RU2537262C2 - Molecular conjugates with polycationic section and ligand for delivery of dna and rna into cell and cell nucleus - Google Patents

Abstract

FIELD: chemistry.

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

Description

FIELD OF THE INVENTION

The invention relates to the field of medicine, pharmacology, biotechnology, genetic engineering, gene therapy, namely the delivery of DNA and RNA to cells for therapeutic, diagnostic and research purposes.

State of the art

In the development of gene therapy technologies, the weak point remains to ensure the effective delivery of genetic constructs based on DNA and / or RNA into the cell.

Currently, a number of approaches are being developed aimed both at increasing the efficiency of delivery of genetic constructs to cells, and ensuring the possibility of their targeted delivery only to certain cells and tissues.

Methods for delivering genes to mammalian cells

The plasma membranes of cells are a reliable barrier to the penetration of foreign nucleic acids - RNA or DNA, unless, of course, the nucleic acids are found in viral particles or are not captured by phagocytic cells. In some cases, it is still possible to make the membrane temporarily permeable to DNA and RNA, changing the physical state of the membrane (fluidity, integrity). The possibility of making such changes depends on a number of factors: the metabolic activity of cells, the supply of energy substances, the ability to phagocytosis, and the stage of the cell life cycle.

If polynucleotide sequences are not able to independently penetrate into cells through a plasma membrane, then relatively low molecular weight oligodeoxynucleotides (ONE) may constitute an exception. The mechanism for the penetration of ODN into mammalian cells has not been fully studied, although it is believed that ODN is absorbed by cells by a combination of adsorption endocytosis and pinocytosis, which are partially activated by binding of ODN to receptor-like surface proteins of cells of a wide range of tissues (Gewirtz A. M. et al., 1996). Most of the ODNs that enter the cells end up in endosomal / lysosomal vesicles and are hydrolyzed by enzymes there.

In general, an important role in the development of gene therapy methods is played by the development of means and methods for transferring “treating” genes (as part of expression vectors) to the body, which should ensure high delivery efficiency of genetic constructs, their selective entry into cells or tissues, and stable functioning transgenes in cells with minimal side effects: toxic, inflammatory, immunogenic, and possibly mutagenic and oncogenic (Culver KW, 1994). In addition, factors such as the cost-effectiveness of creating and using delivery vehicles should be taken into account.

Methods of gene delivery to somatic and reproductive cells of mammals, depending on the conditions of treatment with the cells, can be classified as in vitro, ex vivo, and in vivo transfer methods (Culver K. W., 1994). In the first case (in vitro), gene delivery is carried out in cultured mammalian cells or tissues, most often for experimental purposes, when the target of exposure is normal, mutant, or pathologically altered cells. In the second case (ex vivo), the transfer is carried out into cells that are briefly cultured in vitro taken from the body, and after the fact of the delivery of the “treating” gene to them, 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 is carried out directly into the cells or tissues (targets) of a living organism.

Another classification of the most common means of delivering expression vectors into cells is based on the transfer method:

1. Viral (recombinant adenoviruses, retroviruses, adeno-associated viruses, herpes viruses and smallpox vaccines).

2. Non-viral biological (liposomes, cationic lipids; polycations; viral envelopes, molecular conjugates; microspheres).

3. Physico-chemical, physical (calcium-phosphate transformation of cells in vitro, electroporation, ballistic procedure, microcapillary injection).

Recombinant virus gene transfer

Recombinant retroviruses, adenoviruses, adeno-associated viruses and herpes viruses can be used for in vivo transfer. Delivery of “treating” genes as part of recombinant retroviruses is chronologically one of the first successful experiments in human gene therapy. Treatment projects are being developed in which retroviral particles are injected in vivo to be delivered directly to certain tissues and organs (represented by proliferating cells). In addition, in some cases, retrovirus-producing “packaging” cells are inoculated into the patient’s body, for example, intracranially (stereotactically) in case of malignant brain tumors. The disadvantages of retroviral gene transfer include the risk of disabling the expression of vital genes or activation of oncogenes when randomly inserted into the provirus chromosomes (Temin N.M., 1990). In addition, for not clear reasons, retroviral expression vectors inactivate over time, although they continue to be preserved in the form integrated with chromosomal DNA.

Recombinant adenoviruses are most effective in delivering “treating” genes to mammals, since the infectious titer of such a virus can reach 10 ¹⁰ PFU / ml; in addition, adenoviruses have a wide tropism to various tissues, and when they enter the cells, their genetic material, together with the “treating” gene, moves to the cell nucleus, where it lasts from two weeks to several months (M. Rosenfeld et al., 1991) . The “treating” gene replaces the region of early adenovirus genes in the viral genome, as a result of which such a virus in the cell loses its ability to reproduce and is destroyed after some time. The disadvantages of gene transfer in adenoviruses include a certain toxicity of defective viral particles that can produce some early virus-specific proteins in the body, and, in addition, their high immunogenicity, which makes repeated treatment difficult. To reduce toxicity and immunogenicity, a series of new generation adenoviral vectors are devised that are already devoid of a number of viral genes (Fisher KJ et al., 1996), which avoids the synthesis of virus-specific proteins after delivery of the “treating” gene to the body. In this case, the development of immune responses is delayed and occurs only with repeated injections of adenovirus. Viral particles are injected into the body by intravenous injection or as an aerosol into the respiratory tract to treat hereditary defects of the respiratory epithelium. In particular, for the treatment of patients with cystic fibrosis (a hereditary defect of the CFTR gene), a recombinant adenovirus containing the CFTR gene expression vector is introduced into the patient's airways (Zabner J. et al., 1993; Harvey B. G. et al., 1999; Lerondel S. et al., 2001; Davies JC, 2002; Griesenbach U. et al., 2002; Gaden F. et al., 2004). Projects are underway to transfer “treating” genes as part of recombinant viruses of other species: adeno-associated viruses (Nomura S. et al., 2004; Tietge UJ et al., 2004; Oka K. et al., 2001; Kozarsky KF et al., 1996; Kobayashi K. et al., 1996; Kozarsky KF et al., 1994; Kozarsky K. et al., 1993; Flotte T. R., 2004), herpes simplex viruses (Coffin RS et al., 1996a; Weir JP et al., 1996; Coffin RS et al., 1996b; Wang M. et al., 1998; Wolfe D. et al., 2004; Niranjan A. et al., 2004; Goss JR et al., 2004; Cao B. et al., 2004; Goins WF et al., 2004), vaccinia virus (Guo ZS et al., 2004; Di NM et al., 2003; Walther W. et al., 2000).

Adeno-associated virus delivers genes (of small size) to one of the chromosomes (19th) of a person. The advantages of this delivery include the relative ease of subsequent removal of the viral vector from the cell DNA by co-infection of the target cells with the helper virus. A limitation of vectors prepared on the basis of adeno-associated viruses is the relatively low capacity of their capsid for polynucleotides - about 5 kb, which makes the transfer of large cell genes impossible. Recently, there was a message about the development of a hybrid vector in which the replicative form of the recombinant adeno-associated virus is packaged in an adenovirus capsid. The capacity of such a vector is significantly higher while maintaining the site-specific integrative ability inherent in adeno-associated viruses (Goncalves M. A. et al., 2004).

