InfiniteF200.

1.4 Analysis of cytotoxicity

To determine the cytotoxicity of the peptides used an analytical set of CytoTox-Glo Cytotocity Assay (Promega Corporation, Madison, Wisconsin, USA) according to the Protocol suggested by the manufacturer. Cells were incubated for 48 h in the presence of peptides and siRNAs. Then cells were treated luminogenic peptide substrate to measure the activity of proteases of dead cells that are released from cells that have impaired the integrity of the membrane. The substrate cannot pass through the intact membrane of live cells and does not generate a significant signal when added to a population of living cells. Thus, the level of luminescence in this analysis corresponds to the number of dead cells. Analysis of 96-well plates was performed in the reader to determine luminescence InfiniteF200.

1.5 Synthesis of peptides

Synthesis of peptides was carried out in accordance with accepted protocols ("FastMoc 0.25 mmol) in an automated peptide synthesizer Applied Biosystems ABI 433A using chemical reaction with Fmoc. In the course of repeated cycles collected the relevant peptide sequence by stringing the respective Fmoc-amino acids.

At each stage of the accession of the N-end�th Fmoc-group was removed by treatment of the resin 20% solution of piperidine in N-methylpyrrolidone. Joining was performed using Fmoc-protected amino acids (1 mmol), activated with a solution of hexaflurophosphate-O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium / 1-hydroxybenzotriazole (HBTU/HOBt, 1 mmol of each) and diisopropylethylamine (DIPEA, 2 mmol) in dimethylformamide (DMF). After the accession phase was performed attaching the protective groups to unreacted amino groups by treatment with a mixture of Ac2O (0.5 M), DIPEA (0.125 M) and HOBt (0,015 M) in NMP. Between the two phases of the abundantly resin was washed with N-methylpyrrolidone in DMF. Introduction creating steric obstacles of amino acids was performed using an automated dual-joining. For this, the resin was treated twice with 1 mmole of the activated building block without the intermediate stage of connection of the protective groups. Upon completion of the synthesis of the target sequence were attached to the peptide Fmoc-12-amino-4,7,10-trioxatridecane acid (TEG-spacer), using standard conditions for the reaction. After this was added Fmoc-Cys(Trt)-OH to aminobenzo all peptide sequences. After removing the protective Fmoc group bearing the peptide resin was placed in rittby filter and treated with a mixture triptoreline acid, water and triisopropylsilane (in the ratio of 19 ml : 0.5 ml : 0.5 ml) for 2.5 h. a Solution of fission products were filtered and�was Adali peptides by adding a cooled (0°C) diisopropyl ether (300 ml) to give a colorless solid, which again was washed with diisopropyl ether. The crude product was re-dissolved in a mixture of acetic acid and water, liofilizirovanny and was purified using preparative ortofotos HPLC, using a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid (TFA) (column Chromolith prep RP-18e, 100×25 mm; Merck KGaA, Darmstadt, Germany).

1.6 Analysis using FACS (fluorescence-activated cell sorting)

For analysis with the use of FACS was carried out by detaching cells MCF7 (ATCC no HTB-22) by incubation for 15 min with accurate (solution of the enzyme with proteolytic and collagenolytic activities; available for purchase from different suppliers). After washing with buffer for FACS (phosphate-saline buffer (PBS) containing 5% FCS) cells seeded in 96-well plates with holes having a circular bottom (cat. No. 3799, Corning Inc., Corning, new York, USA), with the final density of 3×105cells/ml and immediately used. Cells were incubated in the presence of, respectively, 1 μm, 5 μm and 10 μm FITC-labeled peptides in OptiMEM medium for 3 h at 37°C. then cells were washed in buffer for FACS and incubated in a buffer solution containing proteinase K (0.02 mg/ml), for 30 min. at 37°C. Cells were washed twice and analyzed on the instrument FACSCanto II (BD Biosciences; San-X�CE, CA, USA) using a channel for FITC.

1.7 Analysis of electrophoretic mobility shift in gel

Analysis of the electrophoretic mobility shift in gel was carried out by mixing 500 mg of duplex siRNAs with the relevant peptide in the specified molar ratio in water. Complexes were formed for 1 h at 37°C, and then analysed by 2.5% agarose gel with ethidium bromide (split: 40 min. at 125). For the treatment of proteinase K was added 1 μl of proteinase K in a standard reaction, as described above.

Example 2: Identification of potential peptides CPP man

2.1 Bioinformatic method

One group of peptides that penetrate into the cells (CPP), examples of which are derived from HIV peptide TAT (Frankel, A. D. and Pabo, C. O. (1988) Cell 55, 1189-1193; Vives; E. et al. (1997), supra) and the model peptide polyalanine (Wender, P. A. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 13003-13008; Futaki, S. et al. (2001) J. Biol. Chem. 276, 5836-5840), characterized by a high content of positively charged amino acids. Other peptides CPP, so-called peptides having a spiral structure with an asymmetric distribution of hydrophobic residues (tilted peptides), a mixture of charged hydrophilic areas and uncharged lipophilic sites of amino acids (Lins, L. et al. (2008) Biochim. Biophys. Acta 1778, 1537-1544). Thus, we can assume that the CPP peptides should possess both of these �features. To identify such sequences in the composition of the human proteome was used bioinformatic method. Fig.1 shows a diagram of the process used to obtain lists of candidate peptides.

To obtain the peptide library was applied the method of the sliding window. Was selected window size of 30 amino acids that applied in respect of all records contained in a database of proteins in SwissProt (Swiss Institute of Bioinformatics; http://www.expasy.org/sprot/). The result was a library of 10459557 individual peptides, of which 1024818 were unique. 163887 peptides were detected more than once, an average of 2.7 times, which reflects the presence in the structure of some proteins repetitive sequences. In the generated library each individual peptide sequence linked to the appropriate source of information (the sequence of the peptide, protein source and position in the structure of this protein), and with annotations in gene ontology (GO) (Ashburner, M. et al. (2000) Nat. Genet. 25, 25-39) about the original protein. Additional information associated with each peptide includes amino acid composition, for example, the amount of the charge, isoelectric point (IEP) and the predicted hydrophobic properties. The rationale for the use of the sliding window size of 30 amino acids was the need for more long� peptides than usually used as peptides CPP, as longer peptides are more likely to form secondary structure (e.g., alpha-helix or beta-layers). It has been shown that peptides containing such secondary structure, have advantages for penetration into the cells, compared to unstructured peptides (Deshayes, S. et al. (2004) Biochemistry 43, 7698-7706). In addition, it was reported that alpha-helical peptides with a length of less than 12 amino acids have a reduced ability to interact with nucleic acids and, thus, transfer them (Niidome, T. et al. (1999) Bioconjug. Chem. 10, 773-780).

In the next phase were filtered peptides containing in its primary amino acid sequence of more than 30% positively charged amino acid residues (i.e., arginine (R), histidine (H) and lysine (K)) for the purpose of obtaining peptides with a higher affinity to the positively charged model peptides such as TAT and poly-Arg. As a result of applying this filter was selected 227307 individual peptides. For the enrichment obtained sample peptides that have a higher potential for portability of the immune system (i.e. that are less likely are immunogenic), the results are compared with the data GO from the records for the respective proteins and limited the selection of only vneck�exact proteins. Ultimately the application of this filter (i.e., selections of candidates derived from extracellular proteins, which, thus, are "visible" to the immune system) have significantly reduced the number of peptides to 8630 from 583 individual proteins.

2.2 Selection of candidate peptides for experimental determination of the parameters

The main factor to reduce the number of candidate peptides was the transition from in silico methods (with which you can analyze a virtually unlimited number of samples) to the experimental analysis in the laboratory (where possible a thorough investigation of only a limited number of candidate molecules). In addition, another practical consideration in favor of reducing the list of candidate peptides was the ease of use, applicability to biological methods of analysis and, finally, the development of pharmacologically active compounds. On the other hand, from further evaluation were excluded peptide sequence, which apparently are poorly soluble or difficult to synthesize.

In order to avoid a situation where the choice of candidate molecules in the last step is defined by one or possibly two strict parameters, the final list of candidate peptides for experimental evaluation received three� different ways.

One subset of candidate peptides is characterized by containing not only at least 30% positively charged ("KHR30"), but also a large number of hydrophobic residues. Therefore, the peptides were ranked with respect to their IEP and their hydrophobicity. The intersection of both subsets of lists, each of which consisted of 1000 peptides, which has the highest score, gave a result of 91 peptide originating from 29 proteins. Finally, 20 were selected peptides derived from the 20 proteins, with the highest values in the composition of the intersection.

The second subset of candidate peptides received on the basis of similarity to the sequence of the TAT peptide. All peptides were subjected to comparison of the degree of homology with the use of the FASTA algorithm (Lipman, D. J. and Pearson, W. R. (1985), supra). Peptides were ranked according to their relative values of E (the lower the value of E, the higher the position in the list). As a result of applying this method took 135 peptides originating from 10 proteins. Finally, for further analysis chose the 5 peptides originating from 5 proteins.

