Molecular conjugates with polycationic section and ligand for delivery of dna and rna into cell and cell nucleus
SUBSTANCE: claimed invention relates to biotechnology and represents molecular conjugates, capable of binding with nucleic acids (DNA or RNA) for their delivery into cells of mammals, expressing transferring receptors. The said molecular conjugates consist of a polycationic sequence, represented by a modified signal of nuclear localisation of the virus SV40 T-antigen, and a ligand. One of two sequences HAIYPRH or THRPPMWSPVWP is used as ligands of cell receptors. Complexes of the molecular conjugate and nucleic acid are used for obtaining medications for genetic therapy or diagnostics of various diseases. To obtain the said complexes solutions of nucleic acid and the molecular conjugate are poured together with a ratio of charges in the reaction medium from 1:0.63 and lower and used for complex formation of solutions with ionic power less than 300 mM.
EFFECT: claimed invention makes it possible to increase the efficiency of delivering genetic constructions into cells.
12 cl, 8 dwg, 1 tbl, 1 ex
The technical field to which the invention relates.
The invention relates to the field of medicine, pharmacology, biotechnology, genetic engineering, gene therapy, namely, the delivery of the cells DNA and RNA with therapeutic, diagnostic and research purposes.
The level of technology
In the development of the technologies of gene therapy weak point remains the effective delivery of genetic constructs based on DNA and/or RNA into the cell.
Currently developing a number of approaches aimed at improving the efficiency of delivery of genetic constructs into cells, and enabling their targeted delivery only in certain cells and tissues.
Methods of gene delivery into mammalian cells
Cell membrane is a reliable barrier to the penetration of alien nucleic acid is RNA or DNA, of course, if nucleic acids are not found in viral particles or not captured faguoqitirute cells. In some cases, still manages to make the membrane temporarily permeable to DNA and RNA, changing the physical state of membrane fluidity, integrity). The possibility of implementation of such changes depends on a number of factors: metabolic activity of cells, energy substances, ability to phagocytosis, the stage of the LM the cycle of cells.
If the polynucleotide sequences are not able to penetrate into cells through the plasma membrane, a relatively low molecular weight oligodeoxynucleotide (ODN), may constitute an exception. The mechanism of penetration of ONE in mammalian cells is not fully understood, although suggest that ONE absorbed by the cells by a combination of adsorptive endocytosis and pinocytosis, which is partly activated by binding ONE with receptorgamma proteins to the cell surface of a wide range of tissues (Gewirtz, A. M. et al., 1996). A large part of trapped in a cage ONE eventually ends up in endosome/lysosomal vesicles and there is hydrolysed by the enzymes.
Overall, a great role in the development of the methods of gene therapy plays a developing means and methods of transfer "treating" genes (comprising the expression vectors) in the body, which should ensure high efficiency of the delivery of genetic constructs, their electoral getting into certain cells and tissues, stable operation of transgenes in cells with minimal side effects: toxic, inflammatory, immunogenic and possibly mutagenic and carcinogenic (Culver, K. W., 1994). Furthermore, it should be taken into account such factors as the efficiency of the creation and use of delivery vehicles.
Methods of delivering genes into somatic and chaff the mammalian cells, depending on the conditions of treatment of the cells can be classified as methods of transfer in vitro, ex vivo and in vivo (K. W. Culver, 1994). In the first case, in vitro gene delivery is carried out in cultured cells or tissues of mammals, mostly for experimental purposes, when target effects are normal, mutant or abnormal cells. In the second case (ex vivo) the transfer is carried out in a short-term in vitro cells taken from the body, and after confirmation of delivery "treating" of a gene, the cells are returned to the body of an experimental animal, donor or sick person to achieve a therapeutic effect. In the third case (in vivo} directed gene transfer for the same purpose are conducted directly into the cells or tissue (target) of a living organism.
Another classification of the most common means of delivery of expression vectors into cells based on the method you prefer:
1. Virus (recombinant adenoviruses, retroviruses, adeno-associated viruses, the viruses of herpes group and ospowiki).
2. Non-viral biological (liposomes, cationic lipids; the polycation; viral membrane, molecular conjugates; microspheres).
3. Physico-chemical, physical (calcium phosphate transformation of cells in vitro, electroporation, ballistic procedure, microcapillary injection).
Gene transfer using recombinant viruses
To migrate in vivo can use is to recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. Shipping "treating" genes in the composition of recombinant retroviruses is chronologically one of the first successful experiments in human gene therapy. Projects are being developed treatment by which retroviral particles injected in vivo with delivery directly to specific tissues and organs (represented proliferating cells). In addition, in some cases, the body of the patient inoculant producing retrovirus "packing" of the cell, for example, intracranial (stereotaxis) in the case of malignant brain tumors. The disadvantages of retroviral gene transfer should be attributed occur at random insertions in the chromosome of provirus danger off the expression of vital genes or activation of oncogenes (Temin H. M., 1990). In addition, no clear reasons, retroviral vectors in gene expression over time are inactivated, although there continues to be integrated with chromosomal DNA.
The most effective in the delivery of "treating" genes in the mammalian organism a recombinant adenovirus, infectious titer of this virus can be up to 1010PFU/ml; in addition, adenoviruses possess a wide trapnest to various tissues, and when injected into cells and their genetic material with "attending" the Yong moves into the cell nucleus, where is saved from two weeks to several months (Rosenfeld, M. A. et al., 1991). "Treating" gene replaces in the viral genome region early genes of adenovirus, resulting in such virus in the cells lose the ability to reproduce and later destroyed. The disadvantages of gene transfer as part of adenoviruses are certain toxicity defective viral particles, capable of producing some early virusspecific proteins in the body, and, in addition, their high immunogenicity, which makes repeated courses of treatment. To reduce toxicity and immunogenicity develop a series of adenoviral vectors of the new generation, already deprived of a whole series of viral genes (K. J. Fisher et al., 1996), which avoids synthesis virusspecific proteins after delivery "treating" of a gene in the organism. In this case, the development of immune reactions delayed and occurs only with repeated injections of adenovirus. Viral particles are injected into the body intravenously by injection or in the form of an aerosol in the respiratory tract for the treatment of hereditary defects of the respiratory epithelium. In particular, for the treatment of patients with cystic fibrosis (a genetic defect of the CFTR gene in the respiratory tract of a patient in an aerosol injected recombinant adenovirus containing the expression vector of the CFTR gene (J. Zabner et al., 1993; Harvey C. G. et al., 1999; S. Lerondel et al., 2001; J. Daies C., 2002; U. Griesenbach et al., 2002; Gaden F. et al., 2004). Projects are being developed for the transfer of "treating" genes in the composition of the recombinant viruses of other species: adeno-associated viruses (S. Nomura et al., 2004; Tietge, U. J. et al., 2004; K. Oka et al., 2001; Kozarsky, K. F. et al., 1996; Kobayashi K. et al., 1996; Kozarsky, K. F. et al., 1994; K. Kozarsky et al., 1993; Flotte T. R., 2004), herpes simplex viruses (Coffin, R. S. et al., 1996a; Weir J. P. et al., 1996; Coffin, R. S. et al., 1996b; Wang, M. et al., 1998; Wolfe D. et al., 2004; Niranjan A. et al., 2004; Goss J. R. et al., 2004; B. Cao et al., 2004; Goins, W. F. et al., 2004), vaccinia virus (Guo Z. S. et al., 2004; Di N. M. et al., 2003; Walther W. et al., 2000).
Adeno-associated virus to deliver genes (small size) in one of the chromosomes (19) person. The advantages of such delivery include the relative ease of subsequent removal of viral vector of the cellular DNA by co-infection of target cells by virus-helper. The constraint vectors prepared on the basis adenoassociated viruses, is the relatively low capacity of their capsid for polynucleotides about 5, etc., ad, making it impossible to transfer the large size of cellular genes. Recently it was reported that the development of the hybrid vector, in which the replicative form of recombinant adeno-associated virus packaged in the capsid of adenovirus. The capacity of this vector is significantly higher when saving a site-specific integrative inherent ability of adenoassociated is authorized viruses (M. Goncalves A. et al., 2004).
Recombinant herpes viruses is recommended for delivery "treating" genes into the cells of the nervous tissue, the nuclei of which herpes virus genome capable of long-term persist in a latent state (in the form of an episome) (Flotte T. R., 2004; Coffin, R. S. et al., 1996; Weir J. P. et al., 1996; Coffin, R. S. et al., 1996; Wang, M. et al., 1998; P. Gregorevic et al., 2004; Gruchala, M. et al., 2004). Recombinant vaccine viruses (genome which is localized in the cytoplasm) is used for treatments of molecular vaccination organism by expression of foreign genes (W. Walther et al., 2000; Di N. M. et al., 2003; Guo Z. S. et al., 2004).