Recombinant herpes viruses are recommended for delivery of “treating” genes to cells of the nervous tissue, in the nuclei of which the herpes virus genome can persist for a long time in a latent state (as an episoma) (Flotte T. R., 2004; Coffin RS et al., 1996; Weir JP et al., 1996; Coffin RS et al., 1996; Wang M. et al., 1998; Gregorevic P. et al., 2004; Gruchala M. et al., 2004). Recombinant vaccine viruses (whose genome is localized in the cytoplasm) are used for the molecular vaccination of the body with products of the expression of foreign genes (Walther W. 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 by plasma nucleases, which is a serious obstacle to transfection (Hashida M. et al., 1996; Crook K. et al., 1996). This problem is partially solved by the condensation of DNA by positively infected molecules, which protects the DNA from the action of nucleases (Sandgren S. et al., 2004; Wiethoff C.M. and Middaugh C.R., 2003; Luo D. and Saltzman W.M., 2000). A less important task for non-viral systems is to facilitate the passage of the plasma membrane. The main pathway for DNA to enter a cell using non-viral delivery systems is endocytosis. In most cases, penetration is due to clathrin- and caveolin-independent endocytosis, as well as clathrin-independent and adsorption endocytosis (Takei K. et al., 2001; Nichols B.J. et al., 2001). In some types of cells, penetration is realized by the mechanism of macropinocytosis or phagocytosis for fairly large particles (Apodaca G., 2001) (Fig. 2-11).

Non-viral delivery vehicles were first used more than 20 years ago. The delivery systems of genetic vectors must perform several functions: to bind to the delivered genetic material, packaging DNA and providing not only penetration through the membrane, but also protecting the DNA both before it enters the cell and immediately after transfection, ensuring exit from the lysosomal compartments, preservation in the cytoplasm and translocation to the cell nucleus. Overcoming not only the plasma membrane, but also avoiding the lysosomal degradation of the introduced DNA is a very difficult task.

It is known that physiological concentrations of salts and proteins in blood serum significantly affect the physicochemical properties of non-viral carriers and often lead to a decrease or complete loss of their properties, which currently severely limits the use of such systems in vivo (Molas M. et al., 2003 ; Ignatovich I. et al., 2003). Active research is currently underway to develop non-viral delivery vehicles that are effective in vivo.

Cationic lipids

The first successful transfection using cationic lipids (lipoplex) was carried out in 1987 (Feigner P.L. et al. 1987). Since then, this method has been widely used not only for research purposes, but also in clinical protocols (Nabel G.J. et al., 1990). Its use is a relatively safe and simple method for local delivery in low doses (Li S. et al., 2000).

The main component of liposome membranes are phospholipids. In addition, to stabilize the particles in the extracellular medium, 40-50 mol. % cholesterol. The tropism of liposomes to cells of different types depends on the charge, which can be changed by varying the phospholipid composition and various additives (Ropert P.1999).

The interaction of DNA with cationic liposomes is a spontaneous process initiated by DNA-mediated liposome fusion and large-scale lipid rearrangement. However, DNA condensation does not occur during the formation of the DNA liposome complex (Li S. et al., 2000, Zuidam N. J. and Barenholz Y., 1998).

The DNA-liposome complex penetrates into cells relatively easily as a result of electrostatic adsorption of a positively charged liposome membrane with the plasma membrane of a eukarotic cell or occurs with the participation of endocytosis (Li S, et al., 2000; Zuidam NJ et al., 1998; Smith RM and Wu GY 1999). In some cases, clathrin-mediated endocytosis plays a significant role in the penetration of DNA complexes with cationic lipids into non-phagocytosed cultured mammalian cells (Zuhom I. S. et al., 2002). Molecules of the cell surface responsible for the interaction with the lipoplex are not yet fully understood, however, it is known that proteoglycans of the cell surface play an important role in this process (Scherman D. et al., 1998, Singh M., 1999).

The possibility of using cationic lipids does not depend on the proliferative activity of cells, so that lipoplex can be used to transform non-dividing or slowly proliferating cells (Zuidam N. J. et al., 1998). The disadvantage of this method is the degradation of a significant part of foreign DNA under the action of lysosomal hydrolases, as well as toxicity when used in high doses (Erbacher P. et al., 1996). The release from endosomal compartments in most cases is achieved due to damage to the membrane of the endosome by replacing part of the lipid components between the membranes of the liposome and endosome (Chi Y. and Szoka F. C., 1996). In accordance with this model, anionic lipids of the endosomal vesicles interact with cationic lipids of liposomes via the “flip-flop” mechanism, providing partial membrane fusion and DNA release. This is in good agreement with the data on an increase in the transfection efficiency upon addition of neutral DOPE lipids to cationic lipid-DNA complexes facilitating membrane fusion (Hui S. et al., 1996; Farhood H. et al., 1995).

Molecular Conjugates

Molecular conjugates are polycations, in most cases covalently linked to ligands of cell receptors (Fajac I. et al., 2000). Compared to cationic lipids, molecular conjugates more efficiently condense DNA into compact structures, which increases the stability of complexes and, in some cases, promotes nuclear localization of transferred genetic constructs (Rudolph C. et al., 2000). Currently, several different types of molecular conjugates have been created and are actively used, based on polycations such as poly-L-lysine, polyethyleneimine and others (Nolan GP et al., 1998; Kost TA, 1999; Fajac I. et al., 2000; Rudolph C. et al., 2000).

Polycations are easily associated with DNA in solution due to electrostatic interactions of positively charged amino groups and negatively charged phosphate groups of DNA. The size of the complex being formed largely depends on the ligand used (Rudolph C. et al., 2000). As a rule, a structure is formed with a diameter of less than 80-100 nm (Fajac I. et al., 2000), which allows the use of such a complex for the delivery of DNA to various types of cells, because most of them have a size of primary endosomal compartments of 100-120 nm. (Cristano R. J. et al., 1995). The DNA conjugate complex selectively binds to cellular receptors using the ligand portion of the conjugate and is transferred into the cell by receptor-mediated endocytosis (Smith R.M., 1999; Gottschalk S. et al., 1996, Zeiphati 0. et al., 1998). The molecules of axialoglycoproteins, insulin, transferrin, epidermal growth factor, and other proteins, as well as low molecular weight compounds (glycoside residues, etc.) are often used as ligands for surface cell receptors (Yanagihara K. et al., 2000).

The main limitation that arises when using polycationic carriers is the rapid inactivation and disappearance from the bloodstream during systemic administration, due to two main reasons - aggregation and absorption of serum proteins in vivo (Dash PR et al., 1999; Ogris M. et al. ., 1999).