As a third sub-group for experimental estimates have selected the 36 candidate peptides derived from 25 individual proteins based on the analysis of literature data about the function (or functions) of proteins from which they originate. Nesootvetstvie� between the number of peptides and proteins is due to the use of a set of sequences from one protein (e.g., CO9_mot1a, CO9_mot1b) or different variants of one sequence (for example, whether or not containing disulfide bridges, such as granulysin WT (wild type), granulysin G8, granulysin G9). In General maintained a length of 30 amino acids, but if it was found that such 30-Mer contains a well-known motif, the length of peptide is increased to 39 amino acid residues, as in the case of the peptide FALL (Yang, Y. X. et al. (2004) Acta Pharmacol. Sin. 25, 239-245), or up to 48 amino acid residues, as in the case LIP_Cons_C_WT, consensus sequences for a variety of lipases (Demant, T. et al. (1988) J. Lipid Res. 29, 1603-1611). The restriction was based on a careful analysis of literary data and the results of BLAST analysis obtained for these sequences. Gave preference: peptides derived from proteins which are known that they are harmful to the membrane, such as factors of the complement system; peptides originating from proteins that are involved in the degradation of lipids (such as lipase); peptides derived from proteins that are precursors of bactericidal factors, such as FALL (Nijink, A. and Hancock, R. E. (2009) Curr. Opin. Hematol. 16, 41-47) or BPIL3 (Mulero, J. J. et al. (2002) Immunogenetics 54, 293-300); and the peptides, similar to the peptides reported that they act as a CPP (Takeshima, K. et al. (2003) J. Biol. Chem. 278, 1310-1315; Arrighi, R. B. et al. (2008) Antimicrob. Agents Chemother. 52, 3414-3417).

All peptides (at the� the number of control), which were subjected to further experimental evaluation are listed in table 1.

To visually demonstrate initial filter plotted the dependence of EEP peptides from their hydrophobicity. When compared with 500 randomly selected peptides (from lots of about the size of 10×106) it is obvious that the initial filter allows you to select a region of the mass of the selected peptides (see Fig.2). For additional comparison, we also included five peptides as a positive control (i.e., TAT, REV, Protamine, poly-Arg and INF7). This analysis shows that the positively charged control peptides, TAT, REV, Protamine and 10×Arg matches the filter criteria, whereas INF7, which is negatively charged and hydrophobic peptide, detected at the other end of the scale.

In addition to the selected candidate of the CPP peptides as controls for screening were added eight peptides, for which it is reported that they have activity CPP (see table 1). These peptides can form non-covalent complexes with nucleotides and have the ability to transfection of TAT, the CPP peptide, derived from transactionhouse protein of HIV (Ignatovich, I. A. et al. (2003) J. Biol. Chem. 278, 42625-42636); REV, a related peptide of HIV TAT peptide (Futaki, S. et al., see above); poly-Arg (Kim, W. J. et al. (2006) Mol. Ther. 14, 343-350) as true �Riemer positively charged peptide; crotalin (Nascimento, F. D. et al. (2007) J. Biol. Chem. 282, 21349-21360). In addition, the truncated Protamine (Song, E. et al. (2005) Nat. Biotechnol. 23, 709-717) and N-terminal fragment of perforin added as examples of the peptides of the person. Two additional controls that are not related to the class of TAT-like peptides CPP, were Inf7 peptide, derived from the HA of influenza virus (negatively charged and hydrophobic peptide; Plank, C. et al. (1994) J. Biol. Chem. 269, 12918-12924) and MTS (mostly hydrophobic peptide; Lin, Y. Z. et al. (1995) J. Biol. Chem. 270, 14255-14258).

Example 3: Screening of peptides that mediate transfection siRNAs

To analyze the potential of the peptides of the RAF on the ability to carry out the transfection siRNAs in mammalian cells was used increasing concentrations of peptides (from 1 µm to 20 µm) at a constant concentration of siRNAs (100 nm). This range of concentrations of the peptides was used because it was shown that positively charged peptides, such as TAT, internalizers inside the cells in the concentration range between 10 µm and remain limited in intracellular membrane compartments without significant release into the cytosol (Duchardt, F. et al. (2007) Traffic 8, 848-866). It is shown that these peptides reach the cytosol only when the concentration is above the threshold of about 5-10 microns. The concentration of 10 nm siRNAs is in the range of saturating concentrations, according to the control experiments the use of transfection reagent DharmaFECT (Dharmacon, Inc., Lafayette, Colorado, USA; data not shown).

The used oligonucleotides siRNAs were designed against Aha1 mRNA (Panaretou, B. et al. (2002) Mol. Cell 10, 1307-1318) or against luciferase mRNA, serving as control.

Complexes siPHK and related peptides were formed in OptiMEM medium with incubation for 15 minutes at room temperature. Cells were incubated in the presence of complexes within three hours in OptiMEM medium. After washing, cells were further incubated for 21 h and 45 h in normal culture medium. Then, using branched DNA method determined the levels of Aha1 mRNA and a control mRNA (i.e. GAPDH mRNA). This analysis was performed on cells that were transfected siAHA1 or control siRNAs (siGL3, against luciferase), respectively.

To quantify specific RNC the lower levels of Aha1 mRNA relative to levels of GAPDH mRNA was compared in cells transfected siAHA1, and in cells transfected siGL3. Transfection mediated by specific siPHK, causes a decrease in the value of the ratio of the levels of Aha1/GAPDH mRNA in cells transfected siAhA1, compared with cells transfected siGL3. Fig.3 shows the results of screening resulting from such experiments: the values of the Aha1/GAPDH mRNA for non-transfected control cells were taken as 100% (�La each experiment) and the values of the Aha1/GAPDH mRNA in transfected siAha1 or siGL3 cells were expressed relative to these values. For this normalized data set was determined by the difference between the relative levels of Aha1/GAPDH mRNA in cells transfected siAha1 and siGL3. Specific peptides caused a significant decrease in transfection siAha1, while a decrease was not observed when transfection siGL3, which leads to positive values of the difference. The more pronounced these values, the more specific is the observed process RNC (see Fig.3).

In the selected samples the lower levels of Aha1 mRNA and/or GAPDH mRNA was observed in all ways transfection. This indicates a tendency to inhibition of growth and the provision of the toxic effects of the peptides used, regardless of the specificity of siRNAs. Peptides that disrupt the integrity of membranes (i.e., peptides CPP), are often the source of their inherent ability to damage cells, and this is confirmed by the fact that some of these peptides also act as bactericidal agents (Arrighi, R. B. et al. (2008), see above). Therefore, the cytotoxic activity of alleged CPP peptides of the person analyzed using analytical kits CytoTox-Glo and CellTiter-Glo (Promega Corporation, Madison, Wisconsin, USA). Mediated by peptide growth inhibition or cytotoxicity determined using analytical set CellTiterGlow, correlated well with the corresponding SN�put forward by the levels of GAPDH mRNA. Based on this observation, the levels of GAPDH mRNA was used as a General indicator of toxicity: the average value of the indicator equal to 100% of GAPDH mRNA relative to control with a nutrient medium, considered evidence of the absence of toxicity values from 99% to 70% - evidence of moderate toxicity and values less than 70% - evidence of toxicity (see Fig.3).

On the basis of the effectiveness of the peptides in mediating transfection and/or growth inhibition/cytotoxicity were allocated to different phenotypic categories: peptides that have demonstrated a lower Aha1-specific siRNAs transfection than the TAT peptide, have been classified as "atransferrinemia" peptides; peptides that showed equal or higher transfection efficiency relative to the TAT peptide, have been named "transferirase" peptides. Similarly, peptides, leading to the values of GAPDH mRNA>70% were classified as "non-toxic", whereas peptides that have caused a weaker decrease in the levels of GAPDH mRNA, were called "toxic".

Based on these categories all peptides were assigned to one of the following four classes:

(a) non-functional peptides (notransferase and non-toxic);

(b) notransferase toxic peptides;

(c) transfairusa toxic peptides;

(d) TRANS�alroumi non-toxic peptides.

3.1 non-Functional peptides

In this category fell from 41 to 61 of the last screening of peptide derived from human proteins (table 1 and Fig.3). These peptides do not show significant toxicity against cells and is not able to mediate transfection siRNAs at concentrations that were applied. Thus, 67% of the peptides that were selected as alleged CPP peptides during in silico method, did not demonstrate any phenotypic manifestations in experiments designed to study the transfection and viability. The group, consisting of 8 control peptides for which previously reported on the existence of the CPP activity, three peptide (crotalin, MTS and perforin) were in this category.

As an example, Fig.4 shows the phenotypic manifestation of the action of the peptide, derived from WNT16 protein that participates in WNT signaling pathway (Mazieres, J., et al. (2005) Oncogene 24, 5396-5400). The sequence of peptide WNT16 was included as a candidate peptide for experimental evaluation, because it fits the search parameters "KHR10 + extracellular localization."

3.2 Notransferase toxic peptides

Fell in this category 11 of 61 passed the screening peptide derived from human proteins that have demonstrated evidence of toxicity (reduction of life�ability and/or > 70% reduction in the level of GAPDH mRNA), but were not able to mediate transfection siRNAs in the concentrations applicable (see table 1 and Fig.3). Thus, their ability to carry out the transfection was lower than that of the TAT peptide. None of the analysed peptides, which are (positive) controls, did not get into this category, which suggests that the controls really have the ability to transfection. Fig.5 presents phenotypic expression for two examples of peptides belonging to this class.