Non-viral gene transfer systems
Naked plasmid DNA is rapidly degraded under the action of nucleases plasma, which is a serious obstacle when conducting transpency (Hashida M. et al., 1996; Crook K. et al., 1996). This problem is partially solved by the condensation of DNA positively infected molecules that protect DNA from nucleases (S. Sandgren et al., 2004; C. M. Wiethoff and Middaugh, C. R., 2003; Luo D. and Saltzman, W. M., 2000). He less important task for non-viral systems - facilitating the overcoming of the plasma membrane. The main route of penetration into the cell DNA using non-viral delivery systems is endocytosis. In most cases, the penetration is due to clathrin - and caveolin is the essential component-independent endocytosis, and clathrin-independent and adsorptive endocytosis (Takei K. et al., 2001; Nichols, B. J. et al., 2001). In some types of cells infiltration is implemented by the mechanism of macropinocytosis or phagocytosis for relatively large particles (Apodaca G., 2001) (Fig.2-11).
First non-viral delivery vehicles have been applied for more than 20 years ago. The system of delivery of genetic vectors must perform several functions: to communicate with the delivered genetic material, DNA packaging and providing not only penetration through the membrane, but also the protection of DNA before getting into the cage, and immediately after transfection, providing access from lysosomal compartments, stored in the cytoplasm and translocation into the cell nucleus. Overcoming not only the plasma membrane but also to prevent lysosomal degradation of the introduced DNA is a very complex task.
It is known that physiological concentrations of salts and proteins in the serum significantly affect physico-chemical properties of non-viral carriers and often lead to a reduction or complete loss of their properties, which currently restricts the use of such systems in vivo (M. Molas et al., 2003; focused on I. et al., 2003). Currently, active research on the development of non-viral delivery vehicles, effective in vivo.
The first successful transfection using cationic lipids (lipoplexes) was conducted in 1987 (Feigner, P. L. et al. 1987). Since then, this method has found wide application not only in research but also in clinical protocols (Nabel G. J. et al., 1990). Its use is a relatively safe and prostin method for local delivery in low doses (Li S. et al., 2000).
A major component of the membranes of liposomes are phospholipids. In addition, to stabilize the particles in the extracellular environment in composition is administered 40-50 mol. % cholesterol. Trapnest liposomes to cells of different types depends on the charge, which can be changed by varying the phospholipid composition and various additives (Ropert S. 1999).
The interaction of DNA with cationic liposomes is a spontaneous process, new initiates DNA-mediated fusion of liposomes and large-scale lipid rearrangeable. However, in the formation of DNA-liposomal complex does not occur condensation of DNA (S. Li et al., 2000, N. J. Zuidam and Barenholz Y., 1998).
DNA-liposomal complex is relatively easy to penetrate into the cells through electrostatic adsorption of the positively charged membrane of liposomes with plasma membranes eukaryotically cells or involving endocytosis (Li S, et al., 2000; N. J. Zuidam et al., 1998; Smith R. M. and Wu G. Y. 1999). In some cases a significant role in the penetration of DNA complexes with cationic lipids in neparazituje cultured mammalian cells plays clathrin-mediated endocytosis (Zuhom I. S. et al., 2002). Molecules on the cell surface, which is responsible for interaction with lipoplexes, is not yet fully understood, but it is known that an important role in this process is played by proteoglycans on the cell surface (Scherman D. et al., 1998, M. Singh,1999).
The possibility of using cationic lipids does not depend on the proliferative activity of the cells, allowing lipoplex can be used to transform non-proliferating or slowly proliferating cells (N. J. Zuidam et al., 1998). The disadvantage of this method is degradation of a significant part of foreign DNA under the action of lysosomal hydrolases, as well as toxicity when used in high doses (P. Erbacher et al., 1996). Exemption from endosomal compartments in most cases is achieved by rupture of endosome by replacing part of the lipid components between the membranes of the liposomes and endosome (Hee Y. and Szoka F. C., 1996). In accordance with this model anionic lipids endosomal vesicles interact with cationic lipids, liposomes on "flip-flop" mechanism, providing a partial fusion of the membranes and the release of DNA. This in good agreement with data about the increase in transfection efficiency when added to the complexes of cationic lipid-DNA neutral DOPE lipids, facilitating the fusion of membranes (Hui S. et al., 1996; H. Farhood et al., 1995).
Molecular conjugates represent a polycation, in most cases, covalently linked with ligands for cellular receptors (I. Fajac et al., 2000). In comparison with the cationic lipid molecular conjugates more effectively condense the shape of the DNA into a compact structure, that increases the stability of the complexes and, in some cases, promotes nuclear localization of transferred genetic constructs (Rudolph S. et al., 2000). Currently created and actively used several different types of molecular conjugates, on the basis of a polycation such as poly-L-lysine, polyethylenimine and others (G. P. Nolan et al., 1998; Kost, T. A., 1999; I. Fajac et al., 2000; Rudolph C. et al., 2000).
The polycation can easily associate with DNA in solution due to electrostatic interactions of the positively charged amino groups and negatively charged phosphate groups of DNA. The size of the formed complex is largely dependent ligand (Rudolph S. et al., 2000). Generally, a structure is formed with a diameter of less than 80-100 nm (I. Fajac et al., 2000), which allows the use of this complex for the delivery of DNA into different types of cells, because most of them have the size of the primary endosomal of compartmental 100-120 nm. (Cristano R. J. et al., 1995). Complex DNA-conjugate selectively binds to cellular receptors with ligand site conjugate and transferred into the cell receptor-mediated endocytosis (Smith R. M., 1999; Gottschalk, S. et al., 1996, Zeiphati 0. et al., 1998). As ligands to cell surface receptors often use molecules asialoglycoprotein, insulin, transferrin, epidermal growth factor and other proteins, and t is the train of low molecular weight compounds (glycosidic residues and others) (Yanagihara K. et al., 2000).
The main limitation encountered when using poly carriers is fast inactivation and disappearance from the bloodstream when the system introduction, due to two main reasons - aggregation and the absorbance of the serum protein in vivo (Dash, P. R. et al., 1999; M. Ogris et al., 1999).
In the formation of complex electrostatic interaction between the DNA molecule and poly-media lead to the formation of a particle with a neutral charge, which allows the particles easily aggregate in aqueous solutions. To reduce aggregation of the complexes and their inactivation adding to the culture medium or systemic administration in the body, using the following approaches: "steric stabilization", adding molecules of the polymer, in particular polyethylene glycol (PEG), keeps from sticking together, which in most cases is attached ligands, providing specific interaction with cellular receptors. Another approach to increase the stability of complexes - "electrostatic stabilization". In this case, the formed complexes have a positive or negative charge that prevents sticking due to electrostatic repulsion (Molas M. et al., 2003). Such systems become more stable in the presence of salt solutions and whey proteins Ahn, S. N. et al., 2002; D. Oupicky et al., 2002). Similar systems are used not only for poly carriers (Ahn et al., 2002; Harada-Shiba et al., 2002; Oupicky et al., 2002), but also for systems based on cationic lipids (Kamps et al., 2000; Ng et al., 2000; Carrion et al., 2001; Bendas et al., 2003).
Complex DNA-conjugate, with no molecular signals getting into the cage, there is in it a short time (24-48 h) and destroyed in the lysosomes (A. Subramanian et al., 1999, Cristano R. J. et al., 1995). In this regard, many conjugates often contain molecular signals destabilization endosome membranes (such as amphipatic peptides of viral origin, is able with decreasing pH to destabilize endosome and to facilitate the release of DNA complexes, the conjugate in the cytoplasm) or (and) signals nuclear translocation, which promotes the penetration of foreign DNA into the nucleus and preserve it in the form of Epsom a long time (Colin M. et al., 2000, K. Yanagihara et al., 2000).
Some molecular conjugates can be used without additional endocarditisjaneway components. It is shown that lizosomaiona degradation of DNA complexes with conjugates synthesized on the basis of polyethylenimine, significantly lower than when used as the polycation poly-L-lysine. Apparently, this fact is connected with the buffer properties of polyethylenimine (Li, S., Huang L. 2000,Nguyen H-K., et al., 2000). The exact mechanism of ensuring the release of DNA from endosome compartments, not well researched, but according to some authors, based on buffering the contents of lysosomes by aminogroup polyethylenimine, and on the action of aminogroup as proton acceptors (the effect of "proton sponge") (Kichler et al., 2001). It remains unclear, does the inhibition of maturation of endosomes direct damage to their membranes and the release of the complex from late endosomes, or increases the probability of their content by delaying maturation of endosomes, though, may be implemented in practice both of these hypotheses (Molas, M. et al., 2003).
There are a large number of works, which tested various strategies to increase the efficiency of the release of complexes of endosome compartments, as by inhibiting degradation, and by introducing specific domains providing destabilization of vesicles (for example, some pore-forming peptides) (Yessine M-A. and Leroux, J.-C., 2004.). For example, the use of chloroquine with various complexes of DNA/polycation, including poly-Ü-lysine and polyethyleneimine, provides increased pH and, consequently, inhibits hydrolytic activity litany enzymes (Gotten M. et al., 1990; I. Fajac et al., 2000; M. A. Zanta et al., 1997).