In the formation of the complex, electrostatic interactions between the DNA molecule and the polycationic carrier lead to the formation of a particle having a neutral charge, which allows the particles to be easily aggregated in aqueous solutions. To reduce the aggregation of complexes and their inactivation when added to the culture medium or systemically introduced into the body, the following approaches are used: "steric stabilization" by adding polymer molecules, in particular polyethylene glycol (PEG), which prevent adhesion, to which in most cases ligands are attached, providing specific interaction with cellular receptors. Another approach to improving the stability of complexes is “electrostatic stabilization”. In this case, the resulting complexes have a positive or negative charge, which blocks their adhesion due to electrostatic repulsion (Molas M. et al., 2003). Such systems become more stable in the presence of saline solutions and serum proteins (Ahn, C.N. et al., 2002; Oupicky D. et al., 2002). Similar systems are used not only for polycationic 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).

The DNA conjugate complex, which does not have any molecular signals, enters the cell, exists in it for a short time (24-48 hours) and is destroyed in lysosomes (Subramanian A. et al., 1999, Cristano R.J. et al., 1995). In this regard, many conjugates often contain molecular signals of destabilization of endosomal membranes (such as amphipathic peptides of viral origin, capable of destabilizing endosomes at lower pH and facilitate the release of DNA conjugate complexes into the cytoplasm) or (i) nuclear translocation signals, which facilitates the penetration of foreign DNA into the nucleus and its preservation as an episode for a long time (Colin M. et al., 2000, Yanagihara K. et al., 2000).

Some molecular conjugates can be used without the use of additional endosomal destabilizing components. It was shown that lysosome-mediated degradation of DNA complexes with conjugates synthesized based on polyethyleneimine is significantly lower than when using poly-L-lysine as a polycation. Apparently, this circumstance is associated with the buffering properties of polyethyleneimine (Li S., Huang L. 2000, Nguyen H-K., Et al., 2000). The exact mechanism that ensures the release of DNA from endosomal compartments has not been sufficiently studied, however, according to some authors, it is based on buffering the contents of lysosomes due to the imino groups of polyethyleneimine, and on the action of imino groups as proton acceptors (the effect of the “proton sponge”) (Kichler et al., 2001 ) It remains unclear whether the inhibition of endosome maturation causes direct damage to their membrane and the release of the complex from late endosomes, or increases the likelihood of their contents coming out by delaying endosome maturation, although both these hypotheses may be implemented in practice (Molas M. et al., 2003).

There are a large number of works in which various strategies are tested that increase the efficiency of complex release from endosomal compartments, both by inhibiting degradation and by introducing specific domains that destabilize vesicles (for example, some pore-forming peptides) (Yessine MA. And Leroux J.- C., 2004.). For example, the use of chloroquine with various DNA / polycation complexes, including poly-L-lysine and polyethyleneamine, provides an increase in pH and, as a result, inhibits the hydrolytic activity of lysome enzymes (Gotten M. et al., 1990; Fajac I. et al. , 2000; Zanta MA et al., 1997).

Despite a significant increase in transfection efficiency, the use of endosome damaging agents significantly disrupts cell function, inhibiting the recirculation of receptors on the plasma membrane and exerting 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 include their toxicity, which limits the use of complexes in high concentrations (Gottschalk S. et al., 1996), as well as insufficiently high efficiency exogenous DNA delivery.

It seems very promising to use low molecular weight ligands and cationic carriers for the synthesis of molecular conjugates. Compared with protein molecules, the use of low molecular weight compounds can increase the ligand / DNA ratio, as well as significantly reduce the size of the formed complex. The latter is extremely important since compact complexes (with a diameter of 10-30 nm) are able to independently penetrate into the nucleus, which contributes to the long-term and effective expression of the gene of interest (Erbacher P. et al., 1996).

Thus, of all the non-viral methods for delivering genes into the mammalian organism that exist today, only molecular conjugates potentially satisfy one of the important criteria for developing systems for transferring genes into human tumor cells - to ensure targeted delivery of DNA to target cells.

Transferrin receptor ligands

Non-viral gene therapy for the healing of superficial damage to mammals involves the creation of molecular conjugates that can efficiently deliver the genes of mitogenic growth factors to tissue cells directly surrounding the lesion sites, primarily to skin fibroblasts. Transferrin receptor is a successful candidate for the role of a receptor that binds DNA / conjugate complexes and ensures their internalization by cells.

The human transferrin receptor is being actively studied as a model of receptor-mediated endocytosis (Thorstensen K. and Romslo I., 1993; Ponka P. and Lok CN, 1999) as a marker of cell proliferation (Ponka P. and Lok C.N., 1999; Inoue, T. et al., 1993) and as a target for the delivery of various pharmacological agents to cells (Jones AR and Shusta EV, 2007). It is a dimer of two identical transmembrane glycoproteins with a molecular weight of 95 kDa, linked covalently due to intermolecular disulfide bonds. The bulk of the iron entering the cells is absorbed by the transferrin receptor. Transferrin receptor ligand is transferrin, an extracellular transport protein with a molecular weight of 80 kDa, capable of efficiently binding four Fe ³⁺ ions (Ponka P. and Lok C.N., 1999). A feature of transferrin is a change in conformation upon interaction with iron ions, which is believed to be critical for interaction with the receptor (Ponka P. and Lok C.N., 1999). Transferrin receptor expression occurs in all cells and tissues of the body, but the level of expression in different tissues varies (Davies M. et al., 1981; Enns S.A., 1982). Maximum expression is observed in actively proliferating cells (10,000-100,000 molecules per cell), while non-dividing cells contain only a few receptor molecules (Inoue T. et al., 1993).

The widespread use of conjugates based on transferrin for delivery to cells of various pharmacological preparations. In particular, there are reports in the literature about the successful delivery to the tumor cells of the cytotoxic agent doxorubicin covalently linked to transferrin (Yeh C. JG and Faulk WP, 1984; Barabas K. et al., 1992; Sizensky JA et al., 1992; Berczi A. et al., 1993a; Berczi A. et al., 1993b), delivery to the cells of fused proteins that are transferrin linked to therapeutic polypeptides (AN S. A. et al., 1999a; All SA et al., 1999b), gene transfer using transferrin-polylysine molecular conjugates to various cultured mammalian cells (Wagner E. et al., 1991; Strydom S. et al., 1993; Taxma n D. J. et al., 1993; Schwarzenberger P. et al., 2001). For gene therapy purposes, transferrin is used not only as part of molecular conjugates with polylysine, but also as a complex with cationic lipids (Penacho N. et al., 2008), as part of nanoparticles based on cationic lipids, etc. (Abela R. A. et al ., 2008). A feature of all of the above gene transfer systems is the use of natural transferrin as a ligand. This approach has significant drawbacks. First, the resulting DNA / conjugate complex has a large size (more than 100 nm), which makes it difficult for DNA to escape from endosomes and transfer to the nucleus (see above). Secondly, the affinity of the interaction of transferrin with the receptor depends very much on the saturation of transferrin with iron ions, which makes it difficult to obtain transfectionally active conjugate-DNA complexes. At the same time, there is a report in the literature on the detection by the phage display method of peptide ligands capable of interacting highly with the transferrin receptor and inducing its internalization (Lee J. H. et al., 2001).