One peptide derived from human protein BPLI3 (Fig.5A; Bingle, C. D. and Craven, C. J. (2004) Trends Immunol. 25, 53-55). In the analysis of CellTiter-Glo in cells exposed to this peptide was observed a dose-dependent decrease in viability. In parallel with this there were dose-dependent decrease in the levels of GAPDH mRNA, which is an evidence in favor of using the average values of the levels of GAPDH mRNA as an indicator of inhibiting the growth and/or toxicity. It is interesting to note that the analysis CytoTox-Glo showed no significant level of apoptosis in cells exposed to peptide BPIL3 (data not shown). Thus, this peptide causes inhibition of growth and/or cell death by a mechanism distinct from apoptosis. Transfectional activity (specific decrease in the levels of Aha1 mRNA relative to levels of GAPDH mRNA) this PE�Chida was observed.

Another peptide with cytotoxic properties (Fig.5B) derived from human protein cathelicidin (Nijink, A. and Hancock, R. E. (2009)/I. above). It was also reported that a peptide called FALL, has antimicrobial functional activity. The peptide showed a significant FALL in the level of toxicity when exposed to cells, which was reflected in the loss of cell viability and decrease in the levels of GAPDH mRNA. Analysis of potential transfectional activity allowed to identify not only allows you to make a final conclusion evidence specific decrease in levels of Aha1 mRNA (compared to GAPDH mRNA) at concentrations of 10 µm or above, in which the toxic properties explicitly. In these (toxic) conditions was observed only a slight decrease in mRNA levels. However, at lower concentrations (5 μm) can be observed a marked and specific decrease in the levels of Aha1 mRNA. This suggests that the peptide FALL, possibly capable of transfection siRNAs, although with a lower transfection efficiency than the TAT peptide, i.e., with efficiency below the threshold limit set for this class transfairusa peptides.

3.3 Transfairusa toxic peptides

This class of peptides does not include any of the control peptide, but contains 5 candidate PEP�species of human. The latter showed clear signs transfectional activity (i.e., specific Aha1 knockdown), and also caused the inhibition of cell growth and/or toxicity.

As an example in Fig.6 shows the phenotype of cells exposed to the peptide derived from protein CU025. CU025 is a protein containing the calcium-binding domain, the functional activity of which is not known (SwissProt, access number: Q9Y426). Experiments on transfection siRNAs showed a significant and specific decrease in the levels of Aha1 mRNA compared to levels of GAPDH mRNA. However, this peptide also caused inhibition of growth and/or toxicity, as shown by the analysis of viability and a significant decrease in the levels of GAPDH mRNA. This reduced viability was evident at concentrations of peptide required to achieve transfection and RNC. Thus, the applicability of this peptide and other members of this class of peptides for transfection with siRNAs significantly limited their toxic phenotypic manifestations.

3.4 Transfairusa non-toxic peptides

This class of peptides is of the greatest interest from the point of view of applications, including transfection siRNAs, as these peptides have transfectional functional activity at concentrations that do not render the negative influence�I on cell viability. Most of the control peptides fell into this class. Of the 61 candidate peptide of the person selected for experimental evaluation, three peptide showed clear evidence transfectional activity at concentrations that do not mediate the negative impact on cell viability or cause only minimal such exposure. These peptides were derived from CPXM2, not previously described carboxypeptidase (the access number in SwissProt: Q8N436), from ASM3B (the access number in SwissProt: Q92485), sour sphingomyelinase-like phosphodiesterase and GDNF-like neurotrophic factor neurturin (NRTN; Kotzbauer, P. T. et at. (1996) Nature 384, 467-470).

Fig.7 shows a comparison of mediated transfection of specific Aha1 knockdown and effects on cell viability peptides CPXM2 (Fig.7B), ASM3B (Fig.7C) and NRTN (Fig.7D) with the same indicators of peptides TAT and poly-Arg (Fig.7A). As CPXM2 and ASM3B caused a significant decrease in the levels of endogenous Aha1 mRNA in their joint application with siRNAs specific against Aha1, without causing substantial toxicity. This phenotypic expression were similar to those observed in the case of the control peptide TAT and poly-Arg (data not shown).

The NRTN peptide was operadonal even more significant decrease in the levels of Aha1 mRNA relative to levels of GAPDH mRNA, providing the e�Ohm only a minor impact on cell viability. The effect on cell growth/viability was observed only at high concentrations (>10 μm). Interestingly, NRTN peptide showed significant transfection activity even at lower, non-toxic, concentrations. At these concentrations the efficiency of transfection of peptides, derived from NRTN was higher than those of all analyzed peptides servants (positive) controls. Given this phenotypic expression, for NRTN peptide undertook a more detailed definition of the parameters.

Example 4: Formation of complexes of peptide/siRNAs is a necessary but not sufficient factor for mediating transfection

Mechanisms that may explain transfection functional activity of charged peptides, such as TAT or Protamine include the formation of complexes between positively charged peptides and the negatively charged nucleic acids. Such systems make possible functional activity by peptide-mediated interaction with membranes and/or the exit from endosomes required for transfer in the complex of nucleic acids into the cytosol of cells (Law, M. et al. (2008) Biotechnol. Prog. 24, 957-963). To determine whether the mechanism described above is applicable also to the case of a peptide derived from NTRN, analyzed �engine electrophoretic mobility in the gel (see Fig.8). To this end, siRNAs were incubated together with increasing peptide concentrations, and then analyzed their migration in the separation by gel electrophoresis. The TAT peptide showed the expected (positive control) concentration-dependent slowing of migration siRNAs (Fig.8A). Thus, the TAT peptide forms complexes with siRNAs molecules. In the case of TAT peptide, the shift in electrophoretic mobility in the gel was observed since the ratio of peptide: siRNAs equal to 10:1, with the most pronounced effect was observed at a ratio of 25:1 or higher.

Parallel to the analysis involving siRNAs subjected to a peptide derived from NTRN shown in Fig.8B. The slow migration of siRNAs was observed at a ratio of peptide : siRNAs. equal to 1:1 or higher. In addition, in contrast to the TAT peptide (which is always included in the gel), at a ratio of peptide NTRN : siRNAs equal to 25:1, were observed retention of the formed complexes in the pocket of the gel. At higher ratios, it was impossible to carry out the detection using ethidium bromide. These results correspond to the observation, which demonstrates that the complexes of nucleic acids with poly-peptides are less accessible to intercalating agents (Wolfert, M. A. and Seymour, L. W. (1996) Gene Ther. 3, 269-273). However, after treatment with protease K in the gel can�about to watch the corresponding signal from siRNAs (see Fig.8C). Thus, a peptide, derived from NRTN, capable of forming stable complexes with siRNAs, which, apparently, have a high degree of compaction.

These results showed that the formation of complexes of peptide/siRNAs associated with transfectional functional activity as the peptide derived from TAT and peptide originating from NTRN. Therefore, we can assume that the complex formation (i.e., formation of complexes) is necessary in order to apply the functional activity of the peptides on interaction with membranes and/or the exit from endosomes for transfection with siRNAs. However, you have to decide whether the observed complexation also sucient for the existence of transfectional functional activity? Accordingly, the ability to form complexes with siRNAs also analyzed for peptide originating from WNT16, which has been neither cytotoxicity nor transfectional functional activity (see above). Analysis of the electrophoretic mobility shift in gel siRNAs subjected to a peptide derived from WNT16, showed a clear dose-dependent slowing of the electrophoretic mobility of siRNAs, even stronger than was observed in the case of the TAT peptide (see Fig.8D). The effective electrophoretic mobility shift in gel �was nabludalsia when ratios of peptide : siRNAs, equal to 1:1 or higher. The effective supervision of complex formation notransferase peptide indicates that the complex formation between the peptide and siRNAs in itself is not sufficient to ensure transfectional functional activity. Thus, complexation with siRNAs, apparently, is a necessary prerequisite for the manifestation of functional activity, but the additional features of the sequence and/or structure of the peptides may also play an important role in mediating transfection.

The above data showed that a peptide derived from NTRN, effectively forms complexes with siRNAs molecules and mediates their transfection. Applied siRNAs was siAha1 which targets the mRNA product of a cellular gene "household", activator homolog 1 ATPase heat shock protein 90 (Panaretou, B. et al. (2002), supra). As control was used siRNAs directed against luciferase mRNA.

To confirm that the peptide NTRN in the General case is applicable for transfection with siRNAs (and its functionality is not limited to the sequence specific siRNAs), investigated its ability to transferout siRNAs than siAha1. Fig.9 shows the results of the mediated peptide NTRN transfection with siRNAs directed against the mRNA of the mitotic kinesin Eg5 (Blangy, A. et al. (1995) Cell 83, 119-1169). Effective reducing levels of cell Eg5 mRNA was detected at concentrations <10 μm. At such doses, the peptide NTRN has no negative impact on cell viability. It is known that the effective decrease in the levels of Eg5 mRNA causes a stop of mitosis, which results in early apoptosis (Blangy, A. et al. (1995), supra). Accordingly, when mediated by peptide NTRN transfection siEg5 observed apoptotic phenotype (Fig.9). Transfection of control siRNAs in the same conditions gave no cytotoxic effect, thus confirming that the apoptotic phenotype was caused by declining levels of Eg5 mRNA.

The above data demonstrate not only what peptide derived from human NRTN, in the General case is applicable for mediating transfection with siRNAs, but also that the efficiency of transfection is sufficient for the existence of cellular phenotypes caused siRNAs.