Despite a considerable increase in efficiency is barb transfection, the use of agents that damage endosome substantially disrupts the cells by inhibiting the recycling of receptors on the plasma membrane, and providing a strong toxic effect (Molas, M. et al., 2003).
The disadvantages of existing molecular conjugates synthesized mainly on the basis of poly-L-lysine or polyethyleneimine, should include their toxicity, which limits the use of the complexes in high concentrations (Gottschalk S. et al., 1996), as well as the relatively low efficiency of delivery of exogenous DNA.
A very promising application for the synthesis of molecular conjugates of low molecular weight ligands and cationic carriers. Compared to belkovymi molecules, the use of low molecular weight compounds can improve the ratio of ligand / DNA, as well as significantly reduce the size of the formed complex. The latter is extremely important, because compact complexes (with a diameter of 10-30 nm) is able to penetrate into the core, which contributes to prolonged and effective expression of the gene of interest (P. Erbacher et al., 1996).
Thus, of all the currently existing non-viral methods of gene delivery into mammalian organism, only molecular conjugates potentially meet one of the important criteria for the design of systems migration g is new in tumor cells of human to ensure targeted delivery of DNA into target cells.
Ligands transferring receptor
Non-viral gene therapy healing of superficial injuries mammals involves the creation of molecular conjugates capable of efficiently deliver genes mitogenic growth factors in tissue cells immediately surrounding the injury, especially in skin fibroblasts. The successful candidate for the role of receptor binding complexes of DNA/conjugate, obespechivayuschego their internalization by cells, is the transferrin receptor.
The transferrin receptor of the person being studied as a model of receptor-mediated endocytosis (Thorstensen K. and Romslo I., 1993; Ponka P. and C. N. Lok, 1999), as a marker of cell proliferation (Ponka P. and Lok C. N., 1999; Inoue T. et al., 1993) and as a target for delivery into cells of various pharmacological agents (A. R. Jones and Shusta, E. V., 2007). He is a dimer of two identical transmembrane glycoproteins with a molecular weight of 95 KD, covalently linked through intermolecular disulfide bonds. Most of the iron entering the cell, absorbed with the participation of the transferrin receptor. Ligand for transferrin receptor is transferrin - extracellular transport protein with a molecular mass of 80 KD, able to effectively connect the four ion Fe3+(Ponka P. and Lok C. N., 1999). About what Bennett transferrin is the change in conformation when interacting with ions of iron, what is believed to be critical for interaction with the receptor (Ponka P. and Lok C. N., 1999). The expression of transferrin receptor occurs in all cells and body tissues, but the expression level in different tissues varies (Davies M. et al., 1981; Enns, S. A., 1982). Maximum expression was observed in actively proliferating cells (10000-100000 molecules per cell), while non-dividing cells contain only a few molecules of receptors (Inoue T. et al., 1993).
Widespread use of conjugates on the basis of transferrin for delivery into cells of various pharmacological agents. In particular, in the literature there are reports of successful delivery in the tumor cells of the cytotoxic agent doxorubicin covalently linked with transferrin (Yeh, S. J. and G. Faulk, W. P., 1984; K. Barabas et al., 1992; Sizensky J. A. et al., 1992; Berczi, A. et al., 1993a; Berczi, A. et al., 1993b), delivery into cells fused proteins, representing transferrin, coupled with therapeutic polypeptides (an S. A. et al., 1999a; All S. A. et al., 1999b), gene transfer using molecular conjugates of transferrin-polylysine in a variety of cultured mammalian cells (E. Wagner et al., 1991; Strydom, S. et al., 1993; Taxman, D. J. et al., 1993; Schwarzenberger P. et al., 2001). In order transferrin gene therapy is not only used in the composition of molecular conjugates with polylysine, but in combination with cationic lipids (Penacho . et al., 2008), in the composition of the nanoparticles based on cationic lipids and other (Abela R. A. et al., 2008). A feature of all the above-listed systems of gene transfer is used as a natural ligand transferrin. This approach has significant drawbacks. Firstly, the resulting complex DNA/conjugate has a large size (>100 nm), which makes the release of DNA from endosomes and transfer to the nucleus (see above). Secondly, the affinity of the interaction with transferrin receptor is highly dependent on transferrin saturation with iron, making it difficult to obtain tranfection active complexes conjugate-DNA. However, in the literature there is a message about the detection method of phage display peptide ligands vysokaye to interact with the transferrin receptor and to induce its internalization (Lee J. H. et al., 2001).
In recent years there have been many publications about the properties of the peptide HAIYPRH and THRPPMWSPVWP. These peptides have been found in several laboratories, using phage library heptapeptides. Shown the presence of these peptides of various properties, including the ability to internalize into the cell as a ligand transferrin receptor.
The possibility of linking with the muscles of a number of peptides, including HAIYPRH (US Patent 6,329,501 - Methods and compositions for targeting compounds to muscle 2001)
In a series of patents and publications group of authors demonstrated the use of peptide HAIYPRH and THRPPMWSPVWP for delivery into cells of various agents.
In the patent (US Patent 6743893 - Receptor-mediated uptake ofpeptides that bind the human transfemn receptor) describes the properties of these two peptides, HAIYPRH and THRPPMWSPVWP, but only patented THRPPMWSPVWP. He is regarded as a ligand transferring receptor, by providing the internalization of delivery into the cell of different agents, such as antigens, chemotherapeutic substances, different labels (fluorescent or without coloring). However, in the formula of this patent is missing the point of delivery into the cell's DNA or RNA, not coding sequence THRPPMWSPVWP for the purposes of gene therapy.
In another patent (US 2008166293 - RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN RECEPTOR TRANSFERRED, 2008-07-10) shows that there are two peptide HAIYPRH and THRPPMWSPVWP), able to communicate and internalservice using transferring receptor human (hTfR). When these peptides are fused with other molecules, such a fused product is absorbed by cells expressing hTfR. As such molecules that can be delivered into the cell, are considered peptides, proteins, antigens, chemotherapeutic agents, antitumor agents, rendered or radioisotope labels, liposomes, including those containing secondary agents. The Pat is the authors indicate the peptide HAIYPRH does not prevent the binding of transferrin to its receptor, and postulate the binding of this peptide in a different site of the receptor than transferrin, which indirectly testifies to the fact that the delivery of different agents in the cell with the help of this peptide may not be mediated by the transferrin receptor. These data cast doubt on previous patents data authors, which is brewed in exactly the receptor-mediated uptake of peptide HAIYPRH and related agents. In addition, in the formula of this patent is missing the point of delivery into the cell DNA or RNA for the purposes of gene therapy.
In one of the patents (SA 2449412, COLLAWAN J., MOORE C., LEE J., JEFFREY. RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR 2002-06-06) it was shown that the peptide HAIYPRH and THRPPMWSPVWP to deliver merged with molecules in cells expressing the transferrin receptor (hTfR) by the internalization of contacting the receptor peptide. Patented this peptide, fused with Lubim other protein or peptide with a chemotherapeutic agent, fluorescent or other type of label, an antigen, and the nucleotide sequence encoding this peptide, including merged with other peptides. This patent describes the use of this peptide, fused with peptide nuclear localization, not shown for the efficiency of the use of the peptide for gene therapy.
In another patent (USPTO Patent Application 20080166293, Receptor-mediated uptake of peptides that bind the human transferrin receptor) proposed to use the peptide HAIYPRH and THRPPMWSPVWP for delivery into the cell peptides, proteins, chemotherapeutic agents, antitumor agents, rendered markers and isotopes. You are not offered the use of this peptide, fused with peptide nuclear localization, not the use of the peptide for gene therapy.
It is proposed to use the peptide HAIYPRH and THRPPMWSPVWP for delivery into the cell peptides, proteins, chemotherapeutic agents, fluorescent markers (WO/2002/044329, RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR). He offered the use of this peptide, fused with peptide nuclear localization, not the use of the peptide for gene therapy.
Feature of the patent (US Patent Application 20060193778 - Receptor-mediated uptake of peptides that bind the human transferrin receptor) is that postulated delivery using peptide HAIYPRH DNA molecule that encodes a peptide HAIYPRH.
The possibility of using these peptides THRPPMWSPVWP HAIYPRH and in a number of designs for the delivery of nucleic acid molecules through cell membranes and the blood-brain barrier (US Patent Application 20050222009 - Dual phase - PNA conjugates for the delivery of PNA through the blood brain barrier, patent WO/2005/035550). However, in this patent discusses only the complexes on the basis of valentich links, including using linker molecules. The nucleic acid molecules that are included in these complexes is limited by the size of 100 nucleotides, peptide HAIYPRH and THRPPMWSPVWP are considered as specific ligands for the receptor. As the nucleic acid is treated DNA, cDNA, RNA, etc. as poly half of the examined peptides, however, the sequence of the receptor-binding peptide and poly-peptide in this patent are separated spatially by molecules of another type (nucleic acids, hydrophobic molecules). I.e., is not considered as peptide delivery of a single peptide molecule consisting of the sequence HAIYPRH and poly-peptide. In addition, the present invention is not applied peptide nuclear localization for delivery structures in the cell nucleus.