In recent years, there have been many publications on the properties of HAIYPRH and THRPPMWSPVWP peptides. These peptides were discovered at once in several laboratories using a phage library of heptapeptides. The presence of various properties of these peptides was shown, including the ability to internalize into the cell as a transferrin receptor ligand.

The possibility of binding to the muscles of a number of peptides, including HAIYPRH (US Patent 6,329,501 - Methods and compositions for targeting compounds to muscle, 2001), has been shown.

A series of patents and publications of a group of authors shows the use of the HAIYPRH and THRPPMWSPVWP peptides for delivery of various agents to cells.

U.S. Patent 6,743,893 - Receptor-mediated uptake ofpeptides that bind the human transfemn receptor) describes the properties of these two peptides, HAIYPRH and THRPPMWSPVWP, but only THRPPMWSPVWP is patented. It is considered as a ligand of the transferrin receptor, which ensures, by internalization, the delivery of various agents to the cell, such as antigens, chemotherapeutic substances, and various labels (fluorescent or colored). However, in the formula of this patent there is no clause on the delivery to the cell of DNA or RNA that does not encode the THRPPMWSPVWP sequence for gene therapy purposes.

Another patent (US 2008166293 - RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRED RECEPTOR, 2008-07-10) shows that two peptides (HAIYPRH and THRPPMWSPVWP) are discovered that are able to bind and internalize using the human transferrin receptor ( . When these peptides are fused to other molecules, such a fusion product is absorbed by cells expressing hTfR. Peptides, proteins, antigens, chemotherapeutic agents, antitumor agents, visualized or radioisotope labels, liposomes, including those containing secondary agents, are considered as such molecules that can be delivered to the cell. Moreover, in this patent, the authors indicate that the HAIYPRH peptide does not interfere with the binding of transferrin to its receptor, and also postulate the binding of this peptide at a different site of the receptor than transferrin, which indirectly suggests that the delivery of various agents into the cell using this peptide may not be mediated by the transferrin receptor. These data cast doubt on the previous patents of the authors' data, in which he insists on the receptor-mediated uptake of the HAIYPRH peptide and related agents. In addition, the formula of this patent does not contain a clause on the delivery of DNA or RNA into the cell for gene therapy purposes.

One patent (CA 2449412, COLLAWAN J., MOORE B., LEE J., JEFFREY. RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR 2002-06-06) shows the use of HAIYPRH and THRPPMWSPVWP peptides for delivery with them molecules into cells expressing a transferrin receptor (hTfR) by internalizing the peptide bound to the receptor. This peptide is patented, fused with any other protein or peptide, with a chemotherapeutic agent, a fluorescent or other type of label, antigen, as well as the nucleotide sequence encoding this peptide, including fused with other peptides. This patent does not describe the use of this peptide fused to a nuclear localization peptide, nor does it show the possibility of using the peptide for gene therapy.

Another patent (USPTO Patent Application 20080166293, Receptor-mediated uptake of peptides that bind the human transferrin receptor) proposes the use of HAIYPRH and THRPPMWSPVWP peptides to deliver peptides, proteins, chemotherapeutic agents, antitumor agents, visualized markers and isotopes to the cell. The use of this peptide fused to a nuclear localization peptide is not proposed, and the possibility of using the peptide for gene therapy is not shown.

It is proposed to use the HAIYPRH and THRPPMWSPVWP peptides for delivering peptides, proteins, chemotherapeutic agents, fluorescent markers to the cell (WO / 2002/044329, RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR). He proposed the use of this peptide fused to a nuclear localization peptide; the possibility of using the peptide for gene therapy has not been shown.

A feature of the patent (US Patent Application 20060193778 - Receptor-mediated uptake of peptides that bind the human transferrin receptor) is that delivery of the DNA molecule encoding the HAIYPRH peptide using the HAIYPRH peptide is postulated.

The possibility of using these peptides THRPPMWSPVWP and HAIYPRH in a number of constructs for the delivery of nucleic acid molecules through cell membranes and the blood-brain barrier has been shown (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 only complexes based on covalent bonds are considered, including those using linker molecules. The nucleic acid molecules that make up these complexes are limited to 100 nucleotide pairs, the HAIYPRH and THRPPMWSPVWP peptides are considered exclusively as specific ligands for the receptor. As nucleic acid is considered DNA, cDNA, RNA, etc. Peptides are considered as the polycationic half, however, the sequences of the receptor-binding peptide and the polycationic peptide in this patent are spatially separated by molecules of another type (nucleic acids, hydrophobic molecules). Those. a single peptide molecule consisting of the HAIYPRH sequence and the polycationic peptide is not considered as a delivery peptide. In addition, the invention does not use a nuclear localization peptide to deliver constructs to the cell nucleus.

All of these patents do not provide conditions for the formation of DNA-peptide complexes for gene therapy purposes.

Another patent of this group of authors has similar characteristics (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) propose the use of HAIYPRH and THRPPMWSPVWP peptide sequences for delivery of hydroperoxides to cells.

All of these patents do not provide conditions for the formation of DNA-peptide complexes for gene therapy purposes.

Disclosure of invention

The above disadvantages of viral vectors have led to an increase in the importance of developing non-viral systems for transferring nucleic acids (NKs) - DNA and RNA - into cells. As such a system for the non-viral transfer of DNA and RNA into a cell, we have developed molecular conjugates, which are two-part peptides.

One part is a ligand for cell receptors and is represented by one of two peptide sequences - SEQ ID NO 1 and SEQ ID NO 2. These sequences provide binding to cell receptors with subsequent internalization. According to reports, binding occurs with the transferrin receptor, although perhaps not only with it. The advantage of using the transferrin cell receptor is its high expression on proliferating cells, which can reach 10,000-100,000 molecules per cell, which largely determines the efficiency of the delivery of gene therapy constructs associated with the ligand of this receptor to the cell. However, the use of natural transferrin as a ligand for gene therapy has significant drawbacks described above. The peptide sequences used by us were detected by phage display of peptide ligands capable of interacting highly with the transferrin receptor and inducing its internalization. Moreover, the latest data indicate that these peptides do not interfere with the binding of transferrin to its receptor, which indicates their binding in a different part of the receptor, different from the site of binding to transferrin (US 2008166293, 2008). This indirectly may also indicate that the delivery of various agents into the cell using this peptide may not be mediated not only by the transferrin receptor.

Another part of the molecular conjugates we developed is polycationic. It is represented by the peptide sequence of the modified nuclear localization signal of the SV40 T-antigen of the virus (SEQ ID NO 3).

Polycations interact with DNA in solution due to the electrostatic bonds of positively charged amino groups and negatively charged phosphate groups of DNA. Typically, structures with a diameter of less than 80-100 nm are formed (Fajac I. et al., 2000), which allows the use of such a complex for the delivery of nucleic acids to various types of cells, because most of them have a size of primary endosomal compartments of 100-120 nm. (Cristano R. J. et al., 1995).

Thus, the polycationic part of the molecular conjugates that we developed ensures their binding to nucleic acids (DNA or RNA) and their transportation to the cell nucleus. The ligand part serves for receptor-mediated internalization of the complex, thus ensuring its penetration into the cell.