Example 5: Able to transfection peptides internalizers inside cells

To determine the ability to internalization of CPP peptides identified during the screening procedure conducted an analysis of FITC-labeled derivatives of the indicated peptides (at concentrations of 1 μm, 5 μm and 10 μm, respectively) using FACS. Used the following peptides: NRTN as transfairusa peptide, WNT16 as notransferase p�of ptid, FALL as toxic peptide and TAT as a standard of comparison (positive control) (see Fig.12). The MCF7 cells were incubated in the presence of fluorescently labeled derivatives of the peptides for 3 hours at 37°C in OptiMEM medium. After that, cells were treated with proteinase K for 30 min. at 37°C to remove associated with the surface of the peptides in order to ensure the study is only the internalized peptides. Then cells were washed in PBS and analyzed with the use of FACS.

The results show that the control peptide TAT internalized into cells MCF (see Fig.12A). The uptake of TAT peptide inside cells shows a linear dependence on the concentration of peptide that is also consistent with the observation, indicating that the TAT peptide functions as a reagent for the transfection of siRNAs (see above). Notransferase non-toxic peptide WNT16 showed no significant uptake into cells MCF7 (Fig.12B), which is consistent with the observed inability of this peptide to carry out the transfection siRNAs. It also demonstrates the lack of simple correlation between the absorption of the peptide and the presence of positively charged amino acids. Toxic peptide FALL was internalservice inside cells MCF, and the process showed a linear dependence on concentration (Fig.12C).

p> Capable of transfection NRTN peptide was also internalservice inside MCF7 cells (Fig.12D). However, unlike other peptides, NRTN did not show a linear dependence of absorption on concentration. In fact, when increasing the concentration from 5 μm to 10 μm showed a strong increase of internalization. These data suggest that there is a threshold value below which the absorption is a significant, but weak. Above this threshold there is a significant increase in the level of absorption by the cells.

Overall, these results demonstrate the ability with transfectional activity of the peptides to interact with cells and be internetizirovannyh in cells. In addition, these data show that the analyzed peptides not only act as reagents for transfection, but also act as peptides that penetrate into the cells.

Example 6: Sorting, filtering and classification of candidate peptides

By combining methods in silico and experimental screening in the human proteome were identified peptide sequence, potentially having the functional activity of the CPP or transfection agents. Found, that among these candidate molecules, three peptides were capable of transfection and were not toxic, i.e., they possessed transfect�approach in functional activity at doses which have no adverse effect on cell viability. These peptides can, among other things, to serve as modules for the development of an agent for the delivery of siRNAs, which is part of drugs on the basis of siRNAs, which will be developed in the future.

6.1 Bioinformatic method

Applied in silico methods were based on a library of 30-dimensional peptides that contained all overlapping peptides present in human proteins. Among these more than 10×106peptides were identified 8630 peptides, derived from 583 extracellular human proteins that match the initial search criteria (>30% positively charged amino acid residues (i.e., H+K+R) in the 30-dimensional peptide).

For the purpose of receiving the final list of peptides that were subjected to experimental laboratory studies, the number of candidate molecules has further restricted the application of negative and positive phases of the selection process: in cases where due to the presence of long main sections there were also a number of peptides located in the same protein, the multiplicity avoided. In most of these cases chose one representative peptide derived from this protein. Peptides that are apparently difficult to synthesize or are difficult to operate, e.g. because of the presence of multiple disulfide bridges or predicted poor solubility, excluded from further consideration. For the purpose of selection (from the remaining list of candidate molecules for experimental evaluation used several parameters of positive selection, including (i) a high value of EEP and a high degree of hydrophobicity; (ii) the similarity of the sequence of the TAT peptide; and (iii) the expected functional activity in relation to interaction with membranes, inherent to the proteins from which the peptides, such as proteins of the complement system, bactericidal factors and lipase.

6.2 experimental Method of evaluation

In experimental evaluation of candidate peptides (selected in the result of the method of screening in-silico) were divided into four classes: (a) non-functional peptides (i.e., notransferase and non-toxic), (b) notransferase toxic peptides, (c) transfairusa toxic peptides and (d) transfairusa non-toxic peptides.

The first method consisted in the fact that the candidate peptides were selected on the basis of their presence in the top positions in the list, ranked on the basis of values of EEP and profiles of hydrophobicity, for example, are able to transfection toxic peptides CU025 and CPXM, not capable of transfection toxic peptides CD026 and MMP25. Therefore, this filter allows you to identify putative peptides that penetrate into the cells (CPP), among many peptides are enriched in positively charged amino acids.

Another filter applied was based on the similarity of sequences of candidate peptides with a peptide comparison - TAT. The experimental evaluation examined five peptides with the most pronounced similarity with the TAT peptide. However, four of these peptides (including peptide PROK2, demonstrating the highest degree of similarity with TAT) showed apparently detected transfectional activity at doses that do not adversely affect cell viability. Peptide, derived from NRTN, was the only functionally active member of the group (i.e., was capable of transfection). This peptide showed the highest transfection functional activity in the course of the research, its efficiency was higher than even the TAT peptide. This suggests that transfection activity of these peptides is determined by not only the primary amino acid sequence, but also defined sequence motifs and, in particular secondary structures.

The third method to restrict the list of candidate peptides used data from the literature and the results of the BLAST analysis. Selected for experimental evaluation of peptides peptides were derived from proteins, which for the manifestation of their activity is interfaced as necessary�e with membranes. Most of the selected peptides did not demonstrate any functional phenotypic manifestations (i.e., ability to carry out the transfection). Even peptides derived from proteins, about which it is well known that they affect the integrity of the membranes (such as the factors of the complement system or perforin), showed transfectional functional activity. These results suggest that giving these peptides activity in violation of the integrity of membranes may be necessary properly structured domains (e.g., MACPF domain (Rosado, C. J. et al. (2008) Cell. Microbiol. 10, 1765-1774)). It is obvious that this functional activity may be reproduced peptides, even if they match the applied search strategy.

On the other hand, in this third group includes transfairusa nontoxic peptides, derived from CPXM2 and ASM3B, respectively, and toxic peptides originating from BPIL3 and FALL39. It is interesting to note that some of the peptides that are classified as toxic, was bactericidal peptides. Such peptides have a negative impact on the integrity of membranes of pathogens. In high concentrations these peptides are toxic to human cancer cells. At least for peptide FALL, it was shown that within a certain range of concentrations, it mediates transfection RNC. One explanation for this result may be the formation of perforations in the plasma membrane, through which may occur nonspecific uptake of siRNAs. Another explanation could be mediated peptide uptake of siRNAs, which is masked by the toxicity of the peptide. In addition, a recently proposed mechanism for DNA repair and membrane involved in uptake of peptides CPP (Palm-Apergi, C. et al. (2009) FASEB J. 23, 214-223), can also be used as a partial explanation of the existence of the functional activity of these peptides.

However, the fact that these peptides reduce cell viability in concentrations that are necessary for transfection, prevents the application of this class of peptides for the delivery of siRNAs.

Example 7: Determination of parameters of a peptide derived from NRTN

7.1 Structural features of a peptide derived from NRTN

Peptide, derived from neurturin (NRTN) was the candidate molecule identified in the present screening, which consistently showed the highest level of ability to carry out the transfection siRNAs at concentrations that do not adversely impact on the viability of the cells. This peptide is able to form non-covalent complexes with siRNAs, wherein the nucleic acid is a highly compactibility. This feature is consistent with the result�m, showed that siRNAs in complex with NRTN, not available for intercalation of ethidium bromide. As determined using the analysis of electrophoretic mobility shift in gel formation of complexes between NRTN and siRNAs was maximum at a ratio of 1:50. This corresponds to a ratio of 100 nm siRNAs to 5 μm of the peptide used in the test system functional activity in vitro (i.e., ability to carry out the transfection). However, if the concentration of NRTN in vitro is increased to the ratio exceeding the ratio at which we observe a saturation of the complexes, there is an additional increase transfectional activity. This result can be explained by the ability of free positively charged peptide NRTN to protect siRNAs complexes-NRTN from destruction anionic proteoglycans on the cell surface.

What may serve as a mechanism by which the NRTN peptide mediates transfection siRNAs? The formation of complexes with nucleic acids is undoubtedly one of the requirements to the functional activity of the peptide, as all capable of transfection peptides show such a feature. However, complexation by itself is not sufficient for mediating transfection, as also identified peptides which form complexes � siRNAs as good or even better than TAT peptide, but do not have transfectional functional activity. Furthermore, it is unlikely that the composition of the primary sequence, i.e., the number of people charged and/or hydrophobic residues, is the only factor mediating the functional activity. It turned out that many of the peptides having sequence similarity with the peptide TAT (including peptides with a very high degree of similarity of sequences), are non-functional.

One possible explanation of the presence transfectional functional activity of the peptide, derived from NRTN, can be detected in its secondary structure. The choice for the screening of the 30-dimensional peptide (unlike most other methods, which use a shorter peptides (Futaki, S. et al. (2001), supra; Crombez, L. et al. (2007) Biochem. Soc. Trans. 35, 44-46; Jafari, M and Chen, P. (2009) Curr. Top. Med. Chem. 9, 1088-1097)) has the advantage consisting in the fact that these peptides with a higher probability to fold into a secondary structure and support it. NRTN is a member of a family of protein growth factor TGF and has similarity to GDNF and artemina, the relevant structures have already been defined (Eigenbrot, C. and Gerber, N. (1997) Nat. Struct Biol. 4, 435-438; Wang, X. et al. (2006) Structure 14, 1083-1092).