In all of these patents does not provide conditions for the formation of complexes of DNA-peptide for the purposes of gene therapy.
Similar characteristics and another patent of this group of authors (US Patent Application 20080038349 - Peptide-Pna Chimera Targeting Inducible Nitric Oxide Synthetase).
The authors of another patent (US Patent Application 20070231300 - COVALENT CONJUGATES BETWEEN ENDOPEROXIDES AND TRANSFERRIN AND LACTOFERRIN RECEPTOR-BINDING AGENTS) suggest the use of a peptide sequence HAIYPRH and THRPPMWSPVWP for delivery into cells of hydroperoxides.
In all these paten the Ah does not provide conditions for the formation of complexes of DNA-peptide for the purposes of gene therapy.
Disclosure of inventions
The above disadvantages of viral vectors has led to an increased importance of developing non-viral systems transfer into cells nucleic acids (NC) - DNA and RNA. As such system non-viral transfer of DNA and RNA in the cell we have developed molecular conjugates, representing peptides, consisting of two parts.
One part is a ligand for a cellular receptor and presents one of the two peptide sequences SEQ ID NO 1 and SEQ ID NO 2. Sequence data provide binding to cellular receptors with subsequent internalization. According to the available data binding occurs with the transferrin receptor, although, perhaps, not only with him. The advantage of using cellular transferrin receptor is its high expression in proliferating cells, which can reach 10000-100000 molecules on the cell, which largely determines the efficiency of delivery into the cell gene therapy constructs related to the ligand of this receptor. However, the use for the purposes of gene therapy as a natural ligand transferrin has significant drawbacks described above. Used the same us peptide sequence found by the method of phage display peptide ligands vysokaye usaimage is consistent with the transferrin receptor and to induce its internalization. Moreover, recent evidence suggests that these peptides do not inhibit the binding of transferrin to its receptor, which indicates linking them to another part of the receptor, distinct from the binding site with transferrin (US 2008166293, 2008). This indirectly also may testify in favor of the delivery of various agents in the cell with the help of this peptide may not be mediated not only by the transferrin receptor.
The other part we have developed molecular conjugates - poly. It presents a peptide sequence modified signal nuclear localization of T-antigen of SV40 virus (SEQ ID NO 3).
The polycation associate interact with DNA in solution due to electrostatic relations positively charged amino groups and negatively charged phosphate groups of DNA. Usually formed structure with a diameter of less than 80-100 nm (I. Fajac et al., 2000), which allows the use of this complex for delivery of nucleic acids into various types of cells, because most of them have the size of the primary endosomal of compartmental 100-120 nm. (Cristano R. J. et al., 1995).
Thus, poly-of the developed molecular conjugates provides linking them with nucleinate acids (DNA or RNA) and transportation of the latter in the cell nucleus. Ligand part is DL the receptor-mediated internalization of the complex, thus its penetration into the cell.
The implementation of the invention
In this work, we used reagents for peptide synthesis, and derivatives of amino acids (Sigma Chemical Co., Fisher Scientific, Bachem, USA; Reanal, Hungary; Saxon Biochemicals GMBH, Germany), 4-(2',4'-acid-Fmoc-aminomethyl)-phenoxyacetamide-narratologically polymer (Gl Biochem, China). Dimethylformamide before using distilled under vacuum and stored over 4 Å molecular sieves. The identity of amino acid derivatives was monitored by TLC on plates Merck F 254 (Germany) in the following solvent system: 1% ammonia-sec-butyl alcohol, 1:3 (A); chloroform-methanol, 9:2 (B); chloroform-methanol-acetic acid, 90:7:3 (In). Visualization of the chromatograms was performed by UV irradiation at 254 nm and the method of charring in sulphuric acid.
Solid phase synthesis of peptides was carried out on semi-automatic synthesizer NPS-4000 (Neosystem Laboratoires, France).
Analysis of the resulting peptides by HPLC was performed using a chromatograph System Gold (Beckman, USA) on columns Bond 300SB-C18 (4.6×150 mm, 5 μm) analytical chromatography and Discovery C18 (10×250 mm, 5 μm) for preparative. The chromatography conditions: UV detection at 230 nm, gradient of acetonitrile in 0.1% H3PO4at a flow rate of 1 ml/min for analytical and gradient of acetonitrile in 0.1% TFA at a flow rate of 5 ml/min for the drug who ate chromatography. Amino acid analysis was performed on the analyzer "Microtechna" T339M (Czechoslovakia) after hydrolysis of peptides 6 N. HCl at 110°C for 24 h Mass spectra were recorded on a time-of-flight mass reflectron MX-5303 with the ion source of the type "Electrospray".
Used in the work of the peptides was synthesized on 4-(2',4'-acid-Fmoc-aminomethyl)-phenoxyacetamide-narratologically polymer containing amino groups 0.5 mmol/g To protect the side of Ser, Thr and Tyr used Butgroup for Arg - Pbf, for Lys and Trp - VOS for His Trt. As a temporary Nαsecurity was Fmoc-group.
The extension of the peptide chain was carried out on semi-automatic peptide synthesizer NPS-4000 (Neosystem Laboratoires, France) according to the following Protocol for each cycle: 1). CH2Cl2(1 min); 2). DMF (1 min); 3). 20% piperidine/DMF (2 and 8 min); 4). DMF (3×1 min); 5). Isopropyl alcohol (3×1 min); 6). DMF (3×1 min); 7). The condensation reaction (2 h); 8). DMF (2 x 1 min); 9). CH2Cl2(1 min).
For condensation used a four-fold excess of activated 1-oxibendazole esters of the corresponding Fmoc-amino acids obtained in situ (1 EQ. Fmoc-amino acid, DIC and HOBt, stirring in DMF at 0°C, 30 min). Upon completion of the reaction carried out the test on the completeness of the acylation. If necessary, re-condensation used the following Protocol:
a). DMF (1 min); b). 5% DIEA/DMF (1 and 2 min) after stage 6) - 9) was repeated.
In the case of partial completion of the condensation reaction when it is repeated 1-2 times, unreacted groups azetilirovanie (after washing CH2Cl2to peptidyl-polymer was added 10 EQ. acetic anhydride, 10 EQ. 10% DIEA/CH2Cl2and was stirred for 10 min).
Cleavage of the synthesized peptide from the polymer with the simultaneous release was performed by the action of a mixture of TIS: N2A: TFA (2.5: 2.5: 95; 5 ml/0.5 g resin) for 2 h the Reaction mixture was diluted with cold ether and the precipitate was filtered on a filter SCHOTT. The peptide was separated from the polymer dissolved in TFA and re-precipitated with ether. The precipitated product was filtered, washed with ether, and dried in vacuum. Further purification was performed using reverse-phase HPLC.
Amino acid composition and molecular weight, calculated from the data of mass spectrometry for all synthesized peptides corresponded to theoretical values.
Plasmid pCMVluc, which was a vector for gene expression-token-luc (encodes the Firefly luciferase) under the control of the promoter of the early genes of human cytomegalovirus, constructed by means of genetic engineering (Ignatovich I. A. and others, 2002; focused on I. A. et al., 2003).
The formation of DNA complexes with molecular conjugates was performed in phosphate-buffered saline (PBS) at various with the respect of the charges of DNA and peptide in the reaction medium according to (Ignatovich I. A. and others, 2002). Identifying dependencies stability of DNA complexes/conjugate from the ionic strength of the reaction mixture is carried out by introducing into the reaction required amount of NaCl. The formation of complexes of DNA/conjugate was controlled by gel-retardation in agarose gel. In experiments to determine the sensitivity of DNA in the complexes to DNase I ("Fermentas", Lithuania) after completion of complex formation in the reaction medium (30 μl) was added MgCl2to a concentration of 3 mm and 3 units Gnkazy I (1 unit Gnkazy I 1 µg DNA) and incubation was performed at 37°C for 30 minutes. Analysis of the treated Dnazol complexes was performed using gel retardation. The study of the stoichiometry of the complexes of DNA/conjugate was performed as described previously (focused on I. A. et al., 2003). Complexes of DNA conjugates were prepared in saturating the excess of the peptide with the subsequent removal of unbound peptide by ultrafiltration with the use of centrifugation on the cones Method YM-10 ("Millipore"). The amount of peptide that is part of the complex was determined by back titration of heparin.