The implementation of the invention

Peptide synthesis reagents and amino acid derivatives (Sigma Chemical Co., Fisher Scientific, Bachem, USA; Reanal, Hungary; Saxon Biochemicals GMBH, Germany), 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) - phenoxyacetamido-norleucylaminomethyl polymer (Gl Biochem, China). Dimethylformamide was distilled in vacuo before use and stored over 4 Å molecular sieves. The identity of amino acid derivatives was monitored by TLC on Merck F 254 plates (Germany) in the following solvent systems: 1% ammonia-sec-butyl alcohol, 1: 3 (A); chloroform-methanol, 9: 2 (B); chloroform-methanol-acetic acid, 90: 7: 3 (B). Chromatograms were visualized by UV irradiation at 254 nm and carbonization in sulfuric acid.

Solid-phase synthesis of peptides was carried out on a NPS-4000 semi-automatic synthesizer (Neosystem Laboratoires, France).

The obtained peptides were analyzed by HPLC using a System Gold chromatograph (Beckman, United States) on Zorbax 300SB-C18 columns (4.6 × 150 mm, 5 μm) for analytical chromatography and Discovery C18 (10 × 250 mm, 5 μm) for preparative. Chromatography conditions: UV detection at 230 nm, a gradient of acetonitrile in 0.1% H ₃ PO ₄ at a flow rate of 1 ml / min for analytical and a gradient of acetonitrile in 0.1% TFA at a flow rate of 5 ml / min for preparative chromatography. Amino acid analysis was carried out on a Microtechna T339M analyzer (Czechoslovakia) after hydrolysis of 6 N peptides. HCl at 110 ° С for 24 h. Mass spectra were recorded on an MX-5303 time-of-flight mass reflectron with an electrospray-type ion source.

The peptides used in the work were synthesized on a 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamido-norlecylaminomethyl polymer with an amino group content of 0.5 mmol / g. To protect the lateral function, Ser, Thr, and Tyr used the Bu ^t group, for Arg — Pbf, for Lys and Trp — BOC, and for His — Trt. An Fmoc group served as a temporary N ^α protection.

Peptide chain growth was carried out on a NPS-4000 semi-automatic peptide synthesizer (Neosystem Laboratoires, France) according to the following protocol for each cycle: 1). CH ₂ Cl ₂ (1 min); 2). DMF (1 min); 3). 20% piperidine / DMF (2 and 8 min); four). DMF (3 × 1 min); 5). Isopropyl alcohol (3 × 1 min); 6). DMF (3 × 1 min); 7). Condensation reaction (2 h); 8). DMF (2 x 1 min); 9). CH ₂ Cl ₂ (1 min).

Four-fold excesses of activated 1-hydroxybenzotriazole esters of the corresponding Fmoc amino acids obtained in situ (1 equivalent of Fmoc amino acids, DIC and HOBt, stirring in DMF at 0 ° С, 30 min) were used for condensation. At the end of the reaction, an acylation test was performed. If necessary, re-condensation used the following protocol:

but). DMF (1 min); b) 5% DIEA / DMF (1 and 2 min), after which stages 6) - 9) were repeated.

In the case of incomplete passage of the condensation reaction when it is repeated 1-2 times, unreacted groups were acetylated (after washing with CH ₂ Cl _2, 10 equivalents of acetic anhydride, 10 equivalents of 10% DIEA / CH ₂ Cl ₂ were added to the peptidyl polymer and stirred for 10 min).

The synthesized peptides were cleaved from the polymer with simultaneous release by the action of a mixture of TIS: H ₂ O: TFA (2.5: 2.5: 95; 5 ml / 0.5 g of resin) for 2 h. The reaction mixture was diluted with chilled ether and the precipitate was filtered off on a Schott filter. The peptide was separated from the polymer by dissolution in TFA and re-precipitated with ether. The precipitated product was filtered off, washed with ether and dried in vacuo. Further purification was performed using reverse phase HPLC.

Amino acid compositions and molecular weights calculated from mass spectrometry data for all synthesized peptides corresponded to theoretical values.

The plasmid pCMVluc, which is the expression vector of the luc marker gene (encodes firefly luciferase) under the control of the promoter of early human cytomegalovirus genes, was constructed by genetic engineering (Ignatovich I.A. et al., 2002; Ignatovich I. A. et al., 2003).

The formation of DNA complexes with molecular conjugates was carried out in phosphate-buffered saline (PBS) at different ratios of DNA and peptide charges in the reaction medium according to (Ignatovich I.A. et al., 2002). The dependence of the stability of the DNA / conjugate complexes on the ionic strength of the reaction mixture was determined by introducing the necessary amounts of NaCl into the reaction. The formation of DNA / conjugate complexes was controlled by agarose gel retardation. In experiments to determine the sensitivity of DNA in the complexes to DNase I (Fermentas, Lithuania), after completion of complexation, MgCl _{2 was} added to the reaction medium (30 μl) to a concentration of 3 mM and 3 units. DNase I (based on 1 unit of DNase I per 1 μg of DNA) and incubation was performed at 37 ° C for 30 minutes. The analysis of the DNase-treated complexes was carried out by gel retardation. The study of stoichiometry of DNA / conjugate complexes was carried out as described previously (Ignatovich IA et al., 2003). DNA complexes with conjugates were prepared in saturating excesses of the peptide, followed by purification of the unbound peptide by ultrafiltration using centric centrifugation YM-10 cones (Millipore). The amount of peptide included in the complex was determined by reverse titration with heparin.

Transfection of HepG2 and VH10 cells with pCMVluc / NLS-TSF7 and pCMVluc / NLS-TSF12 complexes was performed as previously described (Diezhe E. B. et al., 2006). Cells were plated at a density of 10 cells / cm on Petri dishes (30 mm in diameter) and grown to a state of 60-70% monolayer in DMEM supplemented with 10% fetal calf serum (Biolot, Russia) at 37 ° C in an atmosphere of 5% CO ₂ . DNA / peptide complexes with different ratios of DNA and peptide charges were prepared from 12 μg DNA per Petri dish in 500 μl PBS. When added to the cells, CaCl ₂ was introduced into the medium to a concentration of up to 20 mM. After incubation of cells with complexes for 2 h, the cells were washed from non-internalized complexes with a heparin solution (58 μg / ml in PBS) and incubated in DMEM with 10% serum for 24 h.

Luciferase activity was measured using a commercial Promega luciferase activity evaluation system (Luciferase Assay System, Cat. No. E4030) in accordance with the manufacturer's protocol. Luminescence measurements were performed on a Turner Biosystem 20/20 luminometer. Determination of protein concentration in cell lysates was carried out according to the Bradford method. Luciferase activity was expressed in relative light units (RLU), representing the number of flashes per minute per 1 mg of total protein of cell extracts. Background luminescence values did not exceed 120 RLU.