The alignment of the sequence of rat GDNF and sequence human NRTN�and and comparison of estimated secondary structures allows us to see what with transfectional activity period of the NRTN peptide can form secondary structure (see Fig.10). The sequence corresponding functionally active peptide NRTN, partly located on an accessible surface of the protein and contains a positively-charged alpha-helical segment of amino acids. Identification of alpha-helical structures in NRTN is completely consistent with the existing hypothesis that alpha-helical structures are useful from the point of view of penetration into the membrane (Deshayes, S. et al. (2004), supra). Observation showed that the analyzed peptide, derived from NRTN, includes full alpha-helical structure, as well as the surrounding region, provides evidence in favor of the suitability of this method for screening of longer peptides. It remains unclear, is the presence of alpha-helix structure in itself (which is 12 amino acids in the composition of the 30-dimensional peptide) sufficient condition for mediating efficient transfection. However, it seems likely that at least some of the additional residues are also necessary for the existence of the functional activity of the peptide.

With the aim of obtaining experimental evidence for these predictions of the secondary structure has conducted additional analysis of the NRTN peptide in the presence of secondary elements�ow patterns with the use of spectroscopy circular dichroism in the UV range (UV-CD) (see review publication in: Whitmore, L. and Wallace, B. A. (2008) Biopolymers 89, 392-400). This technique allows the identification of the elements of a sequence folding into a secondary structure, based on their specific UV spectrum in comparison with unstructured segments having the form of a statistical coil. This analysis was performed using the spectropolarimeter Jasco J 715 (Jasco, Inc., Easton, Maryland, USA) in the range from 195 nm to 260 nm with a step of 0.1 nm and a bandwidth of 1 nm. Ditch the device had a length of 0.1 cm, the Peptides were used at a concentration of 0.1 mg/ml (see Fig.11).

The peptide FALL, for which it has been previously shown that it folds into an alpha-helical structure, was used as positive control (Agerberth, B. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 195-199). In aqueous solution the peptide FALL took the statistical conformation of a coil. In the presence of 10% triptoreline (TFE) as cerastoides on spectrum showed characteristic depression at 208 nm and 222 nm, respectively, which became even more pronounced when the concentration of TFE (i.e., 25% TFE and 50% TFE; see Fig.11A). It is known that TFE stabilizes and induces the formation of secondary structures in peptides and proteins (Buck, M. (1998) Q. Rev. Blophys. 31, 297-355).

In the analysis of the NRTN peptide in the same experimental conditions that were used for peptide FALL, there was a similar range, i.e., range with troughs at 208 nm and 222 nm, respectively. Consequently, the NRTN peptide actually contains alpha-helical region, which was predicted based on sequence homology (see Fig.11B).

In contrast, the spectrum obtained for the TAT peptide, showed no signs that this peptide folds into a secondary structure. Even in the presence of 50% TFE this peptide was accepted statistical conformation of the coil (see Fig.11C).

In addition, the ability to study the internalization using FACS analysis demonstrated that the NRTN peptide functions not only as a reagent for transfection, e.g., for molecules, siRNAs, but also as a peptide that penetrates into the cells, even in the absence of nucleic acid. These results indicate that the NRTN peptide could also be a suitable carrier for conjugated loads, such as other peptides or proteins. Interestingly, internalization of the NRTN peptide, apparently, does not have a linear relationship to the applied concentration. Most likely, there appeared to be a specific threshold value, above which is absorbed by the cells. This phenomenon of the existence of a threshold is also observed in the case of internalization of other peptides, such as TAT and p�Lee-Arg (Duchardt, F. et al. (2007), see above).

Moreover, FACS analysis revealed a strong accumulation of the peptide FALL in the cells. This result is consistent with the observation of the fact that FALL acts as a cytotoxic peptide. For the manifestation of toxicity requires direct physical interaction of the peptide with the target cell. However, unlike the NRTN peptide, the process of internalization of FALL characterized by a linear dependence on its concentration. Therefore, the toxic properties of the peptide FALL does not exist thresholds, which is consistent with the received data of cell viability, demonstrating the dependence of cytotoxicity on the concentration.

On the other hand, notransferase peptide WNT16 not demonstrated a significant degree of internalization into cells. Thus, only the presence of positively charged amino acids in the primary sequence of the peptides is not a sign of applicability of the peptide as a peptide of the RAF. These results provide additional evidence that the motives of a sequence folding into a secondary structure (as in the NRTN peptide) may be the main determinants for uptake by cells of peptides CPP.

7.2 Peptide, derived from NRTN has transfectional activity in the presence of serum

Therapeutic delivery of siRNAs is one of the promising applications identified CPP-like peptides of the person. Replacement is not owned by the person originating from pathogens, molecules belonging to the human sequence, showing similar or even higher functional activity, is an advantage for therapeutic methods, as it reduces the risk that used for transfection modules can be immunogenic. Therapeutic use of this peptide also requires that the specified peptide had a significant (transactional) activity. Additionally, the observed in vitro activity of the peptide must also occur in vivo.

Most of the methods of analysis described in this paper were carried out in standard conditions in vitro for the detection mediated by peptides transfection, which are well known to those skilled in the art (see, for example, Simeoni, F. et al. (2003) Nucleic Acids Res. 31, 2717-2724; Richard, J. P. et al. (2005) J. Biol. Chem. 280, 15300-15306; Abes, R. et al. (2007) Biochem. Soc. Trans. 35, 775-779; Kumar, P. et al. (2007) Nature 448, 39-43; Mueller, J. et al. (2008) Bioconjug. Chem. 19, 2363-2374; Sugita, T. et al. (2008) Br. J. Pharmacol. 153, 1143-1152). Accordingly, incubation as the initial "phase transfection" is generally carried out in the absence of serum. The addition of serum at this stage prevents able�ti peptides CPP to carry out the transfection (see Fig.13; see also the publication: Ignatovich, I. A. et al. (2003), supra). However, the possibility of therapeutic applications (CPP-like) peptides, obviously, requires coming in contact with serum. It is noteworthy that in the presence of medium (albeit with a low concentration of serum) NRTN peptide retains the ability to mediate transfection (see Fig.13).

7.3 Peptide, derived from NRTN, binds to epithelial cells and is internalized inside of these cells in a model cell culture blood-brain barrier

Earlier it was reported that peptides that penetrate into the cells, not only can be used as media with transfection with the purpose of delivering siRNAs into the cell, but they also, apparently, have functional activity in penetrating barriers such as the blood-brain barrier, for example, the purpose of the mediation process RNC in the brain (Mathupala, S. P. (2009) Expert Opin. Ther. Pat. 19, 137-140). We can assume that the NRTN peptide may also be functionally active in this respect, as NRTN is a neurotrophic factor secreted by glial cells (Sariola, H. and Saarma, M. (2003) J. Cell Sci. 116, 3855-3862), which potentially has a high capacity access to the Central nervous system.

To assess potential interactions of the peptide, derived from NRTN, endothelial cells, forming hematol�cephalic barrier, cells hCMEC/D3 or primary culture of endothelial cells in the human brain were subjected to peptide, derived from NRTN, in a model blood-brain barrier (BBB) (Weksler, V. V. et al. (2005) FASEB J. 19, 1872-1874; Poller, W., et al. (2008) J. Neurochem. 107. 1358-1363). The results of this analysis (see Fig.14) revealed that a peptide derived from NRTN, accumulates (te., internalized) in these experimental conditions in endosomal structures. In endothelial cells of the BBB transfer through the cell layer of hydrophilic molecules effectively prevented their close contacts, and endosomes are the main components of transcytotic mechanisms that allow for the controlled migration of macromolecules through the blood-brain barrier. Thus, a peptide, derived from NRTN, is located in the compartment, which plays an important role in the functioning of the BBB (i.e., in mediation and control of transport through the BBB).

Potential further expansion of the field of therapeutic application of peptides CPP may be associated with the combination with guiding molecules such as antibodies and fragments of antibodies.

Example 8: the Use of a peptide derived from NRTN, for intracellular delivery of Pro-apoptotic peptides

The ability to internalization peptides, derived from NRTN (see above), shows, Thu� these peptides function not only as reagents for transfection, for example, in respect of the siRNAs molecules, but also as a "classic" peptides that penetrate into the cells. These results indicate that the NRTN peptide could also be a suitable carrier for conjugated loads, such as other peptides or proteins.

To determine whether the sequence originating from NRTN, to mediate the uptake by cells of peptides, produced the conjugation of various biologically active peptides with NRTN. For applied as partners for conjugation of the peptides was shown the ability to interact with a cytoplasmic target proteins involved in mediating apoptosis. In other words, when expressed, or active delivery into the cytoplasm of cancer cells, these peptides induce apoptosis (i.e., they are "Pro-apoptotic"). However, these Pro-apoptotic peptides cannot penetrate biological membranes by themselves. Only the conjugation or coupling of known CPP peptides, such as TAT peptide, penetratin and poly-Arg, allows for absorption by the cells and thus induce apoptosis.

To assess the functional activity against the penetration into the cells inherent in the sequences originating from NRTN, used the following molecule partners for conjugation: (i) peptide, derived from the nuclear receptor Nur7, which interacts with the BCL2 protein and turns it into a Pro-apoptotic molecule; and (ii) a peptide derived from 4E-BP1, which interacts with the factor eIF4E translation that communicates with the structure 5'-cap of mRNA and composition, it has been found to modulate apoptosis in cancer cells.