The HepG2 cells and VH10 drugs pCMVluc complexes/NLS-TSF7 and pCMVluc/NLS-TSF12 performed as described previously (Dige E. B. and others, 2006). Cells were sown with a density of 10 cells/cm Petri dishes (diameter 30 mm) and was raised to the status of 60-70% of the monolayer in DMEM with addition of 10% fetal serum is Elat ("Bilat", Russia) at 37°C in an atmosphere of 5% CO2. Complexes of DNA/peptide with different ratios of the charges of DNA and peptide were prepared from the calculation of 12 µg DNA in a Petri dish in 500 ál PBS. When added to cells in medium were introduced CaCl2concentrations up to 20 mm. After incubation of the cells with the complexes for 2 h, the cells were washed from deinternationalise complexes with heparin solution (58 μg/ml in PBS), and incubated in DMEM medium containing 10% serum 24 hours
The luciferase activity was measured using a commercial system evaluate the activity of luciferase production company Promega (Luciferase Assay System, cat. room E) in accordance with the Protocol of the manufacturer. Measuring the luminescence produced by the luminometer Turner Biosystem 20/20". Protein concentration in cell lysates was performed by the method of Bradford. The luciferase activity was expressed in relative light units (RLU), which represents the number of flashes per minute per 1 mg of total protein in cell extracts. Background luminescence values did not exceed 120 RLU.
Testing of the developed system of gene transfer in laboratory animals has been conducted on the treatment of wounds using a plasmid with luciferase, and the use of therapeutic plasmid that encodes a synthetic gene insulin-like growth factor type I (IGF-1), developed by us earlier (for the CAS No. 2007124538/20(026721)). In this work, we used mice of the DBA-2 at the age of 6 weeks (obtained from the kennel "Rapolano RAMS). Before applying RAS skin of experimental animals prepared - away hair. Incised wounds were applied using a surgical scalpel to a depth of 2 mm, capturing the underlying muscle tissue in the lower back. Puncture wounds were inflicted by ten injections insulin syringe in the lower back. Injection experimental treatments were applied with an insulin syringe in the form of 10 injections in the area of the wound. Plasmid DNA and complexes of plasmid DNA with a molecular conjugate was administered in the amount of 50 μg of DNA per mouse in 100 μl of PBS containing 20 mm CaCl2. To measure luciferase activity on day 3 after transfer mice pCMVluc animal was slaughtered and cut out a section of tissue, including the wound. For setting the experiment consisted of 100 mg of tissue for each animal. Samples homogenized in a buffer Reporter Lysis Buffer (Promega, USA, cat. room E), froze and were centrifuged for 3 min at 10000 g. The supernatant was used for measurement of luciferase activity and protein concentration as described above.
Evaluation of expression of a synthetic gene for IGF-1 was performed on the first, third and seventh days after transfer mice pCMVIGF-1 using RealTime RT-PCR. Tissue samples were obtained as described above, and used the for RNA extraction. As a negative control was used mice not treated pCMVIGF-1. Isolation of RNA from tissues was carried out using a set of TRI Reagent" production company "Sigma", USA, per manufacturer's instructions. 100 mg of cells used 2 ml of lyse solution "TRI Reagent". The concentration of RNA was determined spectrophotometrically. The selected RNA was treated with Dnazol 1. The absence of contamination of selected RNA plasmid was verified by PCR using primers for IGF-1. Synthesis of cDNA was performed using the set Revert Aid® First Strand cDNA Synthesis Kit (manufactured by "Fermentas", Lithuania). To measure the relative amount of mRNA used the method of real-time PCR using SYBR GREEN (SYBR Green Supermix (Bio-Rad, USA). All samples were normalized to the expression level of the gene of the 'household' β-actin. The reaction was performed using the IQ5 thermal cycler (Bio-Rad, USA), a preliminary analysis of the data was performed using the accompanying software. The efficiency of amplification was determined by external standard curve using a series dilution of sample 5 ng, 10 ng, 30 ng, 50 ng. The calculated efficiency of amplification used primers was 0.98 srvc. units as a negative control was used in the reaction without added DNA. Normalization of data was performed by Kolichestvennyi, the concentration of RNA and cDNA. The calculations were carried out using the ΔCt method, according to the formula R=(1+E)ΔCtwhere Ct is the threshold cycle, ΔCt=Ct(Mod)norm - Ct(Nat)norm, E is the efficiency of amplification, R - increase in the amount of PCR product after n cycles.
Measuring the area of the wound on the body of experimental animals was carried out using a photograph of the affected area. Analysis of the photographic material is performed using computer-assisted image analysis ImageTool for Windows (version 2,0, USA) according to the basic principles of stereology in morphometrics.
Were synthesized two molecular conjugate, characterized ligand part - NLS-TSF-7 (SEQ ID NO 4) and NLS-TSF-12 (SEQ ID NO 5):
1. NLS-TSF-7: H-Pro-Lys-Lys-Lys-Arg-Lys-Val-β-Ala-His-Ala-Ile-Tyr-Pro-Arg-His-NH2.
2. NLS-TSF-12: H-Pro-Lys-Lys-Lys-Arg-Lys-Val-β-Ala-Thr-His-Arg-Pro-Pro-Met-Trp-Ser-Pro-Val-Trp-Pro-NH2.
Molecular weight NLS-TSF-7 is 1828.26 (average weight); 1827.12 (monoisotopic); NLS-TSF-12 - average molecular weight 2426.00, monoisotopic - 2424.38. Figure 1 presents the results of mass spectroscopic analysis of NLS-TSF-12 (a) and NLS-TSF-7 (b). Molecular weight calculated from the data of mass spectrometry for both conjugates were consistent with theoretical values. As the cationic component of both the synthesized molecular conjugate contained a modified nuclear localization signal of the T-antigen of the virus SV-40 (NLS-peptide). The choice NLS-peptide was determined on their previous studies. Previously we have studied in detail the nature of the complexation of plasmid DNA with cationic peptides K8 and Tat and investigated the mechanisms of internalization of these complexes in mammalian cells (focused on I. et al., 2003; Dige E. B. and others, 2006). It was found that the main route of absorption cells of DNA complexes with cationic peptides - adsorptive endocytosis. Adsorptive endocytosis is a non-specific process that does not depend on the presence on the cell surface of any of the receptors. When designing a system targeted delivery of DNA into mammalian cells the uptake of DNA complexes with molecular conjugates adsorptive endocytosis will degrade transpacificus transfer. In preliminary studies it was shown that, despite the effective binding of plasmid DNA and subsequent compaction, the NLS peptide was not able to deliver DNA in cultured human cells (cells human hepatoma HepG2, fibroblasts of human skin cells, human neuroblastoma Sk-N-SH). Thus, the choice of the NLS peptide to create a conjugate due to its ability to bind nucleic acid and to ensure the delivery of a complex in the cell nucleus. The study of the formation of complexes of DNA/conjugate is represented in Fig.2. Shows the results of gel retardation complexes pCMVluc plasmid DNA with the molecular to Nyugati NLS-TSF-7 and NLS-TSF-12. Both peptides were effectively bound plasmid DNA with isotonic values of the ionic strength of the reaction medium.
Figure 3 presents the dependence of the efficiency of complex formation of the ionic strength of the reaction medium. It is established that both the molecular conjugate is not stable when the ionic strength of the solution exceeding 300 mm. In spite of the same cationic part, NLS-TSF-7 was more stable than NLS-TSF-12, which, apparently, is due to a greater density of positive charge in the case of NLS-TSF-7 (molecular weight of a positive charge 304,71) compared with the NLS-TSF-12 (molecular weight of a positive charge 404,3).
The stoichiometry of complexes of DNA/conjugate was investigated by purification of the complexes prepared in the presence of saturating amounts of peptides (a tenfold excess of charges) from unbound peptide by ultrafiltration. The result was a dedicated complex containing saturating amounts of peptide. It is known that heparin, being more strong polyanion than DNA, is able to displace it from the complex with a polycation (Y. Xu et al., 1996; Ramsey E. et al., 2002). Determination of DNA content in the selected complex was performed spectrophotometrically after its complete destruction by excess heparin. The peptide content in the investigated complex was determined by back titration of heparin. This C is Lew has constructed a calibration curve according to the number of heparin, sufficient for the complete destruction of the complexes of DNA/conjugate, the peptide content in the reaction medium during the formation of the complexes (Fig.4A). It is established that the maximum content of NLS-TSF-7 complexes corresponds to 1.5-fold excess of positive charges NLS-TSF-7 relative to the negative charges of DNA. In the case of NLS-TSF-12 maximum possible content of the conjugate in the complexes corresponds to 1.3-fold excess of positive charges (Fig.4B). Similar results were obtained previously for complexes of DNA with TAT-peptide (focused on I. et al., 2003) and K8 (Dige E. B. and others, 2006).
The interaction of DNA-polycation results in compaction and, as a consequence, the protection from nucleases (T. Niidome et al., 1997; Liu G. et al., 2001). Moreover, the degree of compaction of DNA in the complexes directly correlates with its sensitivity to nucleases. Figure 5 presents the results of the processing of DNA complexes/conjugate prepared at different ratios of the charges of DNA and peptide, Dnazol I. As expected, the increase in the content of the peptide in the reaction medium leads to greater protection of the DNA from degradation. DNA in the complexes prepared at a ratio of negative charges of the DNA to the positive charges of the peptide of 1:1 (electrically neutral complexes), only partially protected from degradation (Fig.4-5, lane 4). Full resistance to D. The Kazi I observed only in the complexes, prepared at the charge ratio is less than 1:1.5. The obtained data are in good agreement with the results of the study the stoichiometry of the complexes of DNA/conjugate (Fig.4).