The developed gene transfer system in laboratory animals was tested on a model of wound treatment using both a plasmid with luciferase and a therapeutic plasmid encoding a synthetic gene of insulin-like growth factor I type (IGF-1), which we developed earlier (Application No. 2007124538/20 (026721)). We used mice of the DBA-2 strain at the age of 6 weeks (obtained from the Rappolovo cattery RAMS). Before applying wounds, the skin of experimental animals was prepared - the hair was removed. The cut wounds were applied with 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 with an insulin syringe into the lower back. Injections of experimental preparations were applied with an insulin syringe in the form of 10 injections into the wound area. Plasmid DNA and plasmid DNA complexes with a molecular conjugate were introduced in an amount of 50 μg DNA per mouse in 100 μl PBS containing up to 20 mM CaCl ₂ . To measure luciferase activity on day 3 after transfer to pCMVluc mice, animals were sacrificed and a tissue site, including a wound, was excised. For the experiment, 100 mg of tissue of each animal was used. Samples were homogenized in Reporter Lysis Buffer (Promega, USA, Cat. No. E3971), frozen and centrifuged for 3 min at 10,000 g. The supernatant was used to measure luciferase activity and protein concentration as described above.

The expression of the synthetic IGF-1 gene was assessed on the first, third, and seventh days after transfer to pCMVIGF-1 mice by RealTime RT-PCR. Tissue samples were obtained as described above and used to isolate RNA. As a negative control, mice not treated with pCMVIGF-1 were used. Isolation of RNA from tissues was performed using the TRI Reagent kit manufactured by Sigma, USA, according to the manufacturer's instructions. Per 100 mg of cells, 2 ml of TRI Reagent lysis solution was used. RNA concentration was determined spectrophotometrically. The isolated RNA was treated with DNase 1. The absence of contamination of the isolated RNA with the plasmid was verified by PCR using primers for the IGF-1 gene. CDNA synthesis was performed using the Revert Aid® First Strand cDNA Synthesis Kit (manufactured by Fermentas, Lithuania). The relative mRNA content was measured using real-time PCR using SYBR GREEN (SYBR Green Supermix (Bio-Rad, USA). All samples were normalized by the expression level of the β-actin “housekeeping” gene. The reaction was carried out using the IQ5 thermal cycler (Bio-Rad, USA), preliminary analysis of the data was carried out using the included software. Amplification efficiency was determined by an external standard curve using a series of sample dilutions of 5 ng, 10 ng, 30 ng, 50 ng. Calculated efficiency the amplification for the primers used was 0.98 conventional units The reaction without the addition of DNA was used as a negative control.The data were normalized by the amount of tissue, RNA and cDNA concentrations.The calculations were performed by the ΔCt method, according to the formula R = (1 + E) ^ΔCt , where Ct - threshold cycle, ΔCt = Ct (Mod) norm - Ct (Nat) norm, E - amplification efficiency, R - increase in the amount of PCR product after n cycles.

The measurement of the area of the wound on the body of experimental animals was carried out by photographing the affected area. Analysis of photographic material is performed using ImageTool for Windows (version 2.0, USA), a computer-based image analysis system, in accordance with the basic principles of stereology in morphometry.

Were synthesized two molecular conjugates that differ in the 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-NH _2.

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-NH _2.

The molecular weight of NLS-TSF-7 is 1828.26 (average weight); 1827.12 (monoisotopic); NLS-TSF-12 - average molecular weight 2426.00, monoisotopic - 2424.38. Figure 1 shows the results of mass spectroscopic analysis of NLS-TSF-12 (a) and NLS-TSF-7 (b). The molecular weights calculated from the mass spectrometry data for both conjugates corresponded to theoretical values. Both synthesized molecular conjugates contained a modified nuclear localization signal of the SV40 virus T-antigen (NLS-peptide) as a cationic component. The choice of the NLS peptide was determined by our previous studies. Earlier, we studied in detail the nature of the complexation of plasmid DNA with cationic peptides K8 and Tat and investigated the mechanisms of internalization of such complexes by mammalian cells (Ignatovich I. et al., 2003; Diezhe E. B. et al., 2006). It was found that the main way for cells to absorb DNA complexes with cationic peptides is adsorption endocytosis. Adsorption endocytosis is a non-specific process that does not depend on the presence of any receptors on the cell surface. When developing a system for targeted delivery of DNA to mammalian cells, the absorption of DNA complexes with molecular conjugates by adsorption endocytosis will lead to a decrease in tissue specificity of transfer. Preliminary studies have shown that, despite effective binding to plasmid DNA and its subsequent compactification, the NLS peptide was unable to deliver DNA to cultured human cells (human hepatoma cells HepG2, human skin fibroblasts, human neuroblastoma cells Sk-N-SH). Thus, the choice of the NLS peptide for creating the conjugate is due to its ability to bind nucleic acids and ensure the delivery of the complex to the cell nucleus. A study of the conditions for the formation of DNA / conjugate complexes is presented in Fig. 2. The gel retardation results of pCMVluc plasmid DNA complexes with the molecular conjugates NLS-TSF-7 and NLS-TSF-12 are shown. Both peptides efficiently bind plasmid DNA at isotonic values of the ionic strength of the reaction medium.

Figure 3 shows the dependence of the efficiency of complexation on the ionic strength of the reaction medium. It was found that both molecular conjugates are not stable when the ionic strength of the solution exceeds 300 mm. Despite the same cationic moieties, NLS-TSF-7 turned out to be more stable than NLS-TSF-12, which is most likely explained by the higher density of the positive charge in the case of NLS-TSF-7 (molecular weight of a positive charge of 304.71) compared with NLS-TSF-12 (molecular weight of a positive charge of 404.3).

The stoichiometry of DNA / conjugate complexes was studied by purification of complexes prepared in the presence of saturating amounts of peptides (tenfold excess in charges) from an unbound peptide by ultrafiltration. As a result, a complex containing saturating amounts of the peptide was isolated. It is known that heparin, being a stronger polyanion than DNA, is able to displace it from the complex with polycations (Xu Y. et al., 1996; Ramsey E. et al., 2002). The DNA content in the isolated complex was determined spectrophotometrically after its complete destruction by an excess of heparin. The peptide content in the studied complex was determined by reverse titration with heparin. For this purpose, a calibration curve was constructed that shows the amount of heparin sufficient for complete destruction of the DNA / conjugate complexes on the peptide content in the reaction medium during the formation of the complexes (Fig. 4a). It was found that the maximum possible content of NLS-TSF-7 in the complexes corresponds to a 1.5-fold excess of positive NLS-TSF-7 charges relative to negative DNA charges. In the case of NLS-TSF-12, the maximum possible conjugate content in the complexes corresponds to a 1.3-fold excess of positive charges (Fig. 4b). Similar results were obtained by us earlier for DNA complexes with a TAT peptide (Ignatovich I. et al., 2003) and K8 (Diezhe E.B. et al., 2006).