8.1 NRTN-mediated uptake into cells Pro-apoptotic peptide, derived from NUR77

Nur77 is a nuclear receptor, an orphan, is able to interact with key intermediaries involved in apoptosis, such as BCLB proteins and BCL2. The interaction of Nur77 with BCL2 protein causes a conformational change in BCL2 protein, which lead to the exposure of the BH3 domain. This converts BCL-2 protein with Pro-apoptotic function (Lin, W. et al. (2004) Cell 116, 527-540; Luciano, F. et al. (2007) Blood 109, 3849-3855). The same transformation can be achieved with the use of peptides derived from related proteins, such as Nor1 (Kolluri, S. K. et al. (2008) Cancer Cell 14, 285-298).

The corresponding sequences interacting with BCL2, consisting Nor1 and Nur77 can be aligned as follows:

Nor1(SEQ ID NO: 70)

Nur77(SEQ ID NO: 71)

Even more short peptide capable of interacting with BCL-2 consists of 12 C-terminal amino acids of the protein Nor1: FSRSLHSLLVDL (SEQ ID NO: 72).

Despite its proven ability to convert BCL-2 into a Pro-apophaticism� molecule, the latest addition of the peptide (i.e., "Nor/Nur"; SEQ ID NO: 72) to cancer cells, even at high concentrations, is not sufficient for the induction of apoptosis as a cellular target protein located in the cytoplasm, but the peptide itself is not able to effectively penetrate through the cell membrane to reach the target molecule. Briefly, breast cancer cells MCF-7 were incubated in the presence of this peptide for 24 h. the Decrease in cell viability or induction of apoptosis was not observed (see description of analysis of cytotoxicity and viability in example 1). Similarly, the impact on these cells peptide CPP NRTN also did not affect cell viability or did not lead to induction of apoptosis (see Fig.15).

In order to analyze whether in fact the NRTN peptide functional activities of the CPP, was obtained hybrid sequence that comprises a segment consisting of a peptide, derived from Nur77, on the N-end attached to a part of the NRTN peptide located at the C-end. When determining a suitable position for connection, it was found that the C-terminal part of Nur77 peptide was similar to the segment of the sequence consisting of the NRTN peptide, and this region was chosen as the point of connection. Amino acid sequence of the resulting chimeric peptide NurNRTN and two and peptide�partner schematically depicted in the lower part of Fig.15. Chimeric peptide retains full sequence cut from Nur77 (practically unchanged) and a shorter N-terminal segment of the NRTN peptide. The resulting peptide has the same length (30 amino acids) as the original NRTN peptide.

NurNRTN has the following amino acid sequence:

(SEQ ID NO: 73).

To determine whether the chimeric peptide NurNRTN properties of binding to BCL2, inherent to the peptide, derived from Nur77, peptides corresponding NurNRTN and NRTN attached to iodoacetyl beads (Pierce Biotechnology, Inc., Rockford, Illinois, USA). The beads were incubated with recombinant BCL2 (Calbiochem/Merck, Darmstadt, Germany) and were washed with PBS and 0.5 M NaCl, 0,025 NaN3, 0,05% tween 20 to remove unbound protein. Specific binding of BCL2 with immobilized chimeric peptide NurNTRN determined elwira associated protein from the beads using elution buffer (pH 2,8), transferring-eluted fraction onto nitrocellulose membrane (Invitrogen Corporation, carlsbad, CA, USA) and specifying a protein with a set for reversible staining of proteins MemCode Reversible Protein Stain Kit (Pierce Biotechnology, Inc., Rockford, Illinois, USA). The results of this analysis showed that BCL2 protein is detected in the eluate from the beads, which were coated with a chimeric peptide NurNRTN. In contrast, with balls, cover�'s the NRTN peptide, was elyuirovaniya very small amount of protein BCL2. Therefore, the chimeric peptide NurNRTN retains the ability to bind to BCL2.

It was further evaluated whether the chimeric peptide NurNRTN to penetrate through cell membranes and thus induce Pro-apoptotic activity in cells. As the model used breast cancer cells MCF-7. The experimental approach was the same as described above (see example 1). At peptide concentrations that did not cause toxic effects in the testing of Nur77 peptides and NRTN, accordingly, a chimeric peptide showed a marked toxic effect, which is also manifested by the induction of apoptosis (see Fig.15). This effect was dose-dependent: the cytotoxicity increased with increasing peptide concentration.

As a control, produced the accession functionally inactive mutant form of the peptide NUR to the NRTN peptide. This peptide did not adversely affect cell viability. Additionally, the accession of the peptide to NUR functionally inactive peptide WNT16 also did not adversely affect cell viability.

These results demonstrate that molecules resulting from the connection of sequences originating from NRTN, with peptides, which themselves are not capable of penetrating into cells, can get EXT� cells and show intracellular activity. In other words, the corresponding NRTN portion of the chimeric peptide functions as a carrier necessary for the existence of the Nur77 peptide (i.e., load) Pro-apoptotic properties inside the cell. Additionally, these results prove the fact that the sequence originating from NRTN, which brings changes in its amino-terminal region retains the functional activity of the peptide CPP.

8.2 NRTN-mediated uptake into cells Pro-apoptotic peptide, derived from 4E-BP1

eIF4E is a factor broadcast that is associated with the structure 5'-cap of mRNA and composition plays an important role in ensuring the viability of the cells. Negative impact on functional activity of eIF4E can lead to apoptosis in cancer cells. Protein 4E-BP1 binds to and thus regulates/inhibits the functional activity of factor eIF4E. In this regard, increased levels of the protein 4E-BP1 leads to inhibition of translation, and thus to the induction of apoptosis in cancer cells due to the ongoing modulation of the functional activity of factor eIF4E (Flynn, A. and Proud, C. G. (1996) Cancer Surv. 27, 293-310; Robert, F. and Pelletier, J. (2009) Expert Opin. Ther. Targets 13, 1279-1293).

Pro-apoptotic functional activity of the full-sized 4E-BP1 can be achieved also by using short peptide length of 20 amino acids (Tomoo, K. et al. (2006) Biochm. J. 140, 237-246). This peptide contains the motif of binding to eI4FE (YXRXXLB, where X is any amino acid and B is a hydrophobic residue; Moerke, N. J. et al. (2007) Cell 128, 257-267). Further analysis showed that the three remaining amino acid residues in the composition of the binding motif (i.e., Y, R, L) are important for the existence of Pro-apoptotic functional activity (Marcotrigiano, J., et al. (1999) Mol. Cell 3, 707-716). Mutations at these residues (for example, replacement of the glycine residues) leads to the transformation of active peptides, derived from 4EBP1, inactive derivative.

The amino acid sequence of active and inactive 4EBP1 peptide used in the present invention, were as follows (for all sequences, see also Fig.16, top):

4E-BP1(SEQ ID NO: 74)
intact intact4E-BP1(SEQ ID NO: 75)

Despite the fact that has been proved the ability of 4E-BP1 to block eIF4E, the simple addition of peptides, derived from 4E-BP1, to cancer cells, even at high concentrations, is not sufficient for the induction of apoptosis as a cellular target protein located in the cytoplasm, but the peptide itself is not able to effectively penetrate through the cell membrane for DOS�izheniya target molecules. Briefly, breast cancer cells MCF-7 were incubated in the presence of this peptide for 24 h. the Decrease in cell viability or induction of apoptosis was not observed (see description of analysis of cytotoxicity and viability in example 1). Similarly, the impact on these cells peptide CPP NRTN also did not affect cell viability or did not lead to induction of apoptosis (see Fig.16, top).

It has been previously shown that the accession of known CPP peptides, such as TAT peptide, peptides, derived from 4EPB1, leads to the absorption of the chimeric peptide cells (Ko, S. Y. et al. (2009) Clin. Cancer Res. 15, 4336-4347). In this premarin used the following chimeric peptides TAT/eIFE4:

TAT4E-BP1(SEQ ID NO: 76)
TATinact4E-BP1(SEQ ID NO: 77)

Breast cancer cells MCF-7, which were incubated for 24 h in the presence of these chimeric peptides TAT/4E-BP1, showed clear evidence of a decline in cell viability and cytotoxicity as a result of induction of apoptosis (see description of analysis of cytotoxicity and viability in example 1) at a concentration of 20 μm. This effect is specifically mediated by functionally active (i.e., having a Pro-APO�toricheskim effect) the peptide sequence 4E-BP1, since the corresponding mutant was completely inactive (see Fig.16, middle part).

In order to analyze whether in fact the NRTN peptide functional activities of the CPP, was obtained hybrid sequence that includes a portion of the NRTN peptide at N-end, attached to the segment is active or inactive peptide, derived from 4EBP1, located at the C-end. The amino acid sequence obtained in the two chimeric peptides shown schematically in the lower part of Fig.16. Chimeric peptides retain a full-sized segment of the sequence of 4E-BP1, as well as full-sized NRTN peptide, which gives in total a length of 50 amino acids. Thus, these molecules are significantly longer than the known peptides CPP.

The amino acid sequence of active and inactive chimeric peptides NRTN/4E-BP1 were as follows:

NRTN4E-BP1 (SEQ ID NO: 78)

NRTNinact4E-BP1 (SEQ ID NO: 79)

It was further evaluated whether the chimeric peptide NRTN4E-BP1 to penetrate through cell membranes and thus induce Pro-apoptotic activity in cells. As the model used breast cancer cells MCF-7. The experimental approach was the same as described above (see example 1). At peptide concentrations that did not cause t�cichecki phenomena when testing peptides 4E-BP1 and NRTN, accordingly, a chimeric peptide showed a marked toxic effect that is also expressed in the induction of apoptosis (see Fig.16, middle part). This effect was dose-dependent: the cytotoxicity increased with increasing peptide concentration.