Evaluation of the sensitivity of DNA complexes/conjugate to DNase I allowed to define another important parameter complexation - cooperatively interactions DNA - polycation. There are two possible models of complex formation. In the case of cooperative interaction of negatively charged complexes of DNA/polycation have a greater affinity for the free polycation than unbound DNA. Therefore, when the charge ratio of DNA and polycation more than 1:1 (excess of DNA), the reaction medium will contain a mixture of electrically neutral (or with a small positive charge) complexes and free DNA molecules. When non-cooperative character of the interaction between the polycation molecules are distributed among DNA molecules random statistical manner, in the reaction medium will contain a more or less homogeneous mixture of complexes, and their charge will be determined by the initial ratio of DNA and polycation (G. Liu et al., 2001). When the cooperative nature of the interactions of DNA-polycation processing Dnazol I complexes prepared at the charge ratio of greater than 1:1 will, on the one hand, to complete degradation nesbatan the th DNA contained in the reaction medium, and on the other hand, to the full protection of DNA, formed complexes with the polycation. In the case of non-cooperative interaction processing Dnazol I negatively charged complexes will lead to partial degradation of the whole DNA, and the size of the products of degradation will increase with increasing the content of polycation complexes. The dependence of the stability of DNA in the complexes with NLS-TSF-7 to action Gnkazy I on the ratio of the charges of DNA and peptide corresponds to the non-cooperative nature of the interactions (Fig.5).
Demonstration of the ability of the synthesized molecular conjugates NLS-TSF7 and NLS-TSF12 to the effective compaction of plasmid DNA with the formation of polyelectrolyte complexes were allowed to move on to the next problem - the study of factors affecting the efficiency of penetration of these complexes in mammalian cells expressing the transferrin receptor. Cells human hepatoma HepG2 and human fibroblasts VH10 was transferrable pCMVluc complexes/NLS-TSF7 and pCMVluc/NLS-TSF12. Both complexes were transfection active. Moreover, a high excess of free molecular conjugates (50x) led to the suppression of the level of transfection, indicating a role transfering receptor binding complexes of DNA/conjugate with the surface of the cells (Fig.6). If this is the molecular conjugate NLS-TSF7 provided a higher level of transfection cells compared with NLS-TSF12 (~ 2.5 fold) (Fig.6). The figure shows the results of luciferase test demonstrating the effect of different factors on the efficiency of transfection of HepG2 cells. In the case of fibroblasts were obtained similar results (data not shown). In previous works we have shown the stimulatory role of free cationic peptides Tat and K8 in the internalization of systems of DNA with cationic peptides. In the same conditions available peptides had no significant influence on the efficiency of transfection of cells with DNA complexes with molecular conjugates containing as a ligand component of the synthetic analogues of luliberin. Seemed interesting to conduct similar studies on the complexes of DNA with NLS-TSF7 and NLS-TSF12. It is established that a moderate excess of free conjugates in the culture medium does not have a significant impact on the level of transfection of cells. In this respect they are similar to molecular conjugates of synthetic GnRH analogues. Apparently, this similarity is explained by a common mechanism of internalization of complexes of receptor - mediated endocytosis, in contrast to complexes of DNA with cationic peptides Tat and K8, where the main role in the internalization plays an adsorptive endocytosis (focused on I. A. et al., 2003; Dige E. B. and others, 2006; Efremov, A. M. and others, 2009).
The role of endocytosis in transfect the cells by DNA complexes with molecular conjugates NLS-TSF7 and NLS-TSF12 tested in experiments, carried out in the absence of ions of CA2+.
It is known that both receptor-mediated and adsorptive endocytosis oppressed in the absence of ions of CA2+. It is established that the destruction of CaCl2from the culture medium results in almost complete suppression of the expression level of the portable gene using both conjugates (Fig.6).
Another important factor influencing the efficiency of transfection of cells polyelectrolyte complexes is the presence in the culture medium serum proteins. Previously, we showed that transfection of cells with DNA complexes with cationic Tat peptide and molecular conjugates based on GnRH analogues in the presence of serum proteins leads to a significant reduction in the level of expression of the transferred gene (focused on I. A. et al., 2003; Efremov, A. M. and others, 2009). This can be crucial when developing systems for gene transfer in vivo. Similar experiments were performed with the molecular conjugates NLS-TSF7 and NLS-TSF12. It is established that the introduction by transfection of cells in culture medium with 10% fetal bovine serum (FCS) led to a fall in the level of expression of the transferred gene (Fig.6). It is interesting to note that in the presence of serum level of transfection of cells with DNA complexes with NLS-TSF12 was higher than in the case of complexes NAM is with NLS-TSF7, despite the fact that in the absence of serum reverse occurred. This fact should be taken into account in the further development of non-viral systems for gene transfer based on the synthesized molecular conjugates. For example, for a system of regenerative gene therapy of superficial tissue damage mammalian more optimal molecular conjugate NLS-TSF7, whereas in the case of transferring the genes requiring systemic injections of drugs into the bloodstream, NLS-TSF12 may be preferable.
Studies have been conducted of the ability of complexes of plasmid DNA with designed molecular conjugates NLS-TSF7 to penetrate into cells in vivo, which may be used for the purposes of gene therapy or diagnosis. As the model used surface wound tissue of mice DBA-2 (male, 6 weeks). PCMVluc complexes/NLS-TSF7 (charge ratio 1:1) was injected either into the skin of healthy mice (10 injections around the area ~60 mm2), or cut away all wound in the same area. The number of complexes was 50 ág of DNA per mouse. Control group mice were injected with the same amount of free plasmid pCMVluc. Before the experiment, the skin of experimental animals prepared - away hair. The wound was applied using a surgical scalpel to a depth of 2 mm, Zech is ativa be muscle tissue, in the area of the lower back. After 5 days after injection, mice were wordplays, the area of skin near the injections were used to obtain lysates, which were evaluated by luciferase activity. The results are presented in Fig.7. Use with injections of pCMVluc complexes/NLS-TSF7 resulted in increased luciferase activity more than 100-fold compared to injections of free DNA. Significant difference between injections to healthy animals and animals with cut wounds were observed (Fig.7). Thus, the synthesized molecular conjugate NLS-TSF7 capable of effectively transferring genes into surface tissues of mammals.
The following example is given for the purpose of illustrating one of the alternative embodiment of the invention and is not intended in whatever was to limit the present invention.
Was performed testing of the developed molecular conjugates in complex with DNA for the purposes of gene therapy for model regenerative gene therapy in laboratory animals. Test the ability of DNA complexes with a molecular conjugate NLS-TSF7 to penetrate the cell surface of mouse tissues was conducted on mice DBA-2 (male, 6 weeks). To this end, mice with lesions in the superficial tissues was using a molecular conjugate NLS-TSF7 was relocated developed by us earlier vector expr the hurt synthetic gene encoding insulin-like growth factor type I human pCMVIGF-1s (application No. 2007124538/20(026721). For the experiment we used mouse strain DBA-2 age 6 weeks (males). Before applying RAS skin of experimental animals prepared - away hair. The wound was applied using a surgical scalpel to a depth of 2 mm, capturing the underlying muscle tissue in the lower back. By the nature of the damage these wounds are classified as "cut". Injection experimental treatments were applied with an insulin syringe in the form of 10 shots in every wound. In the experiment we formed 4 groups of animals. In each group of 10 mice. The experimental group were injected with either free pCMVIGF-1s or complexes pCMVIGF-1s/NLS-TSF7 (charge ratio 1:1). Injection was performed ten times in the area surrounding the wound. For injection used 50 μg of DNA per mouse in 100 μl of PBS containing 4 mm CaCl2. The control group received injections of plasmid DNA with deletionism promoter (pAIGF-1s) and free NLS-TSF7. 3, 5 and 7 day, two mice from each group were killed and the area adjacent to the wound, was isolated RNA, which was used to quantify the expression of IGF-1 using RealTime RT-PCR. The results are shown in Fig.8. The expression level of IGF-1 on 3 and 5 days after the start of the experiment was higher from 64 to 128 times with injections to the complex pCMVIGF-1s/NLS-TSF7 compared to injections of free pCMVIGF-1s. In the control groups injected with plasmid with delegated by the promoter or free molecular conjugate NLS-TSF7, the expression of IGF-1 have not been reported. These results support earlier findings of high efficiency molecular conjugate NLS-TSF7 upon delivery of genes in the surface tissues of mammals.
The rate of regeneration was performed by measuring the area of wounds on 7, 10, 14 and 21 days after the start of the experiment.