The interaction of DNA with polycations leads to its compaction and, as a result, to protection against nucleases (Niidome T. et al., 1997; Liu G. et al., 2001). Moreover, the degree of DNA compaction in complexes directly correlates with its sensitivity to nucleases. Figure 5 shows the results of processing DNA / conjugate complexes prepared at different ratios of DNA and peptide charges with DNase I. As expected, an increase in the peptide content in the reaction medium leads to a greater protection of DNA from degradation. DNA in complexes prepared at a ratio of negative DNA charges to positive peptide charges of 1: 1 (electrically neutral complexes) is only partially protected from degradation (Fig. 4-5, lane 4). Complete resistance to the action of DNase I is observed only in complexes prepared with a charge ratio of less than 1: 1.5. The data obtained are in good agreement with the results of studying the stoichiometry of DNA / conjugate complexes (Fig. 4).

Assessment of the sensitivity of DNA / conjugate complexes to DNase I made it possible to determine yet another important parameter of complexation — the cooperativity of DNA interactions — the polycation. Two models of complexation are possible. In the case of cooperative interaction, negatively charged DNA / polycation complexes have a greater affinity for a free polycation than unbound DNA. Therefore, with a ratio of DNA and polycation charges greater than 1: 1 (excess DNA), the reaction medium will contain a mixture of electrically neutral (or having a small positive charge) complexes and free DNA molecules. If the interactions are non-cooperative, the polycation molecules are randomly distributed among DNA molecules, as a result, the reaction medium will contain a more or less homogeneous mixture of complexes, and their charge will be determined by the initial ratios of DNA and polycation (Liu G. et al., 2001). In the cooperative nature of the interactions of DNA with a polycation, treatment with DNase I of complexes prepared with a charge ratio of more than 1: 1 will lead, on the one hand, to complete degradation of the unbound DNA contained in the reaction medium, and, on the other hand, to complete protection of the DNA that formed complexes with polycation. In the case of non-cooperative interaction, treatment with DNase I of negatively charged complexes will lead to partial degradation of all DNA, and the size of the degradation products will increase with increasing polycation content in the complexes. The dependence of DNA stability in the complexes with NLS-TSF-7 against the action of DNase I on the ratio of DNA and peptide charges 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 efficiently compact plasmid DNA with the formation of polyelectrolyte complexes allowed us to proceed to the next task - the study of factors affecting the efficiency of penetration of such complexes into mammalian cells expressing transferrin receptors. HepG2 human hepatoma cells and VH10 human fibroblasts were transfected with pCMVluc / NLS-TSF7 and pCMVluc / NLS-TSF12 complexes. Both complexes turned out to be transfectionally active. Moreover, high excesses of free molecular conjugates (50x) led to the suppression of the level of transfection, which indicates the role of transferrin receptors in the binding of DNA / conjugate complexes to the cell surface (Fig. 6). Moreover, the NLS-TSF7 molecular conjugate provided a higher level of cell transfection compared to NLS-TSF12 (~ 2.5 times) (Fig. 6). The figure shows the results of the luciferase test, demonstrating the influence of various factors on the efficiency of transfection of HepG2 cells. In the case of fibroblasts, similar results were obtained (data not shown). In previous works, we showed the stimulating role of free cationic peptides Tat and K8 in the internalization of DNA complexes with cationic peptides. Under the same conditions, free peptides did not significantly affect the efficiency of cell transfection with DNA complexes with molecular conjugates containing synthetic luliberins as a ligand component. It was interesting to carry out similar studies regarding DNA complexes with NLS-TSF7 and NLS-TSF12. It was found that moderate excesses of free conjugates in the culture medium do not significantly affect the level of cell transfection. In this regard, they are similar to molecular conjugates based on synthetic analogues of luliberin. Apparently, this similarity is explained by the general mechanism of internalization of complexes - receptor-mediated endocytosis, in contrast to DNA complexes with cationic peptides Tat and K8, where adsorption endocytosis plays the main role in internalization (Ignatovich IA et al., 2003; Diezhe E. B . et al., 2006; Efremov A.M. et al., 2009).

The role of endocytosis in cell transfection with DNA complexes with the molecular conjugates NLS-TSF7 and NLS-TSF12 was tested in experiments conducted in the absence of Ca ^{2+ ions} .

It is known that both receptor-mediated and adsorption endocytosis are inhibited in the absence of Ca ^{2+ ions} . It was found that the removal of CaCl ₂ from the culture medium leads to an almost complete suppression of the expression level of the transferred gene using both conjugates (Fig. 6).

Another important factor that significantly affects the efficiency of cell transfection with polyelectrolyte complexes is the presence of blood serum proteins in the culture medium. Earlier, we showed that transfection of cells with DNA complexes with a Tat cationic peptide and with molecular conjugates based on luliberin analogues in the presence of blood serum proteins leads to a significant decrease in the level of expression of the transferred gene (Ignatovich I. A. et al., 2003; Efremov A. M . and others, 2009). This may be critical in the development of in vivo gene transfer systems. Similar experiments were carried out with the molecular conjugates NLS-TSF7 and NLS-TSF12. It was established that the introduction of 10% embryonic bovine serum (FCS) during transfection of cells into the culture medium led to a decrease in the level of expression of the transferred gene (Fig. 6). It is interesting to note that in the presence of serum, the level of transfection of cells with DNA complexes with NLS-TSF12 turned out to be higher than in the case of DNA complexes with NLS-TSF7, although the opposite was observed in the absence of serum. This circumstance must be taken into account in further development of non-viral gene transfer systems based on synthesized molecular conjugates. For example, the molecular conjugate NLS-TSF7 seems to be more optimal for a system of regenerative gene therapy of superficial tissue damage in mammals, whereas in the case of gene transfer work requiring systemic administration of drugs into the bloodstream, NLS-TSF12 may be preferable.

Studies have been conducted of the ability of complexes of plasmid DNA with the developed molecular conjugates of NLS-TSF7 to penetrate into cells in vivo, which can be used for gene therapy or diagnosis. As a model, surface wounds of tissues of mice of the DBA-2 line (males, 6 weeks) were used. The pCMVluc / NLS-TSF7 complexes (charge ratio 1: 1) were either injected into the skin of healthy mice (10 injections around an area of ~ 60 mm ² ), or they cut a wound of a similar area. The number of complexes was 50 μg of DNA per mouse. A control group of mice was injected with the same amount of free plasmid pCMVluc. Before the experiment, the skin of the experimental animals was prepared - the hair was removed. Wounds were applied with a surgical scalpel to a depth of 2 mm, capturing the underlying muscle tissue in the lower back. 5 days after the injection, the mice were sacrificed, and the area of the skin near the injections was used to obtain lysates in which luciferase activity was evaluated. The results are presented in Fig. 7. The use of pCMVluc / NLS-TSF7 complexes during injection led to an increase in luciferase activity by more than 100 times compared with free DNA injections. There was no significant difference between injections in healthy animals and animals with cut wounds (Fig. 7). Thus, the synthesized molecular conjugate NLS-TSF7 is able to efficiently transfer genes to the surface tissues of mammals.

The following example is given for the purpose of illustrating one embodiment of the invention and is not intended to limit the present invention in any way.