Additionally, a chimeric peptide containing NRTN, showed a significantly higher efficiency compared to its counterpart containing the TAT peptide. In addition, the observed cytotoxic effect is specifically mediated by functionally active (i.e., having a Pro-apoptotic effect) part of the peptide sequence 4E-BP1 as the corresponding mutant variant chimeric peptide was inactive (see Fig.16, middle part). Accession peptide 4E-BP1 to the peptide WNT16 did not adversely affect cell viability.

These results demonstrate that molecules resulting from the connection of sequences originating from NRTN, with peptides, which themselves are not capable of penetrating into cells, can get inside cells and to demonstrate intracellular activity. A direct comparison with chimeric peptides containing the TAT peptide, revealed that the chimeric peptides containing the NRTN peptide, have a higher efficiency. These results also testify to the fact that the sequence originating from NRTN, �Otera brings changes in its carboxy-terminal region, retains the functional activity of the peptide CPP. Finally, using sequences derived from NRTN, you can get a functionally active peptides CPP, which have a length of at least 50 amino acids.

The present invention, illustrative described in this document can be applied in practice in the absence of any element or elements, limitation or limitations, not specifically disclosed in this document. Thus, for example, the terms "containing", "comprising", etc. should be understood in the interpretation and expansion without restrictions. Additionally used in this document, the terms and expressions were used as terms and descriptions, and do not impose restrictions, as well as in the use of such terms and expressions do not have the intention to exclude any of the features, equivalent demonstrated and described features, or parts thereof, and it is recognized that within the scope of the claimed invention, various modifications are possible. Thus, it should be understood that although the present invention has been specifically disclosed with the aid of embodiments and possible features, the person skilled in the art may resort to modifications and variations of the above embodiments of the invention, and it is implied that such modifications and variations I�come in the scope of the present invention.

In this document, the invention has been described broadly and in General terms. Each of the narrower species and subgroups falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with positive and negative signs, which cuts off any object from objects, though, was whether to exclude material specifically listed in this document or not.

Other embodiments are indicated in the following claims. In addition, in cases where properties or features of the invention are described in terms of Markush groups, the person skilled in the art will be understood that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

TABLE 1
Candidate peptides CPP humans and control peptides subjected to experimental evaluation.
Control peptides are highlighted in grey. In the column "Class" is specified functional classification of peptides with respect to their ability to carry out the transfection, as well as their cytotoxicity: "-" - notransferase non-toxic peptides; "tox" - notransferase current�quarter peptides; "+/tox" - transfairusa toxic peptides; "+" - transfairusa non-toxic peptides.
SEQ ID NO:The TITLE PEPTIDEAMINO acid SEQUENCECLASS
1TAT+
2NRTN+
3CPXM2+
4ASM3B+
5FGF12+/tox
6CU025+/tox
7IGS10+/tox
CPXM+/tox
9CD026+/tox
10FALL39, var. 1+/tox
11Poly-Arg+
12INF7+
13REV+
14Trunc_protamine (truncated Protamine)+
15Crotalin-
16MTS-
17Perforin_NT (_NT)-

34
18BPIL3 (38 and.to.)tox
19BPIL3 (30 a.to.)tox
20Defensin-Cons (CI,III,V,VI-S)tox
21LIP_Con_Ntox
22ApoL_cons_1tox
23ApoL_cons_2tox
24FAM5Ctox
25 FAM5Btox
26COOA1tox
27MMP25tox
28NETRtox
29SCUB3tox
30Defensin-Cons (CII,III,IV,VI-S)-
31Granulysin, wild-type-
32_G9-
33CO6_mot2-
CO6_mot1-
35CO8_mot1-
36CO9_mot1a-
37C09_mot1b-
38Perforin_SC (_SC)-
39Originating from futoran-
40LIP_Cons_C_WT-
41LIP_Cons_C_AA-
42ApoL_cons_3 -

43CRSPL-
44ATS7-
45AREG-
46FA20A-
47GNAS3-
48PAP2-
49HISTATIN_1-
50HISTATIN_3-
51NPTX3 -
52PROL4-
53YC002-
54CD029-
55TOR2-
56CO4AB-
57SULF1-
58PROK2-
59WNT16-
60GRAK -
61APLD1-
62CBPN-
63CFAI-
64FGF5-
65LTB1L-
66LFTY1-
67LOXL3-
68PONL-
69PRS23-

<> 1. Composition for delivering nucleic acid molecules into cells containing at least one peptide attached to one or more molecules of nucleic acids, the peptide is able to internalservice inside mammalian cells, where the peptide is:
(a) has an amino acid sequence selected from the group consisting of:
and
amino acid sequence having over its entire length, at least 92% sequence identity with respect to any of the sequences with SEQ ID NO: 2 through SEQ ID NO: 4; and
(b) internalized into the cells of a mammal with efficiency component of at least 200% of the efficiency of internalization of the TAT peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1); and
in this case, the joining is carried out by binding selected from the group consisting of covalent binding and noncovalent binding.

2. A composition according to claim 1, where at least a portion of at least one peptide forms an alpha-helical secondary structure.

3. A composition according to claim 1, wherein the at least one peptide is a mammalian peptide, preferably of human origin.

4. A method of obtaining a composition according to any one of claims. 1-3, containing:
(a) creating at least one service� peptide, defined in claim 1, capable of internalservice inside the cells of a mammal; and
(b) implementation of contact of at least one peptide with one or more nucleic acid molecules in order to ensure the implementation of the merger.

5. The in vitro method of identifying the ability to internalize the composition according to any one of claims. 1-3, including:
(a) introducing the composition according to any one of claims. 1-3 in one or more cells of a mammal; and
(b) detecting the internalization of the peptide or composition.

6. Pharmaceutical composition for delivery of nucleic acid molecules inside one or more cells of the mammal containing the composition according to any one of claims. 1-3 in an effective amount, and optionally further comprising one or more pharmaceutically acceptable excipients and/or additives.

7. Use of the composition according to any one of claims. 1-3 for in vitro transformation or transfection of one or more cells of a mammal.

8. A composition according to any one of claims. 1-3 for use for the prevention or treatment of a condition selected from the group consisting of cancer, immune diseases, cardiovascular diseases, neurological diseases, infections and inflammatory diseases.



 

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FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to a method for analysing the applicability of RNA extracted from a tissue or a cell (cells) fixed by a fixative for analysing gene expression. The method involves performing electrophoresis with the above RNA. The method implies stating, if the above RNA complies with the following equation: B/A≤1, wherein A represents the mass ratio (%) of RNA falling within the range from 1,000 to 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis, while B represents the mass ratio (%) of RNA falling within the range from more than 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis. If the above RNA extracted from the tissue or cell (cells) complies with the above equation, it is considered to be applicable for analysing gene expression.

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FIELD: chemistry.

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FIELD: biotechnology.

SUBSTANCE: characterised method comprises carrying out of PCR with use of specific primers to genes vc0497, vc0502 and vc0514 from the island of pandemicity VSP-II. The characterised test system comprises the components for isolation of DNA, the components for carrying out PCR, including, in particular, a primer mix VSPIIreg-F - 5'-TGGAAAGAAGAGCGTTACTGC-3', VSPIIreg-R - 5'-CCCTGTTGATGATGTGATTTG-3' to the gene vc0497, VSPIIpilin-F - 5'-CTGTGATTCGGGCTTTATCGG-3', VSPIIpilin-R - 5'-GCGTAAACTGAGCCAATAAGC-3' to the gene vc0502, VSPIIchem-F - 5'-CTTGATGGAGCGGAGAAAAC-3', VSPIIchem-R - 5'-CGATGAATAGCCTGTTGAAC-3' to the gene vc0514, taken in the ratio 1:1:1:1:1:1, respectively.

EFFECT: inventions enable to differentiate quickly and reliably the toxigenic genetically modified strains to genovariants with low and high epidemic potential.

2 cl, 1 dwg, 2 tbl

FIELD: biotechnology.

SUBSTANCE: method comprises isolation of DNA from lymphocytes in peripheral blood by the method of phenol-chloroform extraction, carrying out of PCR, amplification of 18 parts of gene MYO7A, detection in the denaturing acrylamide gel and sequencing. PCR is carried out using specially selected sequences of oligonucleotides flanking regions of 18 exons of the gene MYO7A with possible content of different mutations.

EFFECT: invention enables to simplify the method and to improve the accuracy of determining mutations of the gene MYO7A, to reduce the time of the study.

3 dwg, 1 ex

FIELD: biotechnology.

SUBSTANCE: proposed primers comprise endonuclease cleavage sites, flanking genomic regions encoding the glycoproteins Gn and Gc, and the nucleoprotein N, for obtaining libraries of genes encoding glycoproteins Gn and Gc and N nucleoprotein of Rift Valley fever virus.

EFFECT: invention can be used in creating a bank of nucleotide sequences encoding immunodominant proteins of Rift Valley fever virus Gn, Gc and N, which can be used for creation of diagnostic and vaccine preparations based on recombinant technologies.

3 ex

FIELD: biotechnology.

SUBSTANCE: characterised oligonucleotide primers are complementary to a specific region of the mig-gene of Mycobacterium avium and have the following base composition: 5'-CGT CAA AAG CGA ACT GCA-3' and 5'-TAA TTC GTT GCC CGA CTC-3'. The method of detecting DNA of Mycobacterium avium comprises DNA isolation, DNA amplification using oligonucleotide primers, transfer of amplification product on the gel followed by detection of the analysis results on the transilluminator. In case of positive reaction a fragment is synthesised, corresponding to the size of 157 bps.