The results are summarized in table 1. Figure 9 shows pictures of mice 14 days after starting the experiment, injected free NLS-TSF7 and complexes pCMVIGF-1s/NLS-TSF7. Injection as free pCMVIGF-1s and complexes pCMVIGF-1s/NLS-TSF7 resulted in a significant acceleration of the process of wound healing compared with control groups. Through 14 days after the start of the experiment in mice injected complexes pCMVIGF-1s/NLS-TSF7, was complete healing of the wound, whereas in the control groups of the area of the wound was about 10 mm2(~17% of the original size) (table.1). Moreover, in mice injected complexes pCMVIGF-1s/NLS-TSF7, on the 10th day after the start of the experiment the size of the wound was two times lower than in mice injected free plasmid, and 4 times lower than in control groups. The results obtained indicate high efficiency of the developed sistemistica genes for the purposes of gene therapy, including regenerative gene therapy of superficial injuries mammals.
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|Molecular conjugates with poly-a site and a ligand for delivery into the cell and the cell nucleus DNA and RNA|
|The group of animals||Time||1 day||day 7||day 10||day 14||21 days|
The table shows mean values of the area of the wound (in mm2in the experimental and control groups of mice: pCMVIGF-1s - mouse injected free vector expression of insulin-like growth factor type I person; pCMVIGF-1s/NLS-TSF7 - mouse injected complexes pCMVIGF-1s/NLS-TSF7; pΔIGF-1s - mouse injected free vector expression of insulin-like growth factor type I person with delegated by the promoter; NLS-TSF7 - mouse injected free molecular conjugate NLS-TSF7.
1. Molecular conjugate with amino acid sequence SEQ ID NO:4, are able to bind with nucleic acid (DNA or RNA) to deliver it in mammal cells expressing the transferrin receptor, consisting of a poly-a sequence and a ligand, in which the poly-a sequence is represented by the modified nuclear localization signal of the T-antigen of SV40 virus and has amino acid sequence SEQ ID NO:3, and the ligand has an amino acid sequence of SEQ ID NO:1.
2. Molecular conjugate with amino acid sequence SEQ ID NO:5, are able to bind with nucleic acid (DNA or RNA) to deliver it in mammal cells expressing the transferrin receptor, consisting of a poly-a sequence and a ligand, in which the poly-a sequence is represented by the modified nuclear localization signal of the T-antigen of SV40 virus and has amino acid sequence SEQ ID NO:3, and the ligand has an amino acid sequence of SEQ ID NO:2.
3. The complex molecular conjugate under item 1 with nucleic acid (DNA or RNA) used for transfection of mammalian cells expressing the transferrin receptor.
4. The complex molecular conjugate under item 2 with a nucleic acid (DNA or RNA) used for transfection of mammalian cells expressing the transferrin receptor.
5. The complex molecular conjugate under item 3 or 4, characterized in that the nucleic acid is represented by the plasmid.
6. Drug for gene therapy or diagnostics, consisting of complex molecular conjugate with nucleic acid under item 4 in an amount of 0.1 μg (in terms of the content of nucleic acid) and pharmaceutically acceptable carrier, ensuring the stability of the complex, intended for delivery to mammalian cells is affected, expressing transferrin receptors, nucleic acids encoding therapeutic genes or marker genes for diagnostic purposes.
7. Drug for gene therapy or diagnostics, consisting of complex molecular conjugate with nucleic acid under item 5 in an amount of 0.1 μg (in terms of the content of nucleic acid) and pharmaceutically acceptable carrier, ensuring the stability of the complex, intended for delivery into mammalian cells expressing transferrin receptors, nucleic acids encoding therapeutic genes or marker genes for diagnostic purposes.
8. Drug for gene therapy under item 6 or 7, characterized in that is used for regenerative gene therapy.
9. Drug for gene therapy under item 8, wherein the nucleic acid encodes insulin-like growth factor type I human.
10. The drug under item 8 or 9, characterized by the use of the composition of a solution of CaCl2at a concentration of 20 mm.
11. The method of formation of the complex under item 3 or 4, which in the first stage of merging solutions molecular conjugates and nucleic acid, followed by purification of the resulting complexes from unbound molecular conjugate ultrafiltration in the second stage and then at the third stage - a solution of CaCl2.
12. The method of formation of the complex under item 3 or 4, characterized by being merge solutions of nucleic acids and molecular conjugate appropriate charge ratio in the reaction medium is from 1:0,63 and less and using for complexing solutions with ionic strength is less than 300 mm.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to molecular biology and genetic engineering. What is presented is a RNAi molecule for suppressing the thymidilate synthase expression by the action of RNAi, containing a double-stranded RNA domain consisting of a sense chain consisting of a nucleotide sequence presented by SEQ ID NO: 1 hybridised with an anti-sense chain hybridized in the demanding conditions with the sense chain.
EFFECT: molecule can substantially potentiate the antineoplastic action of 5-FU-antineoplastic agent for which reason it can be used in medicine as a part of the antineoplastic therapy.
15 cl, 2 dwg, 3 ex
SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.
EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.
9 cl, 7 dwg, 5 ex
SUBSTANCE: group of inventions relates to the field of biotechnology and is directed to nucleic acid molecules which encode a protein exhibiting properties of a biosensor for detection of hydrogen peroxide in living cells having fluorescence in the red region of the spectrum. The nucleic acid molecules are derived by method of genetic engineering. Protein for detection of hydrogen peroxide has the amino acid sequence shown in SEQ ID NO:2. The host cell and the expression cassette are also provided, comprising the said nucleic acid molecules.
EFFECT: use of the inventions enables to carry out microscopy in thick layers of tissues, and provides the possibility of joint microscopy of several fluorescent structures.
4 cl 7 dwg
SUBSTANCE: invention relates to molecular biology, in particular to short interfering RNA (siRNA), and can be used in antitumor therapy. On the basis of genome analysis the sequences of siRNA against the human gene HIF1A with SEQ ID NO:1-2 are designed, siRNA against the human gene HSP8A with SEQ ID NO:3-4, siRNA against the human gene APEX1 with SEQ ID NO:5-6, and siRNA against the human gene CCND3 with SEQ ID NO:7-8, associated with cell proliferation of human pancreatic adenocarcinoma. Using the obtained siRNA, including as part of a lentiviral vector, results in suppression of cell proliferation of human pancreatic adenocarcinoma and tumor destruction. The invention enables to achieve the level of suppression of proliferation up to 65% using siRNA to genes HIF1A and HSP8A associated with stress and responsible for the major proliferative (CCND3) and reparative (APEX-1) ways of cell division.
EFFECT: improving level of suppression of proliferation.
3 cl, 10 dwg, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and microbiology and represents a method for identifying a cluster of antigens coding staphylococcal proteins that are exotoxins. The present method is implemented by performing a single multiple-primer polymerase chain reaction in two reaction mixtures, first of which contains primers to genes coding such staphylococcal proteins, as thermonuclease, beta-glucosidase in the species S.aureus, S.epidermidis, S.haemolyticus, S.lugdunensis, S.saprophyticus, and the second one - to set1, set2, set3, set4, set5 genes coding the exotoxins. That is followed by a comparative analysis of amplified gene fragments prepared in two sample aliquots and a positive control reference according to the identification table. The present invention also discloses a test system for implementing the above method, which contains DNA recovery components, PCR components, and result analysis components. The PCR components contain a 10-merous buffer solution, pH 8.4, deionised sterile water, one positive control reference, Taq polymerase, mixture of four dNTPs, mixture of primers No.1 containing epi-F, epi-R, aur-F, aur-R, hae-F, hae-R, lug-F, lug-R, sap-F, sap-R in a ratio of 1:1:1:1:1:1:1:1:1:1, and a mixture of primers No. 2 containing set1-F, set1-R, set2-F, set2-R, set3-F, set3-R, set4-F, set4-R, set5-F, set5-R in a ratio of 1:1:1:1:1:1:1:1:1:1.
EFFECT: invention enables recovering the cluster of genes coding the staphylococcal proteins, a molecular weight of which makes 25 to 35 kD consisting of almost 200 amino acid residues and having a tertiary structure high-homologous with some staphylococcus superantigens (enterotoxins, TSST-1 toxins) and with pyrogenous streptococcal exotoxin C.
2 cl, 3 tbl, 1 ex
SUBSTANCE: invention is a recombinant plasmid DNA pET3.54, encoding the polypeptide FN3.54, interacting with human tumour necrosis factor. The present invention also discloses the recombinant strain of bacteria Escherichia coli BL21(DE3)/pET3.54 - producer of the polypeptide FN3.54, interacting with human tumour necrosis factor.
EFFECT: invention enables to obtain the desired protein with high yield, using a smaller amount of the inducer and the expression level of total cell protein.
2 cl, 4 dwg, 6 ex
SUBSTANCE: invention is a recombinant strain of bacteria Escherichia coli N41 (pBpuN4/MR), which is obtained by transformation of the strain Escherichia coli ER2267 of plasmid pBpuN4/MR N41, obtained based on plasmid pUC19 and containing the gene encoding DNA-methyltransferase M.BpuN4I, methylating one of cytosines in position C5 in the sequence 5'-GGNNCC-3', and the restriction endonuclease gene BpuN4I. This recombinant strain of bacteria Escherichia coli N41 (pBpuN4/MR) is the producer of the site-specific endonuclease of restriction BpuN4I which recognises DNA sequence 5'-G^GNNCC-3'/3'-CCNNG^G-5' and cleaves both its chains after the first guanine to form 5'-protruding four-nucleotide cohesive ends.