Example 1

The developed molecular conjugates in combination with DNA were tested for gene therapy in a model of regenerative gene therapy in laboratory animals. A test of the ability of DNA complexes with the NLS-TSF7 molecular conjugate to penetrate into the surface tissue cells of mice was performed on DBA-2 mice (males, 6 weeks). For this purpose, mice with surface tissue injuries were transferred using the NLS-TSF7 molecular conjugate and the expression vector of the synthetic gene encoding the human type I insulin-like growth factor pCMVIGF-1s (application No. 2007124538/20 (026721) was developed by us. mice of a 6-week-old DBA-2 line were used in the experiment. Before applying the wounds, the skin of the experimental animals was prepared - the hair was removed. The wounds were applied using a surgical scalpel to a depth of 2 mm, capturing the subject we tissue in the lower back. According to the nature of the damage, these wounds are classified as “cut." Injections of the experimental preparations were applied with an insulin syringe in the form of 10 injections into each wound. In the experiment, 4 groups of animals were formed. In each group, 10 mice. Experienced the groups were injected with either free pCMVIGF-1s or pCMVIGF-1s / NLS-TSF7 complexes (charge ratio 1: 1) .Injections were performed ten times in the areas surrounding the wound. For injection, 50 μg of DNA per mouse was used in 100 μl of PBS containing 4 mM CaCl ₂ . Control groups received plasmid DNA injections with a deleted promoter (pAIGF-1s) and free NLS-TSF7. On days 3, 5, and 7, two mice from each group were killed and RNA was isolated from regions adjacent to the wound, which was used to quantify the expression of IGF-1 by RealTime RT-PCR. The results are shown in Fig. 8. The level of IGF-1 gene expression on days 3 and 5 after the start of the experiment was higher from 64 to 128 times with injections of pCMVIGF-1s / NLS-TSF7 complexes compared to injections of free pCMVIGF-1s. In control groups injected with a plasmid with a delegated promoter or a free molecular conjugate of NLS-TSF7, no expression of IGF-1 was detected. The data obtained confirm the previously obtained results on the high efficiency of the NLS-TSF7 molecular conjugate in the delivery of genes to the surface tissues of mammals.

The regeneration rate was estimated by measuring the area of the wounds on days 7, 10, 14, and 21 after the start of the experiment.

The results are summarized in Table 1. Figure 9 shows photographs of mice on the 14th day after the start of the experiment, injected with free NLS-TSF7 and pCMVIGF-1s / NLS-TSF7 complexes. Injections of both free pCMVIGF-1s and pCMVIGF-1s / NLS-TSF7 complexes significantly accelerated wound healing compared to control groups. 14 days after the start of the experiment, mice injected with pCMVIGF-1s / NLS-TSF7 complexes underwent complete wound healing, while in the control groups the wound area was about 10 mm ² (~ 17% of the initial size) (Table 1). Moreover, in mice injected with pCMVIGF-1s / NLS-TSF7 complexes, on the 10th day after the start of the experiment, the wound size was two times lower than in mice injected with a free plasmid and 4 times lower than in control groups. The results obtained indicate the high efficiency of the developed gene delivery system for gene therapy, including regenerative gene therapy of surface damage to mammals.

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Table 1 Molecular conjugates with a polycationic site and a ligand for delivery of DNA and RNA into the cell and cell nucleus Group of animals Time 1 day 7 day 10 day 14 day 21 day pCMVIGF-1s 60 ± 13.5 28.5 ± 6 12 ± 3 0 0 pCMVIGF-1s / NLS-TSF7 57 ± 11.2 24 ± 7.5 6 ± 2 0 0 pΔIGF-1s 66 ± 20 42 ± 9 24 ± 5.5 10.5 ± 6 0 NLS-TSF7 62 ± 13.5 46.5 ± 8 28.5 ± 6 10.5 ± 4.5 0

The table shows the average values of the area of the wound (in mm ² ) in the experimental and control groups of mice: pCMVIGF-1s — mice injected with a free expression vector of an insulin-like growth factor I type human; pCMVIGF-1s / NLS-TSF7 — mice injected with pCMVIGF-1s / NLS-TSF7 complexes; pΔIGF-1s — mice injected with a free expression vector of an insulin-like human growth factor I type I with a delegated promoter; NLS-TSF7 — mice injected with the free NLS-TSF7 molecular conjugate.

Claims (12)

1. The molecular conjugate with the amino acid sequence of SEQ ID NO: 4, capable of binding to a nucleic acid (DNA or RNA) to deliver it to a mammalian cell expressing transferrin receptors, consisting of a polycation sequence and a ligand in which the polycation sequence is represented by a modified nuclear localization signal The SV40 virus T antigen has the amino acid sequence of SEQ ID NO: 3, and the ligand has the amino acid sequence of SEQ ID NO: 1. 2. A molecular conjugate with the amino acid sequence of SEQ ID NO: 5, capable of binding to a nucleic acid (DNA or RNA) to deliver it to a mammalian cell expressing transferrin receptors, consisting of a polycation sequence and a ligand in which the polycation sequence is represented by a modified nuclear localization signal The virus T-antigen is SV40 and has the amino acid sequence of SEQ ID NO: 3, and the ligand has the amino acid sequence of SEQ ID NO: 2. 3. The complex of the molecular conjugate according to claim 1 with a nucleic acid (DNA or RNA), designed for transfection of mammalian cells expressing transferrin receptors. 4. The complex of the molecular conjugate according to claim 2 with a nucleic acid (DNA or RNA), designed for transfection of mammalian cells expressing transferrin receptors. 5. The molecular conjugate complex according to claim 3 or 4, characterized in that the nucleic acid is a plasmid. 6. The drug for gene therapy or diagnosis, consisting of a complex of a molecular conjugate with a nucleic acid according to claim 4 in an amount of 0.1 μg (in terms of the content of nucleic acid) and a pharmaceutically acceptable carrier that ensures the stability of the complex, intended for delivery to mammalian cells expressing transferrin receptors, nucleic acids encoding therapeutic genes or marker genes for diagnostic purposes. 7. A preparation for gene therapy or diagnosis, consisting of a complex of a molecular conjugate with a nucleic acid according to claim 5 in an amount of 0.1 μg (in terms of the content of nucleic acid) and a pharmaceutically acceptable carrier ensuring the stability of the complex, intended for delivery to mammalian cells expressing transferrin receptors, nucleic acids encoding therapeutic genes or marker genes for diagnostic purposes. 8. The drug for gene therapy according to claim 6 or 7, characterized in that it is used for regenerative gene therapy. 9. The drug for gene therapy according to claim 8, characterized in that the nucleic acid encodes an insulin-like growth factor type I person. 10. The drug according to claim 8 or 9, characterized in that it uses a solution of CaCl ₂ at a concentration of 20 mm. 11. The method of forming the complex according to claim 3 or 4, which consists in the first stage in the fusion of solutions of the molecular conjugate and nucleic acid, followed by purification of the resulting complexes from unbound molecular conjugate by ultrafiltration in the second stage and then in the third stage - adding a solution of CaCl _2. 12. The method of forming a complex according to claim 3 or 4, characterized at the stage of fusion of nucleic acid and molecular conjugate solutions with an appropriate charge ratio in the reaction medium of 1: 0.63 or less and using solutions with ionic strength less than 300 mM for complexation.

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