EFFECT: inventions can be used in veterinary diagnostic, scientific and practical laboratories for detection of genetic material of Mycobacterium avium in samples.

2 cl, 1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: set is used to recognise mutations of a coding part of NKX2.5, CFC1, GATA4 genes associated with an orphan single-gene pathology underlying familiar congenital heart disease. The mutations are recognised by identifying a nucleotide sequence of the coding part of NKX2.5, CFC1, GATA4 genes. The coding part of NKX2.5, CFC1, GATA4 genes is amplified by means of 15 synthetic base pairs at the same temperature and annealing time; that is followed by sequencing the amplification products by means of one pair of universal primers.

EFFECT: invention enables recognising the mutations of the above genes sensitively and specifically, reducing the amplification reaction time, the number of manipulations, the agent addition time for the sequencing reaction and decreasing a probability of the reaction error.

3 cl, 1 dwg, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: pair of primers has the following structure - PF: 5'-TGGAGAAGGGAATTTCAGATGGG-3' and PR: 5'-TTTGGGTCAAGTGCTACATATTGTGG-3'. The method includes performing of PCR with detection of obtained results by a horizontal electrophoresis method in 1.5% agarose gel. Amplification is performed in the following mode: total denaturation at 95°C for 5 min., denaturation cycle at 95°C for 20 or 60 sec., annealing at 56°C for 20 or 60 sec., elongation at 72°C for 40 or 60 sec. The cycle denaturation - annealing - elongation is repeated 35 times. The final elongation is performed at 72°C for 5 min. In case of existence of an amplificated fragment of nucleotide sequence with the length 366 p.n. the presence of FIV in a cat organism is obvious.

EFFECT: invention allows to perform diagnostics of a virus immunodeficiency of cats with high efficiency.

2 cl, 6 dwg, 3 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to fabrication of oligonucleotide primers and fluorescence-tagged probe for identification of inverse transcriptase polymerase chain reaction in real time.

EFFECT: higher efficiency of identification.

2 dwg, 2 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: method provides DNA extraction, conducting of PCR with the use of synthesized primers for amplification of fragments of intergenic segments wzyE-dapF and YPDF_0064-YPDSF_0065 of the studied strain. Differentiation is conducted by comparison of sizes of produced amplicons of the studied strain with the sizes of similar fragments, typical for strains of the basic (151 p. n. and 374 p. n.), Caucasian (185 p. n. and 434 p. n.), Altai and Hissar (202 p. n. and 374 p. n.), and also Ulegey (185 p. n. and 374 p. n.) subspecies.

EFFECT: invention allows to conduct effectively subspecies differentiation of strains and considerably to reduce time of the analysis execution.

4 ex

FIELD: biotechnology.

SUBSTANCE: method comprises immunoaffinity chromatography using mini-antibodies a-hLF-1 and a-hLF-4, which amino acid sequences are presented as SEQ ID NO:1 and SEQ ID NO:2. The invention may be used to obtain highly purified fraction of human lactoferrin.

EFFECT: invention enables to carry out with high efficiency the separation of proteins of lactoferrin of human and goat consisting in milk, using the single-domain mini-antibodies.

6 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: claimed inventions deal with a modified protein, nucleic acid, coding such protein, a vector, containing nucleic acid, and a carrier for biotin binding, which such protein is immobilised on. The characterised modified biotin-binding protein is obtained by the introduction of a mutation from one to several amino acid residues into a sequence, represented in SEQ ID NO:2, or an amino acid sequence, identical to the said sequence by 98% or more, and the presence of the biotin-binding activity, where at least one residue, selected from the group, consisting of residues from 1) to 4), presented below, is substituted with the residue of acidic amino acid or residue of neutral amino acid; 1) residue of arginine in position 104 SEQ ID NO: 2; 2) residue of lysine in position 141 SEQ ID NO: 2; 3) residue of lysine in position 26 SEQ ID NO: 2 and 4) residue of lysine in position 73 SEQ ID NO: 2.

EFFECT: claimed inventions make it possible to obtain the biotin-binding protein and can be applied for biotin binding.

14 cl, 6 dwg, 11 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.

9 cl, 5 dwg, 1 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to versions of a method for microbiological synthesis of hybrid protein E7-HSP70. Synthesis of protein E7(6)-HSP70, E7(11)-HSP70, E7(16)-HSP70 or E7(18)-HSP70 is carried out by cultivating the respective yeast strain Saccharomyces cerevisiae VKPM Y-3919, yeast strain Saccharomyces cerevisiae VKPM Y-3853, yeast strain Saccharomyces cerevisiae VKPM Y-4057 or yeast strain Saccharomyces cerevisiae VKPM Y-4058 in suitable conditions in a medium containing saccharose as a carbon source. The cultivation process is carried out in two phases. The first phase is carried out at 25-26°C and initial saccharose concentration of 25-30 g/l. Upon achieving saccharose concentration in the medium of 15 mg/l, the second phase of the cultivation process is carried out, where temperature is lowered and kept not higher than 23°C, pH is kept at 5.7-5.9, and saccharose concentration is maintained via exponential replenishment at 15 mg/l.

EFFECT: group of inventions enables to obtain the end product in amount of not less than 550 mg/l and enables to carry out the cultivation process for not more than 65 hours.

4 cl, 12 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a constructed plasmid for expression in a cell of a Chinese hamster, in the following sequence, which mainly contains the following elements: pUC plasmid replication beginning region; an open reading frame (ORF) of beta-lactamase providing immunity to ampicillin; procaryotic gene promoter bla; a section of terminal repetition of Epstein-Barr virus of a human being; a functional gene promoter of elongation factor 1 alpha of the Chinese hamster, 5' non-translated region of this gene and a non-transcribed region flanking this gene, coding Kozak sequence for cap-dependent initiation of translation; ORF of the gene of subunit 1 of complex of 2,3-epoxyreductase of vitamin K (VKORC1) of the Chinese hamster with stop codon; a functional terminator and a signal of polyadenilation of the gene of elongation factor 1 alpha of the Chinese hamster, 3' non-translated region of this gene and a non-transcribed region flanking this gene; a promoter of early genes of virus SV40; gene of immunity to a selective agent; and a polyadenilation signal and terminator of virus SV40. Cells of the Chinese hamster - producer of protein with Gla-domain are transformed by the obtained plasmid, which are used in a method of recombinant obtainment of proteins with Gla-domain.

EFFECT: invention allows increasing productivity of the above cells owing to increasing activity of native VKORC1.

12 cl, 12 dwg, 2 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely to novel analogues of insulin and can be used in medicine. The said insulin analogue is characterised by one of the following structures: Arg(A0), His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin; His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin; Arg(A0), Glu(A15), His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin. The invention also relates to a method of obtaining the said analogue, a pharmaceutical composition and a medication, including its application for treatment of diabetes mellitus.

EFFECT: invention makes it possible to obtain the insulin analogue, characterized by the delayed release and steady and longer action, for instance, in comparison with human insulin or glargine insulin.

18 cl, 2 dwg, 24 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.

16 cl, 19 dwg, 3 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to modified von Willebrand factor (VWF), and can be used in medicine. A recombinant method is used to preparing modified VWF fused in C-terminal of its primary translation product with N-terminal of albumin by the linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS. The prepared modified VWF is used as a part of the pharmaceutical composition for treating or preventing coagulation failure.

EFFECT: invention enables preparing the modified VWF which maintains its ability to N-terminal dimerisation and C-terminal multimerisation with a prolonged half-period of functional blood plasma occurrence as compared to the half-period of functional VWF occurrence.

17 cl, 5 dwg, 4 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biochemistry. Disclosed is L-fucose α1→6 specific lectin, which is extracted from a basidiomycete or an ascomycete or an ascomycete, characterised by peak molecular weight of about 4500 m/z, determined via MALDI-TOF mass spectrometry analysis. The novel L-fucose α1→6 specific lectin has high affinity for a L-fucose α1→6 sugar chain, represented by an association constant of 1.0×104 M-1 or higher (at 25°C), and has an association constant of 1.0×103 M-1 or lower (at 25°C) with high-mannose sugar chains and/or glucolipids which do not contain an L-fucose α1→6 sugar chain. In one version, the disclosed L-fucose α1→6 specific lectin is a protein or a peptide which consists of an amino acid sequence selected from SEQ ID NO:2-6. The L-fucose α1→6 specific lectin is used for specific detection of a L-fucose α1→6 sugar chain and effective purification of the L-fucose α1→6 sugar chain or a sugar chain which does not contain L-fucose α1→6.

EFFECT: obtaining L-fucose α1→6 specific lectin.

16 cl, 38 dwg, 8 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: invention offers recombinant plasmid DNA coding a chimeric antibody against human tumour necrosis factor-alpha (TNF-alpha) based on pOptiVECTM-TOPO® plasmid. Invention refers to eukaryotic cell line as a producer of antibody to TNF-alpha, method of cell line obtainment by transfection of plasmid DNA according to the invention, and method of chimeric antibody obtainment for TNF-alpha by cultivation of cell line according to the invention.

EFFECT: increased synthesis level for antibodies against TNF-alpha by producer cells.

12 cl, 8 dwg, 1 tbl, 3 ex

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