EFFECT: invention enables to obtain restriction endonuclease of the predetermined specificity with high yield.
3 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biological engineering, molecular biology, and biotechnology. What is presented is a yeast cell applicable for detecting an interaction between proteins and their domains co-transformed by two plasmids modified for expression of two proteins in the yeast cells, wherein a nucleotide sequence coding the first protein is cloned into one plasmid, while the nucleotide surface coding the second protein - into the other plasmid, wherein the nucleotide sequences coding the proteins are fused with the nucleotide sequences coding the activation and DNA-binding domains of the protein GAL4; the nucleotide sequences coding the studied proteins are separated from the nucleotide sequences coding the activation or DNA-binding domains of the protein GAL4 by means of a nucleotide sequence coding a peptide representing the sequence GluLeuGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAla which is expressed in the form of a protein bridge between the studied protein and the protein GAL4 domains.
EFFECT: invention enables minimising the mutual interaction of the tested protein domains and DNA-binding/activation domains of a vector and can be used for detecting the mutual interaction between the protein molecules for medical and studying applications.
4 dwg, 2 ex
SUBSTANCE: invention relates to molecular biology, biochemistry and genetic engineering. The invention discloses a nucleic acid characterised by a nucleotide sequence which encodes a protein consisting of an amino acid sequence with deletion, replacement or addition of one or more amino acids in the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 7 and having phosphatidic acid phosphatase activity, a corresponding protein, a recombinant vector, a protein expression cell and a method of producing a fatty acid composition.
EFFECT: invention can be used to produce polyunsaturated fatty acids in the food industry.
9 cl, 6 tbl, 7 dwg, 8 ex
SUBSTANCE: invention refers to biotechnology, more specifically to detecting lung cancer by means of an aptamer and can be used in diagnostics. The aptamers are prepared by selection involving alternating rounds of positive selection of the aptamers to minced human tumour lung tissues sampled from oncological patients after the operation and of negative selection to healthy lung tissues and healthy whole blood to determine a pool of the highest-affinity aptamers to be cloned, sequenced and analysed for a binding specificity to tumour lung cells.
EFFECT: prepared aptamers possess high sensitivity to tumour debris and circulating tumour cells in peripheral blood of the patient suffering lung cancer that enables more effective diagnosis of human lung cancer.
3 cl, 2 dwg, 1 ex
SUBSTANCE: method comprises scaling of diploid cells of line M-20 from cryobank of Institute of Poliomyelitis and Viral Encephalitis n.a. MP Chumakov of RAMS from the ampoule of the seed cell bank of 7 passage with obtaining the working cell bank of 16 passage. At that the cells of 20-33 passages, suitable for use in therapeutic and/or diagnostic purposes are produced by culturing in a nutrient medium containing 10% of human fibrinolytic active plasma (FAP), comprising platelet-derived growth factor PDGF in a concentration of from 155 to 342 pg/ml.
EFFECT: invention enables to increase proliferative activity of human fibroblast diploid cells.
2 cl, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to molecular biology and genetic engineering. What is presented is a RNAi molecule for suppressing the thymidilate synthase expression by the action of RNAi, containing a double-stranded RNA domain consisting of a sense chain consisting of a nucleotide sequence presented by SEQ ID NO: 1 hybridised with an anti-sense chain hybridized in the demanding conditions with the sense chain.
EFFECT: molecule can substantially potentiate the antineoplastic action of 5-FU-antineoplastic agent for which reason it can be used in medicine as a part of the antineoplastic therapy.
15 cl, 2 dwg, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions refers to biotechnology and oncology. A method provides: a) isolating postnatal tissue-specific pluripotent autologous stem cells (ASCs) and/or autologous progenitor cells (APCs) for the following proteome and complete transcriptomic analysis; b) isolating ASCs and/or APCs and/or pluripotent allogenic HLA-haploidentical stem cells (HLA-SCs) for remodelling of their proteome profile; c) isolating cancerous stem cells from patient's tumour; d) carrying out ASC and/or APC and CSC proteome analysis; e) carrying out ASC and/or APC and CSC complete transcriptomic analysis; f) recognising a protein complement each of which is found in the proteome profiles both of ASCs and/or APCs, and of CSCs; g) analysing the recognising protein complement for identifying intracellular signalling pathways in CSCs not subject to the neoplastic transformation as a result of carcinogenesis, and recognising target proteins defined as membrane acceptors of identified signalling pathways; h) analysing the complete transcriptomic profile of the CSC gene expression and confirming the preservation and functional significance of the structural components of the identified signalling pathways in CSCs; i) recognising ligand proteins able to activate the target proteins; j) carrying out a comparative analysis of the ASC and/or APC complete transcriptomic profiles to the transcriptomic profiles contained in the known data bases of transcriptomes for recognising perturbagens able to modify the gene expression profile of ASCs and/or APCs and/or HLA-SCs isolated for remodelling their proteome profile in the line of secreting the pre-recognised ligand proteins; k) remodelling the ASC and/or APC and/or HLA-SC proteome profile with perturbagens to produce the modified transcriptome profile of various cell systems able to have a regulatory influence on patient's CSCs.
EFFECT: preparation produced according to the method includes all the individual peculiarities of the patient's genome and proteome modifications, and has the regulatory influence on patient's cancerous stem cells (CSCs) and malignant cells.
8 cl, 4 dwg, 11 tbl, 4 ex
SUBSTANCE: population of mononuclear cells or non-germ stem cells, which is saturated with cells of monocytic lineage containing promonocytes, is used for treatment of ischemia with a patient.
EFFECT: invention allows effective treatment of ischemia with a patient by injection of the above population of therapeutical cells to ischemic tissue of the patient.
13 cl, 8 dwg, 5 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and medicine. What is described is an active immunostimulating vaccine containing at least one RNA, preferentially iRNA coding at least two antigens evoking the immune response in a mammal and used for treating lung cancer, first non-small cells lung cancer (NSCLC), preferentially specified among three primary subtypes, squamous cell carcinoma, adenocarcinoma and large-cell lung carcinoma, or NSCLS-related disorders.
EFFECT: there are produced kits, first containing the active immunostimulating vaccine.
21 cl, 34 dwg, 1 tbl, 8 ex
SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.
EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.
4 cl, 4 dwg, 3 ex
SUBSTANCE: method comprises isolation of mononuclear cells (MNC) from peripheral blood of a patient, separation of cells to adherent and non-adherent fractions, addition of the adherent fraction to the MNC of growth factors, loading of the dendritic cell with antigens of tumour lysate in vitro, the stimulation of maturation of dendritic cells for the next day. At that, the obtained immature DCs are added to lysate-autologous tumour cells at a dose of 100 mcg/ml, and after 48 hours within the subsequent 24 hours the rf-tumour necrosis factor-alpha is applied at a dose of 25 ng/ml. Then, the co-culture is carried out of mature dendritic cells activated with lysate and the non-adherent fraction of MNC at a ratio of 1:10 in the presence of recombinant human interleukin-12 at a dose of 10 ng/ml and the recombinant human interleukin-18 at a dose of 100 ng/ml.
EFFECT: invention enables to improve the level of cytotoxic and interferon-producing activity of antigen-activated dendritic cells while reducing the duration of their culture.
SUBSTANCE: what is presented is a method for stimulating the post-traumatic spinal cord regeneration consisting in a single-stage transplantation of human umbilical cord blood mononuclear cells pre-transduced with recombinant adenovirus with a cloned gene of glial derived neurotrophic factor (gdnf), in the damage area.
EFFECT: using the invention enables providing a better outcome of the post-traumatic spinal cord regeneration, reduced length of staying in hospital of patients suffering from a spinal cord injury and improving the patients' quality of life.
SUBSTANCE: method involves using a photosensitiser (PS) pre-detected in the tumour by its fluorescence that is followed by tumour irradiation by light emission at a wave length in the spectral range of PS peak absorption. The PS is presented by the genetically coded protein KillerRed by providing its tumour expression by gene insertion into the tumour cells. The tumour is exposed to the light emission at energy density of 180-270 J/cm2 3 times every second day or 7 times daily. The protein KillerRed is preferentially localised either in mytochondria and nuclei, or in nuclei.
EFFECT: method provides high-selectivity PS effect on the tumour, reduced toxic load on the intact organs and tissues, the absence of PS redistribution in the tumour, with no need for the constant radiation control.
2 cl, 6 dwg, 11 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.
EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.
3 cl, 6 ex, 1 tbl, 14 dwg
FIELD: biotechnology, veterinary science.
SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.
EFFECT: valuable biological properties of recombinant virus.
41 cl, 13 dwg